Effect Of Buqing Granules On The Human Lens Epithelial Cells Induced By High Glucose And Expression Of Autophagy-related Genes

ObjectTo investigate the effects of Buqing Granules(BQ) on the activity of proliferation,cell cycle and Autophagy-related genes’ expression in cultured Human lens epithelial cells(SRA01/04)with high glucose, and to provide necessary experimental basis for the follow-up study of pathogenesis and treatment of diabetic cataract.Methods(1)Human lens epithelial cell line(SRA01/04) were cultured in vitro. The cells were exposed to either 0.0(normal control group),or 5.0mmol/L, 10.0mmol/L, 15.0mmol/L, 20.0mmol/L, 25.0mmol/L, 30.0mmol/L, 35.0mmol/L, 40.0mmol/L, 45.0mmol/L, 50.0mmol/L, 55.0mmol/L, 60.0mmol/L, 65.0mmol/L, 75.0mmol/L, 100.0mmol/L glucose for 12 h,24h,48 h. The cell’s activity of proliferation were quantified by CCK-8 assay. Cell cycle analysis was performed by flow cytometry. Scratching wound assay evaluate the ability of cell migration.(2)The serum containing Buqing Granules was prepared by adoping the method of serum pharmacology of TCM.Rats were divided into the blank group(distilled water,10 m L·kg-1),Qiju Dihuangwan(QJ)group,BQ group(1,5,10g·kg-1).There were 15 rats in each group.At the end of the 3th day serum was obtained.SRA01/04 cells were divided into 6 groups:10%blank serum group,high-glucose group+10%blank serum group,high-glucose+10%serum containing BQ 10,5,1g·kg-1group,high-glucose +10%serum containing QJ group.SRA01/04 were cultured in 96-well plates(2×104),the CCK-8 assay was used as a qualitative index of cell proliferation. Another SRA01/04 were cultured in 25 cm2 cell culture bottles(2×104/m L) for cell cycle analysis by flow cytometry.(3)SRA01/04 cells were divided into 5 groups:10%blank serum group,high-glucose group+10%blank serum group,high-glucose+10%serum containing BQ 10,5,1g · kg-1group,high-glucose.Using real-time PCR measure Atg7,LC3 a and Beclin1 m RNA.The expression level of Atg7,LC3 a and Beclin1 in SRA01/04 cell was observed with immunofluorescence analysis.Result(1)Compared with normol control group, high-glucose induced obvious proliferation of the SRA01/04. Especially,the peaks of cell proliferation were in the group of 30.0mmol/L glucose at 48 h. High-glucose(30mmol/L) induced decrease of cells in G0/G1 phase(P<0.01), but increased cells in S phase(P<0.05).Deducting cell proliferation by the cell viability ratio, the number of migrating cells in high-glucose group was significant increased compared with normal control group(P<0.05).(2)Compared with 10%blank group,high-glucose(30mmol/L) group,QJ group(1g·kg-1) and BQ group(1,10 g · kg-1),BQ group(5g · kg-1) suppressed obvious proliferatiion of SRA01/04 cells(P<0.01),Meanwhile,increased of cells in G0/G1 phase(P<0.05), but decreased cells in S phase(P<0.05).(3)Immunofluorescence staining showed that SRA01/04 cells in BQ group(1,5,10g·kg-1) have a gene expression of Atg7,LC3 a and Beclin1.The expression Atg7,LC3 a and Beclin1 m RNA increase in BQ group(1,5,10g·kg-1)(P<0.05).Conclusion(1)It is appreciate to choose 30.0mmol/L glucose at 48 h as the best concentration for establishing model. High glucose may promote the migration and assisted in the cycle at G1/S phase of SRA01/04 cell.(2)The serum containing BQ could arrest with the cell cycle at G1/S phase and inhibit the proliferation of SRA01/04 cells induced by high glucose.(3)The mechanism is likely to be related that QJ may keep the expression of Atg7,LC3 a and Beclin1 maintaining a high-level in SRA01/04 cells induced by high glucose.