Effects Of Tissue Inhibitor Of Metalloproteinase-2 And Insulin-like Growth Factor Binding Protein-7 On Sepsis-induced Acute Renal Injury And Their Possible Mechanisms

Part 1 Objective: To construct a model of acute renal injury(AKI)induced by sepsis in mice,and detect the differential expression of inflammatory factors in serum,cycle-related protein and apoptosis-related protein in renal tissue,IGFBP7 and TIMP-2 in renal tissue and urine by molecular biological methods such as renal histopathology analysis,enzyme-linked immunosorbent assay(ELISA)and Western blot.Methods: Thirty-six SPF grade C57 BL / 6 male mice aged 6-8 weeks and weighing 20-25 g were randomly divided into sham operation(sham)group and cecal ligation and puncture(CLP)group.Each group was further divided into three subgroups(6h,12 h,24h).In the sham group,the abdomen was closed only after laparotomy,and in the CLP Group,50% of the cecum was ligated and perforated.The urine of each group was collected at the preset time point and the levels of IGFBP7 and TIMP-2 were measured by ELISA.Blood was collected by left heart puncture after anesthesia and the TNF-? and IL-6 levels of plasma were measured by ELISA.The expression of cyclin D and cyclin E,Bcl-2 and Bax were detected by Western blot,IGFBP7 and TIMP-2 were detected by RT-PCR and Western blot.Result: 1.Comparison of plasma inflammatory factors Compared with the sham group,the levels of TNF-a and IL-6 began to increase 6 hours after CLP,and the highest levels were 12 hours after CLP.2.Comparison of renal pathology and pathological scores There was no significant change in the renal tissue of the sham group.In CLP Group,there were swelling of tubular epithelium,loss of brush border,vacuole degeneration,tubular necrosis and tubular formation in kidney tissue.Compared with the sham group,the renal histopathological score of the CLP Group was significantly increased.The histopathological score of the kidney increased with the time after CLP.3.Expression of cyclin D and cyclin E and Bcl-2/Bax ratio Compared with the sham group,the expression of cyclin D and cyclin E and Bcl-2/Bax ratio decreased with time in CLP Group.4.Levels of IGFBP7 and TIMP-2 in kidney and urine Compared with the sham operation group,the urinary levels of IGFBP7 and TIMP-2 in CLP Group were significantly higher than the sham operation group.There was no significant difference between sham group and CLP Group at 6h and 12h(p>0.05);the expression of IGFBP7 in the CLP Group was significantly lower than that in the sham group at 24h;compared with sham group,TIMP-2 level in renal tissue increased at 6h and 12h;there was no significant difference between sham group and CLP Group at 24H(p>0.05);compared with sham group,the levels of IGFBP-7 and TIMP-2mRNA in renal tissue in CLP Group were significantly increased.Conclusion: 1.50% cecal ligation and puncture duplicate sepsis-induced AKI and not easy to cause animal death.2.G1/S cell cycle arrest of renal tissue and apoptosis of renal tissue in mice were induced by sepsis.3.The levels of IGFBP7 and TIMP-2 in the urine of mice with sepsis-induced AKI were significantly increased,which was related to the increase of IGFBP7 and TIMP-2 synthesis in the kidney.Part 2 Objective: To construct the model of LPS induced inflammatory response in IGFBP7 and TIMP-2 silenced TCMK-1 cells and investigate the effects of IGFBP7 and TIMP-2 on LPS induced inflammatory response,G1/S cell cycle arrest,apoptosis and autophagy in TCMK-1 cells.Methods: Treated TCMK-1 cells with 0,10,20,50,100,200 and 500 ? g / ml LPS for 6,12 and 24 hours respectively.The inflammatory factors were detected by ELISA and the levels of cyclin D,cyclin E