Новые доказательства фармакологической активности и возможных молекулярных мишеней полисахаридов ягоды Годжи

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Hypolipidemic effects

Based on data from the 2005–2008 National Health and Nutrition Examination Survey, an estimated 71 million (33.5%) US adults aged ≥20 years had high LDL-C, but only 34 million (48.1%) were treated and 23 million (33.2%) had their LDL-C controlled.117 Control of high LDL-C can reduce cardiovascular morbidity and mortality substantially. Luo et al107 investigated the hypolipidemic effect of LBPs on alloxan-induced hyperlipidemic rabbits. LBPs significantly reduced serum total cholesterol and triglyceride concentrations and markedly increased HDL-C levels after treatment with LBPs for 10 days in rabbits.107 LBPs also showed potent antioxidant activities in hyperlipidemic rabbits. These data demonstrate that the hypolipidemic effect of LBPs and anti-oxidation should contribute to this effect.

Immunomodulating effects

Many naturally occurring polysaccharides have been reported to be potent immunomodulators.118,119 These polymers can influence innate and cell-mediated immunity through interactions with T cells, monocytes, macrophages, and polymorphonuclear lymphocytes. LBPs have been found to have a variety of immune-modulatory activities in vitro and in vivo.

T cells, B cells, and splenocytes

Chen et al120 compared the immunomodulating effects of different LBP fractions in mice. Crude LBPs isolated from L. barbarum were separated to obtain five homogeneous fractions, namely LBPF1, LBPF2, LBPF3, LBPF4, and LBPF5. The study showed that LBP, LBPF4, and LBPF5 significantly stimulated mouse splenocyte proliferation. The proliferation proved to be of T cells, but not B cells. Cell cycle analysis indicated that LBP, LBPF4, and LBPF5 markedly reduced sub-G₁ cells.120 LBP, LBPF4, and LBPF5 activated the transcription factors nuclear factor of activated T-cells (NFAT) and activator protein-1 (AP-1), prompted CD25 (ie, IL-2 receptor-α) expression, and induced IL-2 and interferon (IFN)-γ expressions. NFAT proteins (NFATs 1–5) have crucial roles in the development and function of the immune system. In T cells, NFAT proteins not only regulate activation but are also involved in the control of thymocyte development, T-cell differentiation, and self-tolerance.121 AP-1 regulates gene expression in response to a variety of stimuli, including cytokines, growth factors, stress, and bacterial and viral infections. IL-2 is important for the growth and activation of T cells, and IFN-γ is an important activator of macrophages and inducer of class II major histocompatibility complex (MHC-II) molecule expression. IL-2 is mainly produced by T cells122 and IFN-γ is produced predominantly by NK and NK T cells as part of the innate immune response, and by CD4⁺ T helper cells (Th₁) and CD8⁺ CTLs once antigen-specific immune response is triggered.123 Administration of LBPs to mice (intraperitoneal [ip] or oral administration [po]) significantly induced T-cell proliferation.120 These results suggest that activation of T lymphocytes by LBPs may contribute to one of their immuno-enhancement functions.

The in vitro and in vivo immunomodulating effects of LBPF4-OL on mouse splenocytes, T cells, B cells, and macrophages were investigated by Zhang et al.124 LBPF4-OL was the glycan part of L. barbarum polysaccharide–protein complex fraction 4 (LBPF4). Splenocytes were stimulated with LBPF4-OL and cytokine concentrations in the supernatants were determined. In the in vivo study, mice were intraperitoneally injected with 100 µg/mL LBPF4-OL daily for 6 days. The results showed that LBPF4-OL markedly induced the splenocyte proliferation, but could not induce proliferation of purified T- and B-lymphocytes.124 B-cell proliferation occurred in the presence of activated macrophages or lipopolysaccharide (LPS). LBPs obviously induced IL-6, IL-8, IL-10, and TNF-α production in splenocytes in a concentration-dependent manner.124 IL-6 is secreted by T cells and macrophages to stimulate immune response during infection and after trauma (especially burns or other tissue damage) leading to inflammation. IL-8 (also called CXCL8 and neutrophil chemotactic factor) is a chemokine produced by macrophages and other cell types such as epithelial cells, airway smooth muscle cells, and endothelial cells. IL-8 induces chemotaxis in target cells (primarily neutrophils but also other granulocytes) toward the site of infection and induces phagocytosis. IL-10 inhibits the production of IFN-γ, IL-2, IL-3, TNF-α, and granulocyte-macrophage colony-stimulating factor (GM-CSF) by activated macrophages and helper T cells. TNF-α is mainly produced by activated macrophages (M₁), although it can be produced by many other cell types such as CD4⁺ T lymphocytes, monocytes, NK cells, neutrophils, mast cells, eosinophils, and neurons.125 The primary role of TNF-α is in the regulation of immune cells. TNF-α induces fever, apoptotic cell death, cachexia, and inflammation; inhibits tumorigenesis and viral replication; and responds to sepsis via IL-1 and IL-6 producing cells.125 Flow cytometer analysis showed that LBPF4-OL prompted CD86 (B7-2) and MHC-II molecule expression on macrophages and greatly promoted release of TNF-α and IL-1β from macrophages.124 IL-1β is produced by activated macrophages as a proprotein, which is proteolytically processed to its active form by caspase 1. This cytokine is an important mediator of the inflammatory response and is involved in a variety of cellular activities, including cell proliferation, differentiation, and apoptosis.

Vidal et al126 revealed that dietary wolfberry supplementation enhanced both in vivo (delayed-type hypersensitivity) and ex vivo (T-cell proliferation) T-cell response to specific antigens, but it did not affect mitogen-induced T-cell or B-cell proliferation in young and aged mice. Over 44 days, young-adult (2 months) and aged (21 months) C57BL/6J mice were fed ad libitum with a controlled diet and received drinking water supplemented or not with 0.5% (wt/vol) Lacto-Wolfberry. All mice were immunized on day 15 and challenged on day 22 with a T-cell-dependent antigen, keyhole limpet hemocyanin. The study showed that Lacto-Wolfberry supplementation significantly increased in vivo systemic immune markers.126 Both antigen-specific humoral response and cell-mediated immune responses in young-adult and aged mice were enhanced. However, no significant effect of Lacto-Wolfberry supplementation was observed on ex vivo splenocyte proliferative response to mitogens and on splenocyte T-cell subsets.126 These data suggest that dietary intake of Lacto-Wolfberry may favorably modulate the poor responsiveness to antigenic challenge observed with aging.

