Study On The Mechanism Of MTORC1 Regulating Apoptosis And Autophagy Induced By TNF-α In HUVECs

Vascular endothelial cells are essential to maintain the stability of vascular function and structure.The damage of vascular endothelial cells induces the progression of atherosclerosis,vasculitis,microcirculation disorder in sepsis patients,vascular injury-related diabetic syndrome,and so on.Lipopolysaccharide(LPS)is the cell wall component of Gram-negative bacteria,which is also known as endotoxin.After systemic infection with Gram-negative bacteria,LPS causes the damage of vascular endothelial cells by inducing macrophages,neutrophils and platelets to release the cytokines,such as TNF-α.However,the specific molecular mechanisms of vascular endothelial cell injury and cell self-protection induced by TNF-α are not clear.In the present study,we tried to explore the mechanisms of HUVECs apoptosis and autophagy induced by TNF-α,which will provide new evidence for elucidating the mechanisms of increased TNF-α level in blood resulting in the damage of vascular endothelial cells after Gram-negative bacteria infection.The main results are as follows:1.The level of TNF-α in serum of atherosclerotic patients and culture supernatant of human macrophage treated with LPS was detected by enzyme-linked immunosorbent assay.The results showed,(1)The level of TNF-α in serum of vasculitis patients was higher than that of healthy subjects(p<0.01).(2)The level of TNF-α in culture supernatant of human macrophage treated with LPS was significantly increased(p<0.05).2.The proliferation and apoptosis of HUVECs after treatment with TNF-α were examined by MTT assay,Hoechst 33342 staining,western blot,fluorescence spectrophotometry and flow cytometry.The apoptosis pathways induced by TNF-α in HUVECs were confirmed.The results showed,(1)TNF-α could significantly inhibit the proliferation of HUVECs in a concentration-and time-dependent manner.(2)TNF-α could induce the apoptosis of HUVECs.(3)After treatment with TNF-α,the expressions of death receptor TNFR1 and Fas related death domain protein FADD in HUVECs were increased,the expressions of Caspase 8 and Caspase 3 were decreased significantly,the enzyme activities of Caspase 8 and Caspase 3 were increased significantly(p<0.01).There were no significant difference in protein expression and enzyme activity of Caspase 9 compared with the control(p > 0.05).(4)The expressions of TNFR1,FADD,the total proteolysis of Caspase 8 and Caspase 3 in HUVECs induced by TNF-α were blocked by TNFR1 antibody treatment.(5)The expression of MyD88 was increased after treated with TNF-α,but the expression of TIRAP was not affected by TNF-α.These results suggested that TNF-α induces the apoptosis of HUVECs through recognizing membrane receptor TNFR1,which resulted in the up-regulation of MyD88.3.The autophagy of HUVECs induced by TNF-α was detected by MDC staining and Western blot.The results showed,(1)The number of autophagy vesicles in HUVECs cytoplasm increased after TNF-α treatment.(2)The expression of Beclin1 and the ratio of LC3 II/LC3 I increased after TNF-α induction,which indicated that TNF-α could induce autophagy of HUVECs.4.In order to indentify the key regulatory factors on the balance and transformation between apoptosis and autophagy induced by TNF-α,RNA interference technique was used to decrease the expressions of BECN1,Bcl-2,DAP1,IRF1,ATG5 and ATG12.The results showed,(1)Targeted siRNA could inhibit the expression of BECN1,Bcl2,DAP1,IRF1,ATG5 and ATG12 in HUVECs.After silencing BECN1 and Bcl-2,the autophagy in HUVECs induced by TNF-α was decreased and the apoptosis was increased.(2)After silencing BECN1,compared with the control group,the number of shear PARP and the hydrolysis of Pro-Caspase 3 were increased in TNF-α treated group,which promoted the apoptosis induced by TNF-α.At the same time,the ratio of LC3 II/LC3 I was decreased.MDC staining showed that autophagy vesicles were decreased and autophagy was inhibited.(3)After treated with PPX(Beclin1 agonist)for 1 h,compared with the control group,the shear PARP was decreased,Pro-Caspase 3 did not change significantly and apoptosis was inhibited in TNF-α treated 12 h group,while Beclin1 expression and LC3 II/LC3 I ratio were increased,which promoted autophagy.These results suggested that Beclin1 regulated the balance or transformation between apoptosis and autophagy induced by TNF-α in HUVECs,promoting autophagy and inhibiting apoptosis.5.The regulatory effect of mTORC1 on BECN1 was detected by qPCR and Western blot.The results showed that Rapamycin promoted the expression of Beclin1 induced by TNF-α,and the overexpression of Rheb resulted in the decrease of Beclin1.These results suggested that the expression of Beclin1 was regulated by mTORC1.6.After silencing p53,the expression of Beclin1 induced by TNF-α was decreased,indicating that the transcription factor p53 was involved in the expression of BECN1.Rapamycin treatment could promote the expression of p53 induced by TNF-α,and the overexpression of Rheb inhibited the expression of p53 protein,which indicated that mTORC1 regulated p53 expression.It was suggested that mTORC1 might regulate BECN1 expression through transcription factor p53.In conclusion,TNF-α could inhibit the proliferation of HUVECs and induce apoptosis through death receptor-MyD88 pathway.TNF-α could also induce the autophagy in HUVECs,which is regulated by mTORC1.mTORC1 signaling pathway regulated BECN1 expression and Beclin1 protein synthesis through transcription factor p53,and then regulated the autophagy and apoptosis of HUVECs induced by TNF-α to maintain cell homeostasis.These data provided new evidence for understanding the mechanisms of vascular endothelial cells injury induced by TNF-α and cell self-protection.It also provided a new idea for the prevention of atherosclerosis,vasculitis,vascular injury-related diabetic syndrome and other related diseases caused by vascular endothelial cell injury after infection with Gram negative bacteria.