and p-RB were detected by Western blot.The cells were divided into the control group,LPS group,NC group,LPS+IGFBP7 siRNA group and LPS+TIMP-2 siRNA group.The cells were transfected with lipo3000.When the cells were full,the LPS group,NC group,LPS+IGFBP7 siRNA group and LPS+TIMP-2 siRNA group were added with 400 ? l of LPS mother liquor with a concentration of 1mg / ml,so that the final concentration of LPS was 200 ? g / ml.After 6 hours,the cells and culture medium were collected,the levels of inflammatory factors,IGFBP7 and TIMP-2 were detected by ELISA.The levels of IGFBP7,TIMP-2,cyclin D,cyclin E,p-RB,Bcl-2 and Bax,LC 3b and p62 were detected by Western blot.Result: 1.The effects of LPS on the concentration of inflammatory factors and the levels of cyclin D,cyclin E and p-RB in TCMK-1 cells After treatment with LPS for 12 hours and 24 hours,it was found that there was no significant difference in the concentration of inflammatory factors and the levels of cyclin D,cyclin E and p-RB among the different concentration gradients.After treatment with LPS for 6 hours,the concentration of inflammatory factors increased in a dose-dependent manner,and the levels of cyclin D,cyclin E and p-RB decreased in a dose-dependent manner.2.Silencing efficiency of siIGFBP7 and siTIMP-2 After transfection,there was no significant difference between the expression of targeted proteins in the NC group and the control group(p> 0.05).Compared with the control group,siIGFBP7 and siTIMP-2 lead to the decrease of target protein expression in TCMK-1 cells.Compared with the control group,the expression level of the target protein in group I3 and T3 was the lowest(all p<0.001).The lowest silence efficiency was 69.8% and 79.7%,respectively.Therefore,in this study,siIGFBP7-953 and siTIMP-2-884,which were used in I3 and T3 groups,were selected for further study.3.The effect of IGFBP7 and TIMP-2 on the concentration of inflammatory factors in culture medium Compared with the control group,the inflammatory factors in the LPS group and NC group increased significantly;there was no significant difference in the concentration of inflammatory factors between the LPS group and NC group(p>0.05);the concentration of inflammatory factors in siIGFBP7 and siTIMP-2 group decreased significantly compared with NC group.4.The effects of IGFBP7 and TIMP-2 silencing on the levels of cyclin D,cyclin E and pRB Compared with con group,the levels of cyclin D,cyclin E and p-RB in LPS group and NC group were significantly lower;there was no significant difference in the levels of cyclin D,cyclin E and p-RB between LPS group and NC group(p>0.05);the levels of cyclin D,cyclin E and p-RB in LPS+IGFBP7si RNA and LPS+TIMP-2si RNA were significantly higher than NC group.5.The effect of IGFBP7 and TIMP-2 on Bcl-2/Bax ratio Compared with the control group,the Bcl-2/Bax ratio of LPS group and NC group was significantly lower;there was no significant difference between the LPS group and NC group(p>0.05);the Bcl-2 / Bax ratio of LPS+IGFBP7siRNA and LPS+TIMP-2siRNA was significantly higher than NC group.6.Effects of IGFBP7 and TIMP-2 on the levels of LC 3b and p62 Compared with control group,LC 3b ? /I ratio of LPS group and NC group was significantly lower and p62 level was significantly higher;there was no significant difference in LC 3b ?/I ratio and p62 level between LPS group and NC group(p>0.05);LC 3b ?