Zhang et al127 further compared the effect of LBPF4 and LBPF4-OL on the proliferation of splenocytes and mitogen-induced B and T lymphocytes in female Balb/C mice. LBPF4 and LBPF4-OL were isolated in the fruit bodies of L. barbarum through a series of diethylaminoethyl anion exchange cellulose and gel-permeation chromatography. The molecular weight of LBPF4 was 214.8 kDa, and consisting of 17 amino acids and four kinds of monosaccharides. The molecular weight of LBPF4-OL was 181 kDa, consisting of three types of monosaccharides.127 The effects on cytokine secretion, the phagocytic potential of macrophages, and the expression level of intracellular signaling molecules including NF-κB and B-cell-specific activator protein (BSAP, also named Pax5) were also determined. BSAP/Pax5 is essential for commitment of lymphoid progenitors to the B lymphocyte lineage.128 Spleen cells (5×10⁵) were stimulated with 10 µg/mL, 50 µg/mL, and 100 µg/mL LBPF4-OL. Concanavalin A (Con A, 0.5 µg/mL) and LPS (5 µg/mL) were included as positive controls for the proliferation of T and B cells, respectively. The results showed that 50 µg/mL LBPF4 significantly enhanced spleen cell proliferation ∼3.2 fold, while LBPF4-OL enhanced proliferation 7.2 fold. Administration of 10 µg/mL, 50 µg/mL, or 100 µg/mL LBPF4 but not LBPF4-OL, significantly enhanced the Con A-induced T lymphocyte proliferation.127 However, LPS-induced B-cell proliferation was enhanced by 10 µg/mL, 50 µg/mL, or 100 µg/mL of both LBPF4 and LBPF4-OL. Administration of 50 µg/mL LBPF4-OL was more effective on inducing the proliferation of splenocytes and LPS-stimulated B cells than 100 µg/mL LBPF4. LBPF4 appeared to induce lymphocyte proliferation predominantly depending on both B and T cells, and LBPF4-OL induced lymphocytes proliferation only depending on B cells. The stimulation of murine peritoneal macrophages with LBPF4 and LBPF4-OL resulted in a comparable dose-dependent increase of the production of TNF-α and IL-1β.127 In addition, both LBPF4 and LBPF4-OL at concentrations of 10 µg/mL, 50 µg/mL, and 100 µg/mL increased the secretion of NO to comparable levels. Administration of 10 µg/mL LBPF4 and LBPF4-OL showed no significant effects on the phagocytic activity of resting macrophages in mice, but the macrophage chicken erythrocyte phagocytic activity was significantly increased by low concentrations of LBPF4 and LBPF4-OL. About 50 µg/mL (but not 10 µg/mL) LBPF4 and LBPF4-OL significantly promoted BSAP and NF-κB activity.127 These data suggest that LBPF4-OL can only enhance B cell and macrophage functions, but polysaccharide–protein complex LBPF4 can enhance the function of both T and B cells and macrophages.

Recently, Zhang et al129 found that LBPF4-OL acted as an activator of the Toll-like receptor 4 (TLR4)/p38 MAPK signaling pathway using TLR4 knockout mice. LBPF4-OL significantly induced TNF-α and IL-1β production in peritoneal macrophages isolated from wild type (C3H/HeN) but not TLR4-deficient mice (C3H/HeJ). The proliferation of LBPF4-OL-stimulated lymphocytes from C3H/HeJ mice was significantly lower than that of lymphocytes from C3H/HeN mice.129 Furthermore, through a bio-layer interferometry assay, LPS but not LBPF4-OL directly associated with the TLR4/MD2 molecular complex. Flow cytometry analysis indicated that LBPF4-OL markedly upregulated TLR4/MD2 expression in both peritoneal macrophages and Raw264.7 cells.129 LBPF4-OL also increased the phosphorylation of p38-MAPK and inhibited the phosphorylation of JNK and Erk1/2. These data suggest that LBPF4-OL can activate TLR4/p38 MAPK signaling pathway.

Peripheral blood mononuclear cells

IL-2 is necessary for the growth, proliferation, and differentiation of T lymphocytes to become functional T cells.122 Antigen binding to the T-cell receptor stimulates the secretion of IL-2 by T cells and the expression of IL-2 receptors (IL-2Rs). The IL-2/IL-2R interaction stimulates the growth, differentiation, and survival of antigen-specific CD4⁺ and CD8⁺ T cells. IL-2 plays an important role for the development of T-cell-dependent immune memory. An in vitro study reported the effects of LBPs on the expression of IL-2 and TNF-α in human peripheral blood mononuclear cells (PBMCs) from healthy volunteers.130 The LBPs used in this study were the third fraction of LBPs extracted with hot water from L. barbarum planted in Zhongning, Ningxia, People’s Republic of China and isolated by anionic exchange chromatography and gel-filtration chromatography. Administration of 10 mg/L LBPs increased the expression of IL-2 and TNF-α at both mRNA and protein levels in a dose-dependent manner. Treatment of human PBMCs with 5 mg/L, 10 mg/L, 20 mg/L, and 40 mg/L LBPs increased IL-2 mRNA 1.8-, 3.9-, 7.0-, and 7.4-fold, respectively. The activity of IL-2 was increased 4.3-, 7.7-, 14.2-, and 16.0-fold, respectively, compared to the negative control.130 Treatment of PBMCs with 5 mg/L, 10 mg/L, 20 mg/L, and 40 mg/L LBPs increased TNF-α mRNA level 2.4-, 3.9-, 6.1-, and 15.4-fold, respectively. The activity of TNF-α after treatment with 5 mg/L, 10 mg/L, 20 mg/L, and 40 mg/L LBPs for 8 hours was increased 7.1-, 9.1-, 13.6-, and 15.2-fold, respectively, compared to the negative control. LBPs may induce immune responses that contribute to the therapeutic effect in cancer.

Macrophages

Macrophages play a crucial role in innate immunity and also help initiate adaptive immunity.131,132 Macrophages predominantly expressing the killer phenotype are called M₁ macrophages, whereas those involved in tissue repair are called M₂ macrophages.133 The primary role of macrophages is to phagocytose or engulf and then digest cellular debris and pathogens; they also stimulate lymphocytes and other immune cells to respond to pathogens. M₁ macrophages are activated by LPS and IFN-γ and secrete high levels of IL-12 and low levels of IL-10; and M₂ macrophages produce high levels of IL-10, TGF-β, and low levels of IL-12.133 IL-12 is involved in the stimulation and maintenance of Th₁ cellular immune responses and also has an important role in enhancing the cytotoxic function of NKs. Macrophages can be identified by specific expression of a number of proteins including CD14, CD40, CD11b, CD64, F4/80 (mice)/EMR1 (human), lysozyme M, MAC-1/MAC-3, and CD68. LBPs are able to activate macrophages. A study found that LBPs (50 mg/kg, ip) markedly upregulated the expressions of CD40, CD80 (B-lymphocyte activation antigen B7-1), CD86 (B-lymphocyte activation antigen B7-2), and MHC-II molecules on peritoneal macrophages. In vitro studies showed that LBPs activated transcription factors NF-κB and AP-1, induced TNF-α, IL-1β, and IL-12p40 mRNA expression, and enhanced TNF-α production in RAW264.7 macrophage cells in a dose-dependent manner.134 Furthermore, LBPs significantly enhanced macrophage endocytic and phagocytic capacities in vivo. These results indicated that LBPs enhance innate immunity by activating macrophages. The mechanism might be through activation of transcription factors NF-κB and AP-1 to induce TNF-α production and upregulation of MHC-II co-stimulatory molecules.134

An in vitro study by Teng et al135 investigated the inhibitory effects of LBPs on the production of LPS-induced proinflammatory mediators in BV2 microglia. The data showed that LPS induced the activation of NF-κB and its upstream protein caspase 3. NF-κB plays a key role in inflammatory disease and may be involved in autophagy, while autophagy itself may also participate in the pathogenesis of inflammation and inflammatory disease.136 LPS also unregulated the expression of an additional apoptosis-inducing factor with a passive role in the maturation of caspase processing, HSP60, in BV2 microglial cells and increased the release of TNF-α and HSP60 in the culture media. Following treatment with LBPs, the activated caspase 3 were significantly suppressed. Furthermore, the enhanced expression of HSP60 was reduced and the LPS-induced release of TNF-α and HSP60 was inhibited. These results suggest that LBPs may have therapeutic potential for the treatment of neurodegenerative diseases that are accompanied by microglial activation.

