The Effects Of Green Tea Polyphenols On Autophagy Flux Of Endothelial Cells Cultured In High Glucose Conditions

Objectives Chronic hyperglycemia is an important reason for endothelium dysfunction. To observe the effects of high glucose on autophagy of endothelial cells and investigate the benefit effects of green tea polyphenols(GTPs) on protecting endothelial cells from high glucose via autophagy. Methods Bovine aortic endothelial cells were cultured in medium containing 0.4 and 4 mg/L GTPs, 33 mM high glucose and a mixture of 4 mg/L GTPs and 33 mM high glucose. The autophagy indication protein LC3-II, SQSTM1/p62,Beclin-1, mTOR and p-mTOR(2448) were determined by Western Blot after these treatment. The autophagy flux was determined by Western Blot in the presence of bafilomycin A1(BAF). Results(1) GTPs dose(P<0.05) and time(P<0.05) dependent increase the protein level of LC3-II, 4 mg/L GTPs have the strongest autophagy inducing effects(P<0.05). Meantime, the protein level of Beclin-1 were increased(P<0.05) and SQSTM1/p62 were decreased(P<0.05). The protein level of LC3-II induced by GTPs further increased significantly in the presence of BAF(P<0.05).(2) The protein level of LC3-II time dependent increased under high glucose conditions from 6- 24h(P<0.05). And increased protein level of SQSTM1/p62(P<0.05) and decreased Beclin-1(P<0.05) were observed. The protein level of LC3-II induced by high glucose did not further increased in the presence of BAF(P>0.05).(3) The protein level of LC3-II significantly decreased under high glucose conditions when exposed to high glucose for 72h(P<0.05). The protein level of Beclin-1was decreased(P<0.05) whereas the change of protein protein level of SQSTM1/p62 was not observed(P>0.05). BAF treatment increased the protein level of LC3-II(P<0.05) after exposed to high glucose for 72 h, however, it was lower than exposed to normal glucose still(P<0.05).(4) GTPs and high glucose co-treatment for 24 h significantly reduced the increasing protein level of LC3-II induced by high glucose(P<0.05). Besides, GTPs treatment significantly reduced the protein protein of SQSTM1/p62(P<0.05) and increased the protein level of Beclin-1 under high glucose conditions(P<0.05). In comparison with high glucose treatment, the protein of LC3-?significantly increased(P<0.05) after GTPs co-treatment with high glucose in the presence of BAF.(5) GTPs treatment with 72 h high glucose significantly increased the protein protein level of LC3-II under high glucose conditions(P<0.05). Meantime, it significantly reduced the protein protein level of SQSTM1/p62(P<0.05) and attenuate the reducing protein level of Beclin-1 under high glucose conditions(P<0.05). In comparison with high glucose treatment, GTPs significantly increased the protein protein level of LC3-II in the presence of BAF(P<0.05).(6) The protein level of mTOR, p-mTOR(2448) and the ration of p-mTOR(2448) and mTOR were not found changed under 24 h,72h high glucose conditions and GTPs co-treatment with high glucose(P>0.05). Conclusion 4mg/L GTPs enhanced the autophagy flux in endothelial cells and 33 mM high glucose inhibited autophagy flux, 4mg/L GTPs alleviated the autophagy inhibition induced by 33 mM high glucose. The effects of 4mg/L GTPs and 33 mM high glucose on autophagy flux in endothelial cells were independent from mTOR signal pathway. Our results indicated that GTPs might be a promising approach for prevention diabetes cardiovascular complications.