/I ratio of LPS+IGFBP7siRNA and LPS+TIMP-2siRNA group was significantly higher and p62 level was significantly lower than NC group.Conclusion: 1.LPS treatment for 6 hours induced inflammatory response and G1/S cell cycle arrest in TCMK-1 cells in a dose-dependent manner.2.IGFBP7 and TIMP-2 silencing significantly alleviated G1/S cell cycle arrest and inflammatory response,and reduced the level of apoptosis and autophagy caused by LPS.Part 3 Objective: To construct the model of LPS induced inflammatory response in IGFBP7 and TIMP-2 silenced and Ribociclib pretreated TCMK-1 cells,and further explore the role of the G1/S cell cycle in the sepsis-induced AKI.By detecting the levels of ERK 1 / 2,JNK,p38,Akt and mTOR,the possible mechanism of IGFBP7 and TIMP-2 in sepsis-induced AKI was further determined.Methods: the cells were treated with 0,100 nM,500 nM,1 ?M,2 ?M,5 ?M,10 ?M Ribociclib and collected 24 hours later.The levels of p-RB,Bcl-2 and Bax were detected by Western blot.The cells were divided into the control group,NC group,siIGFBP7 group,siTIMP-2 group,si IGFBP7 + R group and siTIMP-2 + R group.The cells in the NC group,siIGFBP7 group,siTIMP-2 group,si IGFBP7 + R group and siTIMP-2 + R group were transfected with lipo3000.When the fusion degree of cells was about 80%,the cells in siIGFBP7 + R group and si TIMP-2 + R group were treated with 5 ? m Ribociclib for 18 hours and then replaced with 5 ? m Ribociclib for 6 hours,the NC group,siIGFBP7 group and siTIMP-2 group were treated with LPS of 200 ?g/ml for 6 hours.Western blot was used to detect the levels of p-RB,Bcl-2 and Bax,ERK 1 / 2,JNK,p38,Akt and mTOR.Result: 1.The effect of Ribociclib on the levels of p-RB,Bcl-2 and Bax in TCMK-1 cells Compared with the control group,the level of p-RB decreased in a dose-dependent manner,24 hours after the treatment of Ribociclib,and there was no significant difference in the level of the Bcl-2/Bax ratio among the concentration gradients.2.The effect of Ribociclib pretreatment on the p-RB level and Bcl-2/Bax ratio in LPS treated TCMK-1 cells after IGFBP7 and TIMP-2 silencing Compared with control group,p-RB level and Bcl-2/Bax ratio in NC group decreased significantly;compared with NC group,p-RB level and Bcl-2/Bax ratio in siIGFBP7 group and siTIMP-2 group increased significantly;compared with siIGFBP7 group and siTIMP-2 group,p-RB level of siIGFBP7+R group and siTIMP-2+R group decreased significantly,and there was no significant difference in Bcl-2/Bax value.3.The effect of Ribociclib pretreatment on the level of LC 3b and p62 in LPS treated TCMK-1 cells after IGFBP7 and TIMP-2 silencing Compared with the control group,LC 3b ? /I ratio in the NC group decreased significantly,and p62 level increased significantly;compared with the NC group,LC 3b ?/I ratio in si IGFBP7 group and si TIMP-2 group increased significantly,and p62 level decreased significantly;compared with siIGFBP7 group and siTIMP-2 group,LC 3b ?/I ratio of siIGFBP7+R group and siTIMP-2+R group increased significantly,and p62 level decreased significantly.4.The effects of IGFBP7 and TIMP-2 silencing on the Akt/mTOR and MAPK pathways in LPS treated TCMK-1 cells Compared with the control group,the levels of p-38,p-JNK,p-ERK1/2 and p-Akt in LPS group and NC group were significantly increased;there was no significant difference in the levels of P-38,p-JNK,p-ERK1/2 and p-Akt between LPS group and NC group(p>0.05);compared with NC group,the levels of p-38,p-JNK,p-ERK1/2 and p-Akt in LPS+IGFBP7 siRNA and LPS+TIMP-2 siRNA group were significantly increased.Conclusion: 1.Ribociclib induced the G1/S cycle arrest of TCMK-1 cells in a dose-dependent manner without inducing apoptosis.2.The effect of IGFBP7 and TIMP-2 silencing on decreasing the LPS induced apoptosis did not depend on cell cycle arrest.3.Ribociclib increased the autophagy level in IGFBP7 and TIMP-2 silenced cells 4.IGFBP7 and TIMP-2 alleviated the inflammatory injury of TCMK-1 cells through MAPK and Akt / mTOR pathway.