Peng et al137 investigated the effect of Lycium ruthenicum polysaccharides (LRGP3) on inflammatory reactions induced by LPS in mouse macrophage RAW264.7 cells. The results showed that LRGP3 treatment significantly inhibited the LPS-induced NO production and the mRNA expression of iNOS, as well as the level of TLR4. Furthermore, LRGP3 treatment prevented IκBα degradation and reduced phospho-NF-κB p65 protein expression in LPS-stimulated RAW264.7 cells.137 Meanwhile, the levels of proinflammatory cytokines, such as IL-1α, IL-6, and TNF-α, were suppressed by LRGP3 in LPS-stimulated RAW264.7 cells. LRGP3 attenuated LPS-induced inflammation via inhibiting TLR4/NF-κB signaling pathway.

Natural killers

NK cells are major effectors of the innate immunity, providing rapid responses to virally infected cells and respond to tumor formation.138,139 Cytokines involved in NK activation include IL-12, IL-15, IL-18, IL-2, and CCL5. NK cells are activated in response to IFNs or macrophage-derived cytokines. NK cells control viral infections by secreting IFN-γ and TNFα.138,139 IFN-γ activates macrophages for phagocytosis and lysis, and TNFα acts to promote direct NK tumor cell killing. NKs express the surface markers CD16 (FcγRIII) and CD56 in humans. A recent study by Huyan et al140 reported the effects of LBPs on primary human NK cells under normal or simulated microgravity conditions. The results demonstrated that LBPs markedly promoted the cytotoxicity of NK cells by enhancing IFN-γ and perforin secretion and increasing the expression of the activating receptor NKp30 under normal conditions. Meanwhile LBPs enhanced NK cell function under simulated microgravity conditions by restoring the expression of the activating receptor NKG2D and reducing the early apoptosis and late apoptosis/necrosis.140 In addition, the antibody neutralization test showed that the complement receptor CR3 may be the critical receptor involved in LBP-induced NK cells activation. These findings indicate that LBPs are potent immune regulators and can promote the immune functions of the public and astronauts during space missions.

Dendritic cells

Dendritic cells (DCs) are potent antigen-presenting cells that play pivotal roles in the initiation of the adaptive (T and B cell) immune response.141,142 The principal function of DCs is to present antigens, and only DCs have the ability to induce a primary immune response in resting naïve T lymphocytes. DCs also play a role in the maintenance of B cell function and recall responses. DCs express a variety of adhesion molecules including CD11a (integrin lymphocyte function-associated antigen-1, namely LFA-1), CD11c/CD18, CD50 (ICAM-2), CD54 (ICAM-1), CD58 (LFA-3), and CD102 (ICAM-3).141,142 CD11a/LFA-1 plays a central role in leukocyte intercellular adhesion through interactions with its ligands, ICAMs 1-3, and also functions in lymphocyte costimulatory signaling. DCs also express costimulatory molecules including CD80 (B7-1) and CD86 (B7-2), which are upregulated during DC activation. CD86 tends to be a marker of early DC maturation, while CD80 only appears in mature DC.141,142

The effects of LBPs on the phenotypic and functional maturation of murine bone marrow DCs (BMDCs) were investigated in vitro by Zhu et al.143 The co-expression of MHC-II, CD11c, and secretion of IL-12 p40 by BMDCs stimulated with 100 mg/L LBPs increased significantly. LBPs are capable of promoting both the phenotypic and functional maturation of murine BMDCs in vitro.

Chen et al144 reported that LBPs induced phenotypic and functional maturation of DCs with strong immunogenicity. LBPs can upregulate the expression of CD40, CD80, CD86, and MHC-II molecules on DCs, downregulate DC uptake of antigen, enhance allostimulatory activity of DCs, and induce the production of IL-12p40 and p70 in DCs.144 LBP-treated DCs can enhance both Th₁ and Th₂ responses in vitro and in vivo. LBPs may serve as a potent adjuvant for the design of DC-based vaccines.

Chen et al145 investigated the effect of LBPs on differentiation and maturation of healthy human peripheral blood-derived DCs cultured in different tumor microenvironment in vitro. Peripheral blood-derived DC precursor cells were obtained by the density-gradient centrifugation method, and the tumor-cell supernatants were used to prepare conditioned medium. The GM-CSF and IL-4-induced DC precursor cell differentiation to DCs, the TNF-α promoted the immature DCs developed to mature DCs. In LBP-treated groups, the molecular phenotype of DCs, their capacity to stimulate allogeneic lymphocyte proliferation, and the levels of IL-12p70 and IFN-γ secretion were higher than the untreated group.145 Meanwhile, the expression of NF-κB of the DCs in the medium treated with LBPs was higher than the untreated group.145 Between the two different tumor microenvironment groups, the nuclear NF-κB expression was obviously different. LBP could increase the expression of the phenotype of DCs via NF-κB signaling pathway.

Follicular helper T cells

Follicular helper T (Tfh) cells are recognized as a subset of helper T cells that regulate the multiple stages of B-cell maturation and function.146 Tfh cells retain intense expression of CXCR5, which directs these cells toward CXCL13-rich areas within the germinal center. Tfh cells express a number of costimulatory molecules, such as inducible costimulator and CD40L that have the capacity to restrain their interaction with B cells and antigen-presenting cells, including CTLA-4 and PD-1, which may reflect their discriminating role in the germinal center. Tfh cells also express a number of cytokines that facilitate antibody production including IL-4, IL-10, and IL-21.

A recent study by Su et al147 reported that LBPs were able to activate CXCR5⁺/PD-1⁺ Tfh cells and induced IL-21 secretion in female Balb/C mice. Mice were immunized once with ip injection of 0.2 mL of 10⁸ TCID50 rAd5VP1. LBPs were given to mice daily for 7 days by gastric gavage at 5 mg/kg, 25 mg/kg, or 50 mg/kg body weight. After 7 days, mice were sacrificed, the splenocytes were harvested, and the number of CXCR5⁺/PD-1⁺ Tfh cells was determined by three-color flow cytometry.147 Mouse splenocytes were also analyzed by flow cytometry to determine the counts of B220⁺/GL-7⁺ B cells. The study showed that LBP treatment increased the percentage of CXCR5⁺/PD-1⁺ Tfh cells within total CD4⁺ T cells; 5 mg/kg (2.17%±0.07%), 25 mg/kg (3.93%±0.74%), and 50 mg/kg (3.84%±0.20%). Administration of 5 mg/kg LBP for 7 days exhibited a minor effect on the production of IL-21, whereas 25 mg/kg and 50 mg/kg LBPs significantly increased the production of IL-21 when compared with mice treated with phosphate buffered saline only.147 LBPs also promoted the formation of germinal centers and production of B220⁺/GL-7⁺ germinal center B cells in mice. The fraction of B220⁺/GL-7⁺ B cells was significantly increased with 25 mg/kg and 50 mg/kg LBPs compared with mice receiving phosphate buffered saline only (1.80%±0.49%). Moreover, LBPs as an adjuvant increased generation of rAd5VP1-induced Tfh cells in mice.147 There was a marked increase in the number of CXCR5⁺/PD-1⁺/CD4⁺ Tfh cells and B220⁺/GL-7⁺ germinal center B cells in mice immunized with 10⁸ TCID50 rAd5VP1 plus LBPs. These results indicate that LBPs may enhance T-cell-dependent antibody responses by acting as an adjuvant for the generation of Tfh cells.

LBP as a vaccine adjuvant

LBPs stimulated moderate immune responses, and therefore, could potentially be used as a substitute for oil adjuvants in vaccines. Subfractions of polysaccharides, 12.5 mg/kg, 25 mg/kg, or 50 mg/kg LBP3a, were mixed with a DNA vaccine encoding the major outer membrane protein of Chlamydophila abortus.148 Balb/C mice were inoculated at days 0, 14, and 28, and challenged on day 44. Serum antibody levels, in vitro lymphocyte proliferation, the levels of IL-2, IFN-γ, and TNF-α, and Chlamydia clearance in the spleen were monitored. A combination of DNA vaccine plus LBP3a induced significantly higher antibody levels in mice, higher T-cell proliferation, and higher levels of IFN-γ and IL-2. Mice immunized with DNA vaccine and LBPs showed significantly higher levels of Th₁ immune response and Chlamydia clearance in mouse spleen. The immunoenhancing effect induced by 25 mg/kg LBP3a was more effective than that induced by 12.5 mg/kg and 50 mg/kg LBP3a. These results suggest that LBPs may be used as an effective adjuvant with a DNA vaccine against swine C. abortus.

