| Objective: To investigate the effect of ursolic acid(UA)in the process of endoplasmic reticulum(ER)stress induced by ochratoxin A(OTA)in human renal tubular epithelial cells(HK-2)and the mechanism of Lonp1 and Sig-1R in UA alleviating OTA-induced ER stress(ERS)in HK-2 cells.Methods:1.This experiment applys HK-2 cells as the experimental model,using the method of CCK-8 to determine cell survival rate under the different concentration of UA after pretreatment,then decide the subsequent experimental concentration of UA.2.Set experimental groups: Control group(C group,0 μmol/L UA and 0 μmol/L OTA treatment for 26 h),UA group(UA group,4 μmol/L UA treatment for 2 h),OTA group(5 μmol/L OTA treatment for 24 h),UA group and OTA group(UO group,4 μmol/L UA for 2 h and 5 μmol/L OTA for 24 h).Then we detected the morphological changes,cell survival rate,intracellular ROS level and ERS-related protein expression by means of inverted microscope,CCK-8 method,DCFH-DA probe and western blot.3.Research the expression of related proteins in the experimental groups of HK-2 cells treated by Lonp1 inhibitor CDDO-me.Then we set the control groups(CDDO-me-groups)and inhibitor-treated groups(CDDO-me + groups).The expression of related proteins was measured by western blot in CDDO-me-groups and CDDO-me + groups according to the grouping method in 2.4.The experimental groups of HK-2 treated with Sig-1R agonist AVex-73 hydrochloride.We divided them into the control groups(AVex-73-groups)and the agonist-treated groups(AVex-73 + groups).They were grouped according to the method in 2,and the expressionof related proteins was determined by western blot.5.The cells were transiently transfected with Sig-1R si RNA for 5 hours.After 24 hours under cultured by complete medium,the inhibitory effect of Sig-1R si RNA on Sig-1R was detected by western blot.Then we set the control si RNA groups and Sig-1R si RNA treated groups.According to the method in 2,the expression of related proteins was determined by western blot.Results:1.CCK-8 results showed that the survival rate of HK-2 cells was significantly increased after pretreatment with 4 μmol/L UA for 2 h(P<0.05).Compared with OTA group,UO group significantly reversed the inhibitory effect of OTA on cell viability(P<0.05).2.Intracellular ROS levels in the group were significantly higher than those in the control group(P<0.05),and a small amount of ROS was induced in the UA group(P<0.05).ROS content in UO group was significantly lower than that in OTA group(P<0.05).3.Inverted microscope observation: the cell shape in OTA group were badly demaged and UO group could improve their morphology and growth rate.4.Western blot results indicated that the repressed expression of Lonp1 protein and Sig-1R protein under OTA treated could be reversed by UA.Compared with group C,the expression of Lonp1 and Sig-1R in OTA group was significantly decreased(P<0.05).The expression of Sig-1R in UA group was significantly increased(P<0.05),while there were not a distinct change in the expression of Lonp1 protein(P>0.05).Compared with OTA group,the expressions of Lonp1 and Sig-1R were significantly increased in UO group.UA pretreatment can alleviate ERS induced by OTA.The expression of p-perk,p-EIF2α,IRE1α,CHOP and GRP78 in OTA group were significantly increased in OTA group(P<0.05).The expressions of ERS related proteins p-PERK,p-EIF2α,IRE1α,CHOP and GRP78 were significantly decreased in UO group compared with OT group(P<0.05).UA pretreatment could alleviate OTA induced apoptosis.Compared with group C,the expression of anti-apoptotic protein Bcl-2 was significantly decreased and the expression of pro-apoptotic protein Bax was markedly increased in OTA group(P<0.05),but the expression of Bcl-2 and Bax was not significantly changed in UA group(P>0.05).Compared with OTA group,the expression of anti-apoptotic protein Bcl-2 was significantly increased(P<0.05)and the expression of pro-apoptotic protein Bax was significantly decreased(P<0.05)in UO group.5.Pretreatment with 0.75 μmol/L CDDO-me for 2 h could effectively inhibit Lonp1 protein expression(P<0.05).Inhibition of Lonp1 can significantly reduce the expression of Sig-1R(P<0.05)and increase the expression of GRP78 and CHOP(P<0.05).6.40 μmol/L AVEX-73 Hydrochloride pretreated for 2 h could effectively stimulate the expression of Sig-1R protein(P<0.05).Upregulating Sig-1R can increase the expression of Lonp1(P<0.05),which can significantly inhibit the expression of GRP78 and CHOP(P<0.05).7.Sig-1R si RNA could effectively inhibit the expression of Sig-1R(P<0.05).After inhibiting Sig-1R,the expression of Lonp1 decreased(P<0.05),and the expression of CHOP and GRP78 increased significantly(P<0.05).Conclusion: 1.UA can alleviate renal cytotoxicity caused by OTA-induced ERS and apoptosis in HK-2 cells.2.Lonp1 and Sig-1R promote each other and the positive feedback regulation plays an important role in UA alleviating OTA-induced ERS in HK-2 cells. |
PDF Full Text Request