Effect Of Omeprazole On Nephrotoxicity Of Adriamycin And Its Preliminary Mechanism

Acute kidney injury（AKI）is a common syndrome in clinic,which with high mortality and the risk of developing chronic kidney disease and end stage injury disease.Nephrotoxic drugs are a common cause of acute injury injury.Adriamycin（ADM）is a chemotherapeutic drug often used in the clinical treatment of malignant tumors,but with many side effects,nephrotoxicity is one of the serious adverse reactions that can not be ignored in chemotherapy.Although the basic process of ADM-mediated nephrotoxicity is still unclear,there is evidence that the contributing factors include oxidative stress and apoptosis.At the same time,ADM chemotherapy with gastrointestinal adverse reactions is widely used in combination with proton pump inhibitors（PPIs）to alleviate gastrointestinal adverse reactions.It is reported that PPIs can also cause kidney damage,and the molecular mechanism of PPIs nephrotoxicity may be related to mitochondrial damage.Mitochondria are the main sites to product reactive oxygen species（ROS）.ROS is extremely important in maintaining the stability of internal and external environment and signal transduction.When the mitochondria function is damaged,the accumulated ROS will in turn attack the maladjusted mitochondria to aggravate the damage of mitochondria and form a vicious circle.At the same time,excessive intracellular ROS leads to oxidative stress,and some ROS can induce the activation of NF-κB,activate apoptosis-related signal pathways,and lead to disease.At present,the effect and mechanism of PPIs on adriamycin-induced kidney injury are not clear.Therefore,on the basis of clinical reports,the purpose of this study is to establish a rat model of minimal lesion of ADM to investigate the effect of PPIs on nephrotoxicity of ADM,and to explore the effect of mitochondrial oxidative stress pathway mediated by F₁F₀-ATPase on kideny cell apoptosis in this process,so as to provide some experimental basis for clinical rational drug use.OBJECTIVE1.To determine the effect of omeprazole on the kidney injury of adriamycin in Wistar rat.2.To reveal the preliminary mechanism of omeprazole increases kidney cell apoptosis through F₁F₀-ATPase-mediated mitochondrial oxidative stress pathway,which in turn aggravates adriamycin-induced nephrotoxicity in rats.METHODS1.The rat model of kidney damage induced by adriamycin was established and intervened with omeprazole for 42 days.Then the blood of rats was collected to prepare serum and24-hour urine,and all the kidneys were collected.Kidney function indexes（serum creatinine and endogenous creatinine clearance）,kidney pathological changes and kidney cell apoptosis（TUNEL staining）were detected in each group.To explore the effect of omeprazole on the rat model of mild kidney damage induced by adriamycin,the oxidative indexes（GSH and MDA）of rat kidney were detected,the activity of F₁F₀-ATPase was mensured by micromethod,the expression of apoptosis-related protein and p-P65 were detected by WB（Western blot,WB）.2.The effects of different concentrations of omeprazole and adriamycin on the proliferation of NRK-52E cells were detected by CCK-8,and the best concentration and time of omeprazole and adriamycin to stimulate NRK-52E cells were selected.3.The effects of omeprazole and adriamycin on apoptosis of NRK-52E cells were detected by flow cytometry.The effects of omeprazole and adriamycin on the expression of apoptosis-related proteins and p-P65 in NRK-52E cells were detected by Westernblot assay.Colorimetry and fluorescence probe were used to detect the effects of omeprazole and adriamycin on mitochondrial function of NRK-52E cells.4.Discovery Studio 2019 Client software was used to dock the small molecule omeprazole and F₁F₀-ATPase protein to observe whether omeprazole and F₁F₀-ATPase can interact directly.The effects of omeprazole,oligomycin and adriamycin on F₁F₀-ATPase activity of NRK-52E cells were detected by micromethod,then the mitochondrial function and ROS of NRK-52E cells induced by omeprazole and oligomycin were detected by colorimetry and fluorescence probe method,and the apoptosis of NRK-52E cells was detected by flow cytometry.To explore the effect of omeprazole on mitochondrial function on ROS accumulation and apoptosis induced by adriamycin by regulating F₁F₀-ATPase.5.ROS scavenger N-acetyl-L-cysteine（NAC）was used to remove the accumulation of ROS in NRK-52E cells induced by omeprazole and adriamycin,then the apoptosis rate of NRK-52E cells was measured by flow cytometry,the expression of apoptosis-related proteins and the activation of NF-κB in NRK-52E cells were detected by Westernblot,and the changes of mitochondrial function in NRK-52E cells were detected by colorimetry and fluorescence probe.To explore the role of ROS/NF-κB in apoptosis induced by omeprazole and adriamycin.RESULTS1.Effect of omeprazole on adriamycin-induced kidney injury in rats.The