Vaccination is the most efficient strategy to prevent influenza infection. However, vaccine efficacy is significantly diminished in the elderly due to the age-related impairment of both innate and adaptive immune responses. A recent study149 has examined whether dietary wolfberry supplementation enhanced the protective effect of influenza vaccine against influenza challenge in aged male C57BL/6J mice (20–22 months old). The mice were fed a 5% milk-based preparation of wolfberry (Nestec) or fed with 5% corn starch (control) for 30 days, then immunized with an influenza vaccine or saline (control) by ip injection on days 31 and 52 of the dietary intervention, and finally challenged with influenza A/Puerto Rico/8/34 virus (Sino Biological, Bejing, People’s Republic of China) with an aluminum adjuvant at a ratio of 1:1. The milk-based preparation of wolfberry contained 530 mg/g of wolfberry fruit, 290 mg/g of skimmed milk, and 180 mg/g of maltodextrin. At day 73, mice were infected with influenza A/Puerto Rico/8/34 virus and were monitored daily for weight loss and mortality.149 Mice fed with wolfberry diet had higher influenza IgG titers, less weight loss, and improved survival rate in influenza-infected mice when compared with the mice treated by influenza vaccine alone.149 Furthermore, an in vitro study showed that administration of 100 mg/L, 200 mg/L, 400 mg/L, or 800 mg/L wolfberry supplementation enhanced maturation and activity of antigen-presenting DCs isolated from the bone marrow of aged mice. Wolfberry extract dose-dependently increased the percentages of DCs expressing MHC-II and T-cell costimulatory molecules CD40, CD80, and CD86 and their expression. Wolfberry enhanced the production of proinflammatory IL-12 and TNF-α.149 With improved maturation of DCs, the endocytic capability of DCs was significantly reduced when treated with wolfberry extract. Adoptive transfer of wolfberry-treated bone marrow DCs loaded with ovalbumin323–339 to recipient mice promoted antigen-specific T-cell proliferation as well as IL-4 and IFN-γ production in CD4⁺ T cells.149 Wolfberry may enhance the antigen-presenting function of DCs, leading to a higher level of antigen-specific T-cell effector function involving at least Th₁ and Th₂ responses. These data indicate that dietary wolfberry potentiates the efficacy of influenza vaccination, resulting in better host protection to prevent subsequent influenza infection via improved DC function.

Clinical studies

Amagase et al150 investigated the systematic effects of consuming 120 mL/day GoChi for 30 days on immune function, general well-being, and safety in a randomized, double-blind, placebo-controlled clinical study in 60 older Chinese healthy adults (55–72 years old). The GoChi group showed a statistically significant increase in the number of lymphocytes and levels of IL-2 and IgG compared to pre-intervention and the placebo group, whereas the number of CD4, CD8, and NK cells or levels of IL-4 and IgA were not significantly altered. The placebo group showed no significant changes in any immune measures, whereas the GoChi group showed a significant increase in general feelings of well-being, such as fatigue and sleep, and showed a tendency for increased short-term memory and focus between pre- and post-intervention; the placebo group showed no significant positive changes in these measures.150 GoChi was well tolerated. No adverse reactions, abnormal symptoms, or changes in body weight, blood pressure, pulse, visual acuity, urine, stool, or blood biochemistry were noted in either group.150 Daily consumption of GoChi significantly increased several immunological responses and subjective feelings of general well-being without any adverse reactions in the elderly.

A recent study by Vidal et al151 reported that elderly persons who consumed Lacto-Wolfberry for 3 months (13.7 g/day in the form of the same milk-based preparation of wolfberry) had higher serum influenza-specific IgG concentrations and seroconversion rate after receiving an influenza vaccine compared with age-matched elderly individuals in the placebo group. The study was conducted in 150 healthy community-dwelling Chinese elderly (65–70 years old) supplemented with Lacto-Wolfberry or placebo (13.7 g/day). No serious adverse reactions were reported during the trial, neither symptoms of influenza-like infection nor changes in body weight and blood pressure, blood chemistry or cells composition, and autoantibodies levels were observed.151 Lacto-Wolfberry supplementation had no significant effect on delayed-type hypersensitivity response and inflammatory markers. These data show that chronic dietary supplementation with Lacto-Wolfberry in the elderly enhances their capacity to respond to influenza vaccine challenge.

Summary of immunomodulating effects of LBPs

A number of in vitro and in vitro studies have revealed the immunomodulating activities of LBPs (Figure 9). LBPs promote the proliferation and activity of splenocytes, T cells, B cells, macrophages, and NK cells. LBPs induce IL-6, IL-8, IL-10, and TNF-α production in splenocytes. LBPs stimulate PBMCs to produce IL-2 and TNF-α. IL-2 stimulates growth and differentiation of T cells. LBPs promote T lymphocytes and macrophages to release important cytokines such as IL-10 and TNF-α. LBPs activate macrophages and upregulate the expressions of CD40, CD80, CD86, and MHC-II molecules. LBPs activate transcription factors NF-κB and AP-1, induce TNF-α, IL-1β, and IL-12p40 expression in macrophages. LBPs significantly enhance macrophage endocytic and phagocytic capacities. LBPs promote the cytotoxicity of NK cells by enhancing IFN-γ and perforin release and the expression of the activating receptors NKp30 and NKG2D. LBPs also stimulate macrophages and NK cells to release TNF-α and IL-1β. LBPs activate the transcription factors NFAT and AP-1 and prompt CD25 (IL-2 receptor-α) expressions. LBPs induce the maturation of DCs and improve their antigen-presenting function. LBPs can upregulate the expression of CD40, CD80, CD86, and MHC-II molecules in bone marrow- and peripheral blood-derived DCs, downregulate DC uptake of antigen (Ag), enhance allostimulatory activity of DCs, and induce the production of IL-12p40 and p70 in DCs. LBP-treated DCs can enhance both Th₁ and Th₂ responses. LBPs potentiate the immune responses of DNA vaccine against C. abortus in mice. LBPs activate CXCR5⁺PD-1⁺ Tfh cells and induce IL-21 secretion. Dietary wolfberry supplementation enhances both in vivo and ex vivo T-cell response to specific antigens. Elderly persons who consume Lacto-Wolfberry for 3 months show higher serum influenza-specific IgG concentrations and seroconversion rate after receiving an influenza vaccine.

Figure 9

Possible mechanisms for the immunomodulating effects of LBPs.

