| Proanthocyanidins (PC) are a group of naturally occurring polyphenolic bioflavonoids which are widely present in fruits, vegetables, nuts, flowers, seeds and bark, especially grape seeds. At present, PC from grape seeds has been reported to possess a wide range of physiological and pharmacological activities including antibacterial, antiviral, anti-inflammatory, anti-tumor, anti-oxidative, anti-allergic and cardio-protective actions at home and abroad. However, so far, there was little research of PC on INS-1 cells. Therefore, in this experiment, to investigate the protection and repairment effects of PC on STZ-injured INS-1 cells, we adopted cell culture in vitro. It may provide experimental basis of natural antioxidants for prevention and treatment of diabetes mellitus (DM).INS-1 cells were cultured with complete RPMI 1640 medium, which contains 10% new born calf serum,1 mmol/L Pyruvic acid sodium,2 mmol/L L-Glutamine,50μmoL/Lβ-Mercaptoethanol, 5.6mmol/L glucose, 1×105 IU/L penicillin and 100 mg/L streptomycin. After subcultured 2-3 times, INS-1 cells with a density of 1×105/ml were seeded into culture plates, and incubated for 24 h in CO2 inhubator.The experiment was divided into several groups with six replications per group:(1) normal control group(2) STZ group:final concentration of STZ was 3 mmol/L(3) PC group:the final concentrations of PC were 6.25,12.5,25,50 and 100μg/ml, respectively(4) PC protection group:after adding 3 mmol/L STZ into INS-1 cells, and then added PC at the final concentrations of 6.25,12.5,25,50 and 100μg/ml at the same time(5) PC repairment group:after adding 3 mmol/L STZ into INS-1 cells, and then added different concentration PC described above in 6 hours(6) Atropine group:final concentration of Atropine was 1×10-6mol/L(7) Atropine+PC group:after adding 1×10-6mol/L Atropine into INS-1 cells, and then added different concentration PC described above(8) Verapamil group:final concentration of Verapamil was 1×10-7mol/L(9) Verapamil+PC group:after adding 1×10-7 mol/L Verapamil into INS-1 cells, and then added different concentration PC described above.The cell proliferation was measured by MTT method. Supernatant insulin levels were measured by radio-immunoassay. Supernatant malondialdehyde (MDA) and total anti-oxidation capability (T-AOC) were determined by Colorimetric assay, and cell cycle was assayed by flow cytometry. Cell morphology was observed under inverted microscope.Experimental data were expressed by mean±standard (x±s). Statistical comparisons were made using analysis of variance (ANOVA), andχ-test with software SPSS16.0. Differences with P<0.05 were considered statistically significant.Our experimental results are as follows:1.The stimulation effect of Glucose on INS-1 cellsCompared with normal control group, glucose (8.4mmol/L～22.4mmol/L) obviously improved insulin secretion of INS-1 cells (P<0.05). The 16.8mmol/L was the optimal concentration of stimulation effect.2. Effect of proanthocyanidin on INS-1 cell proliferation viabilityDifferent concentrations of PC obviously increased INS-1 cell proliferation viability after 12,24, and 48h treatment as compared with normal control group, while STZ (3 mmol/L) inhibited cell proliferation (P<0.01). Compared with STZ group, PC (25,50, and 100μg/ml) had protection and repairment effects on STZ-injured INS-1 cell proliferation viability (P<0.01).3. Effect of proanthocyanidin on insulin secretion of INS-1 cellsCompared with normal control group, PC increased insulin secretion of INS-1 cells, while STZ decreased insulin secretion (P<0.01). Cmpared with STZ group, protection and repairment groups of PC (25μg/ml,50μg/ml and 100μg/ml) had an obvious increase on insulin secretion of INS-1 cells (P<0.01).4. Effect of proanthocyanidin on MDA and T-AOCCompared with normal control group, MDA generation was significantly increased, but T-AOC level was lowered in STZ group (P<0.01). Compared with STZ group, The MDA production in protection and repairmen groups of PC was lowered (P<0.01), while T-AOC level was elevated (P<0.01)5. Effect of proanthocyanidin on INS-1 cell insulin secretion inhibited by Verapami and AtropineCompared with normal control group, Verapamil and Atropine obviously decreased INS-1 cell insulin secretion (P＜0.01). Compared with Verapamil group, Verapamil+PC markedly increased insulin secretion (P＜0.01). However, compared with Atropine group, Atropine+PC did not affect insulin secretion (P>0.05)6. Effect of proanthocyanidin on INS-1 cell morphological change, cell cycle and apoptosis Under inverted microscope, The INS-1 cells of normal control group were spindle and tightly adhered on the bottom of cultural plates, while most INS-1 cells of STZ group became round and were loosely adhered on the bottom, and the cell numbers were fewer than those of normal control group. The morphological change had no difference between STZ group, protection and repairment groups of proanthocyanidin.STZ increased G1 phase and decreased S phase in INS-1 cells as Compared with normal control group, while PC partly recovered cell cycle induced by STZ; Apoptosis rate in STZ guoup was increased, but it was lowered in PC+STZ group (P＜0.01)In conclusion, our experimental results showed:1. Glucose (8.4mmol/L-22.4mmol/L) could obviously stimulate insulin secretion of INS-1 cells. The 16.8mmol/L of glucose was the optimal concentration of stimulation effect.2. PC could increase INS-1 cell proliferation viability and insulin secretion.3. PC has protection and repairment effects on STZ-injured INS-1 cells, and its mechanisms may be related to T-AOC elevation, MDA reduction, and apoptosis inhibition.4. PC could partly recovered INS-1 cell insulin secretion inhibited by L-type Calcium ion channel blocker (Verapami), but it has no affect on INS-1 cell insulin secretion inhibited by cholinergic M receptor blocker (Atropine). |
