Molecular Mechanism Of Rhein In The Treatment Of Ischemia-reperfusion Induced Acute Kidney Injury Based On Apoptosis Mediated By P38 MAPK/JNK/Caspase-3 Signaling Pathway

OBJECTIVE(1)To study the potential pathway mechanism of rhein in the treatment of acute kidney injury(AKI)by network pharmacology.(2)A rat model of ischemia-reperfusion induced acute kidney injury(IR-AKI)was established by bilateral renal artery occlusion.In vivo experiments were conducted to study the renal protective effect and inhibitory effect on apoptosis of rhein on IR-AKI rats,and to explore its mechanism;(3)HK-2 cells were induced by hypoxia/reoxygenation(H/R)to simulate the process of renal ischemia-reperfusion in vivo.Through in vitro experiments,the optimal dose and intervention time of rhein to improve HK-2 cell viability were screened,and SB203580 was used as a positive control drug to further study the inhibitory effect of rhein on HK-2 cell apoptosis of HK-2 cell induced by H/R and its mechanism..METHOD(1)The structure and drug targets of rhein were searched through Pubchem,Swisstargets,SEA,and genecards databases.The disease targets of AKI were obtained through Genecards,NCBI,TTD,OMIM,Dis Ge NET databases.Use related software to screen drug-disease common targets,and construct a "disease-targetcomponent" network graph and a PPI network of protein interactions to screen core targets.GO enrichment analysis and KEGG enrichment analysis were performed on the core targets to predict the potential pathway mechanism of rhein in the treatment of AKI.(2)The IR-AKI rat model was established by the method of bilateral renal artery clipping and divided into sham group,model group and rhein group.After the model was established,normal saline or rhein was injected through the tail vein for intervention.The methods of biochemical analysis,HE staining,WB,PCR and TUNEL were used to detect serum creatinine,urea,renal tissue pathological damage, cell apoptosis and protein and m RNA expression of Bax,Bcl-2,JNK,P38-MAPK,Caspase-3.(3)The HK-2 cell model of H/R injury was established and divided into control group,model group,and rhein group with different concentration gradients.Cell viability was detected by CCK8 method to screen the optimal concentration and intervention time.According to the screened rhein concentration and intervention time,a HK-2 cell model with H/R injury was established and divided into control group,model group,rhein group and SB203580 group.Flow cytometry was used to detect the apoptosis of HK-2 cells.The protein and m RNA expression levels of P38-MAPK,JNK,Bax,Bcl-2 and Caspase-3 were detected by Western Blot and PCR.RESULT(1)Through database search,212 drug targets of rhein and 7614 disease targets of AKI were screened,and 190 drug-disease common targets were obtained after taking the intersection.After constructing the PPI network,through topological analysis and cluster analysis,core targets such as STAT3,TP53,MAPK3,MAPK1,IL6,CASP3,GRB2,MAPK8,TNF,APP,8 gene clusters and 7 core genes were screened.The core targets were enriched by GO and KEGG,and the intersection targets were enriched into 1944 biological processes,142 molecular function-related processes and 40 cellular composition-related processes.A total of 133 signaling pathways were obtained,mainly enriched in apoptosis and other signaling pathways.Further analysis of the apoptosis pathway showed that 21 targets were enriched in the apoptosis pathway,mainly including the Bcl-2 family,the Caspase family and the MAPK family,which may represent the targets of rhein to interfere with apoptosis.In previous studies,the m RNA expressions of P38 MAPK and JNK in H/R-injured HK-2 cell models were significantly different,which were related to the decrease of cell viability.Based on this,we believe that the effect of rhein in the treatment of AKI is related to the inhibition of apoptosis induced by P38 MAPK/JNK/Caspase-3.