Notes: LBPs have been found to have a variety of immune-modulatory activities in vitro and in vivo. LBPs promote the proliferation and activity of splenocytes, T cells, B cells, macrophages and NK cells. LBPs induce IL-6, IL-8, IL-10, and TNF-α production in splenocytes. LBPs stimulate PBMCs to produce IL-2 and TNF-α. IL-2 stimulates growth and differentiation of T cells. LBPs promote T lymphocytes and macrophages to release important cytokines such as IL-10 and TNF-α. IL-10 inhibits the production of IFN-γ, IL-2, IL-3, TNF-α, and granulocyte-macrophage colony-stimulating factor (GM-CSF) by activated macrophages and by helper T cells. LBPs activate macrophages and upregulate the expressions of CD40, CD80, CD86, and MHC-II molecules. LBPs activate transcription factors NF-κB and AP-1, induce TNF-α, IL-1β, and IL-12p40 expression in macrophages. LBPs significantly enhance macrophage endocytic and phagocytic capacities. LBPs promote the cytotoxicity of NK cells by enhancing IFN-γ and perforin release and the expression of the activating receptors NKp30 and NKG2D. LBPs also stimulate macrophages and NK cells to release TNF-α and IL-1β. LBPs activate the transcription factors NFAT and AP-1 and prompt CD25 (IL-2 receptor-α) expression. LBPs induce the maturation of DCs and improve their antigen-presenting function. LBPs can upregulate the expression of CD40, CD80, CD86, and MHC-II molecules in bone marrow- and peripheral blood-derived DCs, downregulate DC uptake of Ag, enhance allostimulatory activity of DCs, and induce the production of IL-12p40 and p70 in DCs. IL-12 is involved in the stimulation and maintenance of Th₁ cellular immune responses and also has an important role in enhancing the cytotoxic function of NKs. LBP-treated DCs can enhance both Th₁ and Th₂ responses. LBPs potentiate the immune responses of DNA vaccine against Chlamydophila abortus in mice. LBPs also activate CXCR5⁺PD-1⁺ Tfh cells and induce IL-21 secretion. Dietary wolfberry supplementation enhances both in vivo and ex vivo T-cell response to specific antigens. Elderly persons who consume Lacto-Wolfberry for 3 months show higher serum influenza-specific IgG concentrations and seroconversion rate after receiving an influenza vaccine.

Abbreviations: Ag, antigen; AP-1, activator protein-1; GM-CSF, granulocyte-macrophage colony-stimulating factor; DCs, dendritic cells; IFN-γ, interferon-γ; IL, interleukin; LBP, Lycium barbarum polysaccharide; MHC-II, class II major histocompatibility complex; NF-κB, nuclear factor κB; NFAT, nuclear factor of activated T-cells; NK, natural killer; PD, programmed death; TNF, tumor necrosis factor; JNK, Jun N-terminal kinases; Nrf2, nuclear factor erythroid 2-related factor; PI3K, phosphatidylinositol 3-kinase; p38 MAPK, p38 mitogen activated protein kinase; GLUT4, glucose transporter type-4; IRS-1, insulin receptor substrate-1; HO-1, heme oxygenase-1; SOD, superoxide dismutase; GSK3β, glycogen synthase kinase 3β; Tfh, T follicular helper.

Neuroprotective effects and effects on cognitive and memory deficits, AD, and stroke

As the aged population dramatically increases in these decades, there is a great increase in the prevalence of age-associated neurodegenerative diseases such as cognitive and memory deficits, AD, and Parkinson’s disease. There is increased interest in seeking new therapeutic agents for these devastating diseases from herbal medicines. LBPs possess neuroprotective effects in various in vitro and in vivo models152–154 but the mechanisms have not yet been fully elucidated. In the nervous system, LBPs can protect against neuronal injury or loss induced by I/R,155,156 Aβ peptide,157,158 glutamate excitotoxicity, and other neurotoxic insults.154 LBPs also enhance neurogenesis.154,159

Ischemic brain disease and MCAO

Ischemic stroke has become one of the most devastating diseases, which cause high rates of disability and mortality in aged people.160–162 Acute excitotoxicity, oxidative stress, and inflammation are the three primary mechanisms involved in cell death during ischemic stroke.163 Cerebral edema is a detrimental feature after ischemic stroke and is one of the impact factors of clinical deterioration within the first 24 hours after stroke onset. Cerebral ischemia and reperfusion triggers a cascade of cellular events including cell death, oxidative stress, and inflammation, which all contribute to the breakdown of blood–brain barrier (BBB).160–162 Neuronal cell apoptosis plays an important role in the development of ischemic injury in the brain tissue. Mitochondrial apoptotic pathway is a major apoptotic pathway, and a large number of apoptosis-related proteins in mitochondria play an important role in the initiation and development of neuronal apoptosis.164 Pro-apoptotic and anti-apoptotic Bcl-2 family proteins play important roles in mitochondrial apoptotic pathway. Bax is a pro-apoptotic and Bcl-2 is an anti-apoptotic protein in the Bcl-2 family. Cytochrome C binds and activates apoptotic protease-activating factor-1 as well as procaspase-9, forming an apoptosome together with ATP. Apoptosome then activates caspase-9, leading to caspase-3 activation and eventually cellular apoptosis. Caspase-3 has been identified as a key mediator of apoptosis and cleaves the substrate PARP-1, which is a multifunctional nuclear enzyme whose activity is rapidly stimulated by DNA breaks.

The protective effect of LBPs was investigated in primary cultured rat hippocampal neurons subject to oxygen–glucose deprivation/reperfusion by Rui et al.153 Cultured hippocampal neurons were exposed to oxygen–glucose deprivation for 2 hours followed by a 24-hour re-oxygenation. Treatment with LBPs (10–40 mg/L) significantly attenuated neuronal damage and inhibited LDH release in a dose-dependent manner.153

Yang et al155 investigated the protective effect of LBP pre-treatment in an experimental stroke (MCAO) model in male C57BL/6N mice. To gain LBPs, dry L. barbarum residues were dissolved in water at 70°C, and the supernatant was concentrated, precipitated with 95% ethanol, and then vacuum dried to produce the extracts. The mice were administered 1 mg/kg or 10 mg/kg LBPs daily for 7 days, and then subjected to 2-hour transient MCAO by the intraluminal method followed by 22-hour reperfusion upon filament removal. LBP pre-treatment dose-dependently improved neurological deficits; decreased infarct size, apoptotic neurons in ischemic penumbra area, and cerebral edema; and protected the brain from BBB disruption as indicated by reduced Evans Blue dye leakage into the ipsilateral hemispheres and an upregulation of occludin expression.155 Occludin, one of the proteins located at tight junctions, plays an important role in maintaining the integrity of BBB. Pre-treatment with 10 mg/kg LBPs for 7 days also profoundly suppressed the upregulation of AQP4 expression in ipsi-lateral penumbral areas.155 Furthermore, 10 mg/kg LBPs suppressed GFAP activation in ipsilateral penumbral areas. Pre-treatment with 10 mg/kg LBPs reduced both nitrosative stress and lipid peroxidation in cerebral ischemic penumbra after MCAO. LBPs at both doses attenuated the expression of matrix metalloproteinase-9 (MMP-9) in ipsilateral penumbral areas.155 These findings clearly demonstrate the beneficial prophylactic effects of LBPs against ischemic damage and cerebral edema in a murine experimental stroke model. The neuroprotective effects of LBPs on ischemic stroke include reduction of neuronal damage and infarct, maintenance of BBB integrity, and alleviation of cerebral edema through antioxidation, suppression of upregulated MMP-9 and AQP4, anti-apoptosis, and inhibition of glial activation.

In a study using male Kunming mice, Wang et al165 examined the effect of intragastric administration with LBPs on brain injuries in MCAO mice. The study demonstrated that LBPs at doses of 20 mg/kg and 40 mg/kg significantly decreased the neurological deficit scores and the infarct area in MCAO mice. LBPs also significantly decreased MDA content, and increased SOD, GPx, CAT, and LDH activities in the ischemic brain.165 These findings suggest that LBPs might act as potential neuroprotective agent against the cerebral reperfusion-induced brain injury through reducing lipid peroxides, scavenging free radicals, and improving the energy metabolism.

