Lycium barbarum увеличивает калорийность расходы и уменьшает окружность талии у здоровых полных мужчин и женщин: пилотное исследование

3.1. Effect of Taurine and LB Extract on Cell Viability in ARPE-19 Cells Cultured in Normal Glucose Condition

To determine whether LB extract and taurine component influenced viability in normal glucose-treated ARPE-19 cells, MTS assay was performed for 24 h, 48 h, and 72 h of incubation. Incubation of LB extract at 0.001, 0.01, 0.1, 0.5, and 1 mg/mL in normal glucose-treated APRE-19 cells for 24 h and 48 h had little or no cytotoxicity (>90% viability remained). At 5 mg/mL, LB extract was cytotoxic at all incubation times (Figure 1(a)).

Incubation of APRE-19 cells with taurine for 24 h, 48 h, and 72 h had little or no effect on cytotoxicity (>90% viability remaining). At 5 and 10 mM, taurine was slightly cytotoxic (approximately 80% viability remaining) at all incubation times (Figure 1(b)).

3.2. Cytoprotective Effects of Taurine and LB Extract in ARPE-19 Cells Exposed to High Glucose

As it is well established that hyperglycemia induces cell death in retinal epithelial cells [37], ARPE-19 cells were treated with 33.3 mM glucose for 48 h in the presence of LB extract (0.1, 0.5, and 0.75 mg/mL) or taurine (0.001, 0.1, and 1 mM), and their cytoprotective effect was examined by MTS assay. Since high glucose increases osmolarity, ARPE-19 cells were also exposed to an osmotic control (27.5 mM mannitol + 5.5 mM glucose). Cell viability in 33.3 mM glucose was significantly decreased by 59.9%; the loss of viability was reversed by adding LB extract dose dependently (by 67.9, 74.4, and 81.0%, resp.) (Figure 2). Likewise cell viability in 33.3 mM glucose condition was reversed by adding taurine in a dose-dependent manner (69.4, 82.6, and 89.3%, resp.) (Figure 2). Loss of cell viability in high-glucose culture was similarly reversed by adding the positive controls RG (by 73.8%) and PG (by 89.1%). The osmotic control did not show a significant reduction in cell viability.

3.3. Effect of Taurine and LB Extract on Cell Apoptosis in High-Glucose-Treated ARPE-19 Cells

To investigate whether the cytoprotective effects of LB extract and taurine were due to the attenuation of high-glucose-induced apoptosis, characteristic morphological staining was performed using Hoechst 33342 and flow cytometry with Annexin V/PI double staining to identify and quantify the apoptotic cells.

Incubation with high glucose for 48 h induced a significant increase in the number of apoptotic cells (26.8% of total cells), as identified by Hoechst staining, compared with normal glucose culture (Figure 3(a)). The inhibitory effect of LB extract and taurine on high-glucose-induced apoptosis was demonstrated in characteristic morphological staining (Figure 3(b)). Treatment with LB extract (0.1, 0.5, and 0.75 mg/mL) dose dependently decreased the number of apoptotic cells by 18.3, 15.0, and 8.7% of total cells, respectively, compared to the high-glucose control, reaching values comparable to those in the control cultures (5.2% of total cells) (Figure 3(a)). Taurine (0.001, 0.1, and 1 mM) dose dependently decreased the number of apoptotic cells, by 14.0, 12.0, and 8.6% of total cells, respectively, compared to the high-glucose control, approaching values in the control cultures (5.2% of total cells) (Figure 3(a)). The number of apoptotic cells as identified by Hoechst staining in high-glucose condition was similarly decreased by the positive controls RG (9.4%) and PG (7.6%).

To further determine whether LB extract and taurine decreased apoptosis in high-glucose-induced ARPE-19 cells, apoptosis was measured as Annexin V binding to positive cells and quantified by flow cytometry, as Annexin V binds specifically to PS, allowing the discrimination between viable and apoptotic cells [38]. Representative dotplots of control and treated high-glucose-induced ARPE-19 cells stained with Annexin V and PI used for quantitation of apoptosis are shown in Figure 4(b). LB extract (0.1, 0.5, and 0.75 mg/mL) dose dependently decreased the number of Annexin V positive cells (14.8, 10.2, and 6.4% of the control) (Figure 4(a)). Taurine dose dependently decreased the number of Annexin V-positive cells (14.8, 9.3, and 6.0% of the control) (Figure 4(a)). The number of apoptotic cells in high-glucose culture was decreased by adding the positive controls, RG (by 10.0%) and PG (6.1%). The osmotic control did not enhance apoptotic cell death, excluding the involvement of osmotic effects of high-glucose concentrations.

3.4. Effect of Taurine and LB Extract on Caspase-3 Protein Expression in High-Glucose-Treated ARPE-19 Cells

To explore the mechanisms of the anti-apoptotic effect of LB extract, protein levels of active caspase-3 were examined by western blot analysis. LB extract (0.1, 0.5, and 0.75 mg/mL) dose dependently downregulated caspase-3 protein expression (by 6.6-, 4.7-, and 2.7-fold, resp.) (Figure 5(a)).

Taurine (0.001, 0.1, and 1 mM) dose dependently downregulated caspase-3 protein expression (by 7.0-, 5.2-, and 3.3-fold, resp.) (Figure 5(b)).

The protein levels of caspase-3 in high-glucose-treated cells were decreased by adding the positive controls, RG (4.4-fold) and PG (4.3-fold). The osmotic control did not enhance active caspase-3 protein level, excluding the involvement of osmotic effects of high-glucose concentrations.

3.5. Effect of Taurine and LB Extract on Caspase-3 Activity in High-Glucose-Induced ARPE-19 Cells

To confirm the findings of the downregulating effect of LB extract and taurine on apoptosis, activated caspase-3 in apoptotic cells was determined by fluorometric enzyme assay. LB extract (0.1, 0.5, and 0.75 mg/mL) dose dependently downregulated caspase-3 activity (by 5.0-, 4.2-, and 3.9-fold, resp.) (Figure 6).Taurine (0.001, 0.1, and 1 mM) dose dependently downregulated caspase-3 activity (by 5.7-, 4.2-, and 3.1-fold, resp.) (Figure 6).

The activated caspase-3 levels in high-glucose condition were reversed by adding the positive controls, RG (2.8-fold) and PG (3.1-fold). The increased osmotic control did not enhance active caspase-3 activity, excluding the involvement of osmotic effects of high-glucose concentrations.