(2)After modeling,intravenous injection of normal saline or rhein solution was administered for intervention.Compared with the sham group,the renal index of each model group was increased,and the increase was the most obvious in the 24 h group.In the rhein group,the renal index decreased gradually with the increase of reperfusion time.At 12 h,24h,and 48 h after modeling,the serum creatinine and urea in the model group were higher than those in the sham group,and the increase was the most obvious at 48 h.Compared with the model group,the urea and serum creatinine were significantly decreased in the rhein group at 48 h.HE staining results showed that there was no obvious pathological damage at each time point in the sham group;the rats in model group showed different degrees of renal tubular damage,and the Pallers pathological damage score was significantly increased.Compared with the model group,the pathological damage of rhein group was relieved and the improvement was most obvious at 48 h.The results of TUNEL staining showed that,compared with the sham group,the percentage of apoptosis in the kidney tissue of the model group increased significantly at each time point;compared with the model group,the apoptosis of rhein in the kidney tissue was significantly improved at 24 h and 48 h.After 48 hours of reperfusion,compared with the sham group,the protein and m RNA expressions of Bax and Caspase-3 in the kidney tissue of the model group were significantly increased,and the protein and m RNA expressions of Bcl-2 were significantly decreased.After intravenous injection of rhein,compared with the model group,the expressions of Bax and Caspase-3 in the kidney tissue of the rats in the rhein group were down-regulated,and the expression of Bcl-2 was up-regulated.The expression of P38 MAPK and JNK was further detected.Compared with the sham group,the protein expression levels of P38 MAPK and p-P38 MAPK,and the m RNA of P38 MAPK in the model group were significantly increased.Compared with the model group,the protein expression levels of P38 MAPK and p-P38 MAPK and the m RNA of P38 MAPK in the kidney tissue of the rats in the rhein group were significantly decreased.Compared with the sham group,the protein expression levels of JNK and p-JNK,and the m RNA of JNK in the model group were significantly increased.Compared with the model group,the protein expression levels of JNK and p-JNK and the m RNA of JNK in the kidney tissue of the rats in the rhein group were significantly decreased.(3)After establishing the H/R-injured HK-2 cell model,the cell viability was significantly decreased.Compared with the model group,rhein could significantly improve the viability of HK-2 cells,and the effect was most obvious at 80 μM for 24 h.Further detection of cell apoptosis,compared with the model group,the result showed that the cell apoptosis rate in the rhein group and SB203580 group was significantly decreased.Compared with the control group,the protein expression levels of Bax and Cleaved-Caspase-3 in the model group were significantly increased,the protein expression of Bcl-2 was significantly decreased,and the ratio of Bax/Bcl-2 was increased,while the protein expression of Caspase-3 was not obvious.Compared with the model group,rhein and SB203580 could significantly inhibit the protein expression of Bax and Cleaved-Caspase-3,and reduce the ratio of Bax/Bcl-2.After establishing the H/R injury HK-2 cell model,the expression of P38 MAPK and JNK did not change significantly,but the expression of p-P38 MAPK and p-JNK increased.Compared with the model group,the expression of p-P38 MAPK decreased in the rhein group and P38 MAPK inhibitor groups,while the expression of P38 MAPK did not change.Compared with the model group,the expression of p-JNK decreased in the rhein group and P38 MAPK inhibitor groups,while the expression of JNK did not change.CONCLUSION(1)The results of network pharmacology study found that rhein in the treatment of AKI has the characteristics of multiple targets,and the apoptosis signaling pathway may be related to the renoprotective effect of rhein in the treatment of AKI,and is closely related to the MAPK,BCL-2,and Caspase families.Combined with previous studies,we believe that the effect of rhein in the treatment of AKI may be related to the inhibition of apoptosis induced by P38 MAPK/JNK/Caspase-3.(2)Combined in vivo and in vitro experiments,IR-AKI is characterized by renal tubular damage,and the induction of renal tubular epithelial cell apoptosis by P38 MAPK/JNK/Caspase-3 signaling pathway is one of the important pathological mechanism of IR-AKI.Rhein can inhibit the activation of P38 MAPK/JNK/Caspase-3 signaling pathway,inhibit the apoptosis of renal tubular epithelial cells and play a renal protective effect.