In a similar study, Wang et al156 used male Imprinting Control Region mice to make the model of MCAO and investigated the protective effect of intragastric administration of 10 mg/kg, 20 mg/kg, and 40 mg/kg body weight LBPs or 0.4 mg/kg nimodipine for 7 days on MCAO-induced brain injuries. The results showed that intragastric administration of 20 mg/kg and 40 mg/kg LBPs markedly decreased the neurological deficit scores and the infarct volume in MCAO mice.156 Administration of 10–40 mg/kg LBPs also reduced neuronal morphological damage and neuronal apoptosis in ischemic penumbra of the left cortex. About 40 mg/kg LBPs significantly suppressed cortex overexpression of Bax, cytochrome C, caspase-3, -9, and cleaved PARP-1, and reduced the downregulated Bcl-2 expression in MCAO mice.156

In summary, the protective effects of LBPs on MCAO-induced brain injuries are mainly attributed to the reduction of oxidative stress, inhibition of apoptosis, and increase in the integrity of BBB. LBPs treatment reduces the oxidative stress via increasing the SOD, GPx, CAT, and LDH activities, but decreasing the content of MDA and lipid peroxidation. LBPs also inhibit the apoptosis via decreasing the expression of cytochrome C, cleave caspase-9, caspase-3, Bax, and cleaved PARP-1, but increasing the expression level of Bcl-2. In addition, LBPs increase the integrity of BBB expression through the upregulation of expression of occludin, but downregulation of the expression of MMP-9 and AQP4 (Figure 10).

Figure 10

Possible mechanisms for the neuroprotective effects of LBPs against MCAO-induced brain injuries.

Notes: LBPs treatment protects neurons against MCAO-induced brain injuries mainly via reduction of oxidative stress, inhibition of apoptosis, and increase in the integrity of BBB in mice. LBPs increase the activities of SOD, GPx, CAT, and LDH, but decrease the content of MDA and lipid peroxidation, resulting in a reduction in oxidative stress. LBPs inhibit the expression of cytochrome C, cleave caspase-9, cleaved caspase-3, Bax, and cleaved PARP-1, but increase the expression level of Bcl-2, leading to inhibition of apoptosis. In addition, LBPs increase the expression of occludin but decrease the expression of MMP-9 and aquaporin-4, increasing the integrity of BBB.

Abbreviations: LBPs, Lycium barbarum polysaccharides; SOD, superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase; LDH, lactate dehydrogenase; MCAO, middle cerebral artery occlusion; MDA, malondialdehyde; PARP, poly(ADP-ribose) polymerase; MMP-9, matrix metalloproteinase-9; BBB, blood–brain barrier; MHC-II, Class II major histocompatibility complex; TNF, tumor necrosis factor; IL, interleukin; IgG, immunoglobulin G; IFN, interferon; NK, natural killer; Tfh, follicular helper T cell; NKp30, natural killer cell p30-related protein.

Aβ-induced neuronal injury and AD

Aβ peptides are thought to be associated with the progressive neuronal death observed in AD. The effect of LBPs was investigated by Yu et al157 on the neuronal injury induced by Aβ1-42 and Aβ25-35 peptides in primary rat cortical neurons. Remarkable apoptosis and necrosis in primary rat cortical neurons were observed when exposed to Aβ peptides. Pre-treatment with LBPs significantly reduced the release of LDH. In addition, LBPs attenuated Aβ peptide-activated caspase-3-like activity.157 Aβ peptides induce a rapid activation of c-JNK by phosphorylation. Pre-treatment of LBPs markedly reduced the phosphorylation of JNK-1 at Thr183/Tyr185 and its substrates c-Jun-I at Ser73 and c-Jun-II at Ser63.157 LPBs elicit dose-dependent neuroprotective effects via regulation of JNK-1 pathway.

Yu et al158 also investigated the effects of LBPs on the phosphorylation of the double-stranded RNA-dependent protein kinase (PKR) in rat cortical neurons exposed to Aβ peptides. PKR is an intracellular sensor of stress and can arrest protein synthesis by phosphorylating the alpha subunit of the translation initiation factor eIF2. Pretreatment of LBPs effectively protected neurons against Aβ-induced apoptosis by reducing the activity of both caspase-3 and -2, but not caspase-8 and -9. LBPs markedly reduced Aβ-induced PKR phosphorylation.158

In summary, LBPs protect neurons against Aβ-induced apoptosis by reducing the activity of both caspase-3 and -2, but not caspase-8 and -9 (Figure 11). LBPs inhibit the phosphorylation of JNK-1 at Thr183/Tyr185 and its substrates c-Jun-I at Ser73 and c-Jun-II at Ser63 in neurons. LBPs reduce the phosphorylation of Erk1/2m but not GSK3β. LBPs also markedly reduced Aβ-induced PKR phosphorylation. LBPs also significantly reduce homocysteine-induced phosphorylation of Tau-1 at Ser198/199/202, pS396 at Ser396, and pS214 at Ser214 as well as cleavage of Tau.

Figure 11

Possible mechanisms for the neuroprotective effects of LBPs against Aβ-induced neurotoxicity and Alzheimer’s disease.

Notes: LBPs protect neurons against Aβ-induced apoptosis by reducing the activity of both caspase-3 and -2, but not caspase-8 and -9. LBPs inhibit the phosphorylation of JNK-1 at Thr183/Tyr185 and its substrates c-Jun-I at Ser73 and c-Jun-II at Ser63 in neurons. LBPs reduce the phosphorylation of Erk1/2m but not GSK3β. LBPs also markedly reduced Aβ-induced PKR phosphorylation. PKR is an intracellular sensor of stress and can arrest protein synthesis by phosphorylating the alpha subunit of the translation initiation factor eIF2. LBPs also significantly reduce homocysteine-induced phosphorylation of Tau-1 at Ser198/199/202, pS396 at Ser396, and pS214 at Ser214 as well as cleavage of Tau.

Abbreviations: Aβ, amyloid-β; LBPs, Lycium barbarum polysaccharides; JNK, Jun N-terminal kinases; GSK3β, glycogen synthase kinase 3β; PKR, protein kinase; eIF2, eukaryotic initiation factor 2.

Scopolamine-induced brain injury

A recent study by Chen et al154 reported the therapeutic effects of LBPs on learning and memory and neurogenesis in scopolamine (SCO)-treated adult male Sprague–Dawley rats. SCO was used to induce learning and memory deficits. LBPs were administered 0.2 mg/kg or 1 mg/kg body weight per day via gastric perfusion for 14 days before the onset of subcutaneous SCO treatment for a further 4 weeks. LBPs used were extracted with boiling water, followed by precipitation with ethanol, protein hydrolysis, dialysis, and fractionation with a diethylaminoethanol-Sepharose CL-6B column. An osmotic pump containing SCO solution at 440 mg/mL was subcutaneously embedded in the abdominal wall of rats and SCO release at a rate of 0.25 µL/h was maintained for 28 days and administration of LBPs was continued as before, throughout SCO treatment. LBPs at both doses almost restored the memory and learning abilities in SCO-treated rats.154 LBPs prevented SCO-induced reduction in neuronal proliferation and enhanced neuroblast differentiation in the hippocampal dentate gyrus of rats.

LBP treatment also protected the dendrites from damage by SCO. LBPs dose-dependently decreased the SCO-induced oxidative stress in hippocampus and reversed the increased ratio of Bax/Bcl-2 induced by SCO treatment.154 LBPs significantly increased hippocampal SOD and GPx activity and reduced MDA level in SCO-treated rats. However, LBPs did not affect the SCO-induced elevation of hippocampal acetylcholinesterase activity and decrease of brain-derived neurotrophic factor level.154 These results suggest that LBPs prevent SCO-induced cognitive and memory impairments and reductions in hippocampal cell proliferation and neuroblast differentiation. Anti-oxidation and anti-apoptosis are the two major mechanisms for the neuroprotective effects of LBPs in SCO-treated rats (Figure 12).

Figure 12

Possible mechanisms for the neuroprotective effects of LBPs against SCO-induced neurotoxicity.

Notes: LBPs protect neurons against SCO-induced neurotoxicity through the reduction of the oxidative stress and apoptosis. LBPs increase the activities of SOD and GPx, restore the balance of Bcl-2 to Bax, but decrease the content of MDA.

Abbreviations: LBPs, Lycium barbarum polysaccharides; SCO, scopolamine; SOD, superoxide dismutase; GPx, glutathione peroxidase; MDA, malondialdehyde.

Glutamate-induced neuronal injury

Glutamate excitotoxicity is involved in many neurodegenerative diseases including AD. Attenuation of glutamate toxicity is one of the therapeutic strategies for AD. LBPs were administrated to detect if they can prevent neurotoxicity elicited by glutamate in primary cultured neurons.166 The glutamate-induced cell death as detected by LDH assay and caspase-3-like activity assay was significantly reduced by LBPs at concentrations ranging from 10 µg/mL to 500 µg/mL. LBPs provided neuroprotection even 1 hour after exposure to glutamate. In addition to glutamate, LBPs attenuated N-methyl-D-aspartate-induced neuronal damage, and glutamate-induced phosphorylation of JNK was reduced by treatment with LBPs (Figure 13). LBPs exerted significant neuroprotective effects on cultured cortical neurons exposed to glutamate.

Figure 13

Possible mechanisms for the neuroprotective effects of LBPs against glutamate-induced neurotoxicity.

Notes: LBPs attenuate glutamate- and NMDA-induced neuronal damage. LBPs decrease the activity of LDH and inhibit the phosphorylation of JNK and the expression of caspase-3, resulting in a decrease in apoptosis.

Abbreviations: LBPs, Lycium barbarum polysaccharides; NMDA, N-methyl-D-aspartate; LDH, glutathione peroxidase; JNK, Jun N-terminal kinases; p, phosphorylated.

Manganese-induced neuronal injury

Manganese could induce multiple organs injury especially in brain and show obvious cognitive and memory deficits. A study focused on the therapeutic effect of LBPs on neurogenesis and learning and memory of manganese poisoned mice. Healthy adult Kunming mice were used. The spatial learning and memory capacity of mice was determined by the Morris water maze training test. The neurogenic cells were labeled with bromodeoxyuridine (BrdU) and detected by immunohistochemistry. The average escape latency was significantly higher and the times of passing through platform were lower in the manganese treated group. BrdU-positive cells in the LBPs-treated group were significantly more than those in the manganese-treated group. The author suggested that LBPs could enhance the learning and memory capability of the manganese poisoned mice by promoting neurogenesis in the hippocampus.167

Homocysteine-induced neuronal injury

Previous clinical and epidemiological studies have suggested that elevated plasma homocysteine levels increased the risk of AD.168 Homocysteine damages neurons by inducing apoptosis, DNA fragmentation, and Tau phosphorylation.169 Ho et al170 conducted in vitro and in vivo studies to study the beneficial effects of LBPs on neurotoxicity caused by homocysteine. LBA treatment significantly attenuated homocysteine-induced neuronal cell death and apoptosis in primary rat cortical neurons as determined by LDH release and caspase-3 activity assays. LBPs also significantly reduced homocysteine-induced phosphorylation of Tau-1 at Ser198/199/202, pS396 at Ser396, and pS214 at Ser214 as well as cleavage of Tau.170 LBP treatment suppressed elevation of both phosphorylated extracellular-signal-regulated kinases (Erk1/2) and phosphorylated JNK. However, the phosphorylation level of GSK3β at Ser9/Tyr 216 remained unchanged among different treatment groups. The data demonstrated that LBPs exerted neuroprotective effects on cortical neurons exposed to homocysteine via modulation of JNK and Erk1/2 pathways (Figure 14).

Figure 14

Possible mechanisms for the neuroprotective effects of LBPs against homocysteine-induced neurotoxicity.

Notes: LBPs exert neuroprotective effects on cortical neurons exposed to homocysteine via modulation of JNK and Erk1/2 pathways. LBPs suppress the phosphorylation of Erk1/2 and JNK, resulting in an inhibition of phosphorylation of Tau; LBPs also reduce the expression level of caspase-3 and decrease the activity of LDH.

Abbreviations: LBPs, Lycium barbarum polysaccharides; LDH, glutathione peroxidase; JNK, Jun N-terminal kinases; Erk1/2, extracellular signal-regulated kinase 1/2; p, phosphorylated.

High ambient temperature

Yang et al171 investigated the effects of LBPs on the expression of neuropeptide Y (NPY) mRNA level in the hypothalamus, plasma concentration of corticotropin-releasing hormone (CRH), cortisol, HSP70, and epinephrine in rats subject to high ambient temperature. Compared to the control group, the plasma levels of CRH, cortisol, HSP70, and epinephrine were markedly increased, and the level of NPY mRNA was downregulated in the high ambient temperature-exposed rats.171 These effects were significantly reversed by LBP treatment in rats. LBPs have a potentially protective function against high temperature by increasing the expression of HSP70 and NPY.

Traumatic neuroma

Traumatic neuromas are tumors produced by a reactive process to regenerate injured nerves that result in a disordered proliferation of nerve bundles. These tumors are usually related to previous surgery or trauma. Fan et al172 investigated the effects of LBPs on the formation of traumatic neuroma and pain after transection of sciatic nerve in rats. LBPs were intraperitoneally injected to the rats for 28 days. The study showed that there was less neuroma formed in the LBP-treated group than in the control group. Data from transmission electron microscopy showed that there were numerous axons in nerve tumor, more fusoid fibroblasts, more collagen fiber, and hyperplasia and degenerated myelin sheath in the control group, while in the LBP-treated group, there was less myelin sheath in the proximal end of injuring nerves, less Schwann cells and fibroblasts, and sparsed collagen fibers. LBPs can inhibit autophagy and the formation of traumatic neuroma after transection of sciatic nerve in rats.

Protective effects against irradiation- or chemotherapy-induced organ toxicities

Both irradiation and chemotherapy can induce severe organ toxicities.173,174 LBPs could serve as a very useful adjunct to the cancer therapies such as chemotherapy and radiotherapy. Therapeutic effects of LBPs on mitomycin C-induced myelosuppressive mice were investigated by Hai-Yang et al.175 Mice were intravenously injected with 150 mg/kg mitomycin C for 2 consecutive days to produce severe myelosuppression, and then treated by subcutaneous injection of 100 mg/kg/day or 200 mg/kg/day LBPs for 6 days. Blood samples were collected from the tail veins of mice on days 7, 10, 12, 14, 17, 19, 21, 24, and 27, and peripheral white blood cells, red blood cells, hemoglobin, and platelet counts were monitored. Administration of 100 mg/kg LBPs (LBP-L) on day 14 and 200 mg/kg LBPs (LBP-H) on days 10, 14, 17, 19, and 21 significantly increased peripheral red blood cells, hemoglobin, and hematocrit of myelosuppressive mice compared to mice treated with mitomycin C only.175 LBP-L on days 12 and 14 and LBP-H on days 10, 12, 14, 17, 19, and 21 significantly promoted peripheral platelet recovery of mitomycin C-treated mice compared with the control mice. LBP-H on days 12, 17, 19, and 21 also significantly inhibited the increase of mean platelet volume of myelosuppressive mice compared to the control.175 These results indicate that LBPs significantly enhanced platelet recovery of myelosuppressive mice compared to the control, but did not significantly affect white blood cell recovery.

Gong et al176 investigated the effects of LBPs on irradiation or chemotherapy-induced bone marrow suppression in mice and cultured PBMCs. In the in vivo experiment, mice were irradiated with X-ray or intraperitoneally injected with carboplatin to produce severe myelosuppression. LBPs significantly increased peripheral white blood cell, red blood cell, and platelet counts compared to mice receiving irradiation only. LBPs also significantly increased peripheral white blood cell and red blood cell counts of chemotherapy-induced myelosuppressive mice. This study demonstrates that LBPs promoted the peripheral blood and bone marrow recovery from irradiation or chemotherapy-induced myelosuppression in mice, and the effects may be due to the release of GM-CSF from PBMCs.

Protective effects on the reproductive system

Wolfberry was described to exhibit pro-sexual effect by the Chinese herbalist Li Shizhen, and thus it was included in sexual-enhancing Chinese herbal remedies. Daily consumption of wolfberry juice in healthy subjects improves the well-being feeling toward sexuality, including increase in sexual activity and ability.27 Animal studies have demonstrated that LBPs exert beneficial effects on sexual performance and fertility, although the underlying mechanisms remain largely elusive.

Bisphenol A-induced sperimatogenic damage

LBPs showed protected effects against spermatogenic injuries induced by bisphenol A in mice.177 Bisphenol A was subcutaneously injected into mice at a dose of 20 mg/kg body weight for 7 consecutive days and LBPs were administered simultaneously with bisphenol A by gavage daily for 7 days. The results showed that the weights of testis and epididymis were all increased after supplementation with different dosages of LBPs compared with bisphenol A alone group, and the activities of SOD and GPx were significantly increased in LBP-treated groups, while MDA contents were gradually decreased.177 LBPs also showed significant positive effects on the expression of Bcl-2/Bax in bisphenol A-treated mice. The authors concluded that LBPs might be one of the potential ingredients protecting the adult male animals from bisphenol A-induced reproductive damage (Figure 15).

Figure 15

Possible mechanisms for the protective effects of LBPs against bisphenol A-induced sperimatogenic damage.

Notes: LBPs exhibit protective effect on then reproductive system via the regulation of oxidative stress, apoptosis, and cell proliferation. LBPs increase the activities of SOD and GPx and restore the balance of Bcl-2 to Bax. LBPs promote cell proliferation but decrease the expression level of cytochrome C and the content of MDA.

Abbreviations: LBPs, Lycium barbarum polysaccharides; SOD, superoxide dismutase; GPx, glutathione peroxidase; MDA, malondialdehyde.

Corticosterone-induced inhibition of sexual behavior

In a recent study,159 the effects of LBPs on male sexual behavior of young adult male Sprague–Dawley rats were investigated. Oral administration of 1 mg/kg or 10 mg/kg LBPs for 21 days significantly improved the male copulatory performance including increase of copulatory efficiency, increase of ejaculation frequency, and shortening of ejaculation latency. Furthermore, sexual inhibition caused by chronic corticosterone was prevented by administration of 40 mg/kg LBPs for 21 days. Simultaneously, treatment of rats with corticosterone suppressed neurogenesis in the subventricular zone and hippocampus in adult rats, which could be reversed by LBPs.159 In the subventricular zone, the number of BrdU-positive cells in the corticosterone-treated animals was significantly lower than LBP-treatment groups. The neurogenic effect of LBPs was also shown in vitro using mouse C17.2 neural stem cells derived from the cerebellum of neonatal mice and immortalized by retrovirus-mediated v-myc gene transfection. Corticosterone treatment suppressed the cell proliferation of C17.2 cell line, while co-incubation with 10 µg/mL LBP reversed the growth suppression. Blocking neurogenesis in male rats abolished the pro-sexual effect of LBPs. These results demonstrate the pro-sexual effect of LBPs on normal and sexually inhibited rats, and LBP may modulate sexual behavior by regulating neurogenesis.

Heat- or H₂O₂-induced testicular cell damage

Luo et al178 investigated the effect of LBPs on rat testis damage induced by a physical factor (43°C heat exposure), on DNA damage of mouse testicular cells induced by a chemical factor (H₂O₂), and on sexual behavior and reproductive function of hemicastrated male rats. The results showed that LBPs provided a protective effect against the testicular tissue damage induced by heat exposure. When compared with negative control, a suitable concentration of LBPs significantly increased testis and epididymis weights, improved SOD activity, and raised sexual hormone levels in the damaged rat testes.178 LBPs exhibited a dose-dependent protective effect against DNA oxidative damage of mouse testicular cells induced by H₂O₂. LBPs also improved the copulatory performance and reproductive function of hemicastrated male rats, such as shortened penis erection latency and mount latency, regulated secretion of sexual hormones and increased hormone levels, raised accessory sexual organ weights, and improved sperm quantity and quality.178

LBPs could provide some protective effect against heat stress (HS)-induced apoptosis of germ cells in rats.179 Ninety male Sprague–Dawley rats were randomly divided into five groups of 18 each: control, HS, high-dose LBPs, median-dose LBPs, and low-dose LBPs. The rats of the three LBP groups were given LBPs by intragastric administration. Compared with the HS group, the three LBP groups showed statistically significant decreases in the apoptosis index, the expression level of caspase-3 in germ cells, and the concentration of cytochrome C in the cytosol.179 LBPs protected germ cells against apoptosis via modulation of the mitochondrial pathway.179

Radiation-induced spermatogenic damage

Zhang et al180 explored the protective effects of LBPs on ⁶⁰Co-γ-induced spermatogenic disturbance in mice and found that LBPs exhibited almost complete recovery from reproductive endocrine disorder and spermatogenic damage. Luo et al181 further confirmed the protective effects of LBPs on radiation-induced spermatogenic damage in male rats exposed to local subchronic ⁶⁰Co-γ-irradiation. In this study, the effects of LBPs on sperm quantity and motility, sexual ability, serum hormone levels, oxidative status, and testicular tissue DNA damage on days 1, 7, and 14 postdosing were determined. The results showed that LBPs significantly increased the sperm quantity and motility; shortened the erection, capture, and ejaculation latencies; increased the number of captures and ejaculations; and improved the sexual ability of male rats.181 LBPs also played a significant role in the recovery of serum testosterone levels, increased superoxide dismutase activity, decreased MDA levels, promoted oxidative balance, and rescued testicular DNA damage. LBPs have significant protective effects against damage induced by local subchronic exposure to ⁶⁰Co-γ irradiation, allowing rats to achieve full recovery with LBP treatment.

Aging

Wei et al182 studied the protective mechanism of LBP administration for 30 days on the function of ovarian tissue in 14-month-old female senile rats. Radioimmunoassay was used to determine the blood levels of estrone and progesterone, and enzyme immunoassay was used to determine the ovarian levels of IGF-1. Daily oral LBPs (20 mg/kg, 40 mg/kg, or 60 mg/kg body weight) for 30 days significantly recovered uterine atrophy and restored serum IGF-1 level, estrone and progesterone levels that were decreased in older rats, and reduced the expression of IGF-binding protein-1 (IGFBP-1) in ovarian tissue that was increased in older rats.182

Summary of protective effects of LBPs on the reproductive system

The protective effect of LPBs on the reproductive system is, at least in part, ascribed to antioxidation, promotion of cell proliferation, and anti-apoptosis. It has been shown that LBPs protect mice from bisphenol A-induced reproductive system damage by increasing the activities of SOD and GPx, and that LBPs increase sexual organ weight in rats (Figure 15). Moreover, LBPs decrease the ratio of Bcl-2/Bax, the expression level of caspase-3, and the concentration of cytosolic cytochrome C, and they increase cell proliferation in vitro.

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