results showed that the urine protein,serum creatinine and endogenous creatinine clearance rate of adriamycin-induced rats were significantly higher than those of normal rats on the 14th day,indicating that the model was successful.After 56 days,compared with normal rats,the serum creatinine increased significantly in adriamycin-induced rats,and further increased in the combined administration group（omeprazole and adriamycin）.At the same time,compared with adriamycin group,the creatinine clearance rate of rats treated with adriamycin and omeprazole decreased significantly.The pathological results showed that there were histopathological changes in the kidneys of rats in each drug group.compared with the normal group,Mesangial hyperplasia appeared in the glomeruli of rats in the adriamycin group,and the epithelial cells of the kidney tubules formed vacuoles and fell off to form a colloid protein tube type.there was multifocal inflammatory cell infiltration in the kidney interstitium,in addition to the pathological characteristics of the adriamycin group.New pathological features of renal tubule dilatation and renal interstitial edema also appeared.The results of TUNEL staining showed that there were no obvious green fluorescent spots in the normal group,slight green fluorescent spots in the omeprazole group and more green fluorescent spots in the adriamycin group,while the green fluorescent spots in the combined group increased further,and the distribution of green fluorescent particles in the kidney tissue was annular,and the green fluorescence distribution was mainly concentrated in the nucleus of kidney tubular epithelium.The results of WB showed that the ratio of Bcl-2 to Bax in omeprazole group and adriamycin group was significantly higher than that in normal group,while the ratio of Bcl-2 to Bax in combined group was further increased,and the expression levels of Cyt C and cleaved caspase8 in adriamycin group were significantly higher than those in normal group,while the expression levels of Cyt C and cleaved caspase8 in combined group were further increased.Omeprazole inhibited the decrease of F₁F₀-ATPase activity and ATP content in kidney tissue.Both omeprazole and adriamycin could cause kidney lipid peroxidation damage.At the same time,the kidney lipid peroxidation damage induced by combined group.Both omeprazole and adriamycin can increase the expression level of p-P65 in kidney,and the expression level of p-P65 in rat kidney further increased in combined group.2.Selection of optimal concentration and time of omeprazole and adriamycin stimulating NRK-52E cells.CCK-8 results showed that omeprazole 20μM was selected as the routine intervention concentration,which had little effect on NRK-52E cell activity and was consistent with the drug concentration in patients.adriamycin 0.1μM was selected as the model concentration,and the NRK-52E cell activity decreased by about 30%at this concentration,and the stimulation time was 24 hours.3.Effects of omeprazole and adriamycin on apoptosis,mitochondrial function,ROS accumulation and NF-κB activation in NRK-52E cells.The results of Annexin V/PI double staining and WB method showed that.Compared with Control,the apoptosis rate of ADM group and OPZ+ADM group was significantly higher,and OPZ+ADM group was significantly higher than ADM group.Compared with Control group,the ratio of Bcl-2 to Bax in OPZ group,ADM group and OPZ+ADM group increased significantly,the expression level of Cyt C in ADM group and OPZ+ADM group increased significantly,and the expression level of cleaved caspase8 in OPZ+ADM group increased significantly compared with ADM group.Compared with ADM group,the ratio of Bcl-2 to apoptosis-promoting protein Bax,Cyt C and cleaved caspase8expression were significantly increased in OPZ+ADM group.The results of colorimetry showed that compared with Control group,ATP content in OPZ group,ADM group and OPZ+ADM group decreased significantly,while ATP content in OPZ+ADM group decreased significantly compared with ADM group.The results of fluorescence probe（JC-1）method showed that the mitochondrial membrane potential（MMP）of OPZ group,ADM group and OPZ+ADM group was significantly lower than that of Control group,and that of OPZ+ADM group was significantly lower than that of ADM group.The results of fluorescence probe（DCFH-DA）method showed that the release of ROS in OPZ group,ADM group and OPZ+ADM group was significantly higher than that in Control group,and the release of ROS in OPZ+ADM group was significantly higher than that in ADM group.The results of WB showed that compared with Control group,the expression of p-P65 in OPZ group,ADM group and OPZ+ADM group was significantly increased,and the level of p-P65 expression in OPZ+ADM group was higher than that in ADM group.4.Omeprazole regulates the activity of F1F0-ATPase in NRK-52E cells and its effect on mitochondrial function.The results of molecular docking show that OPZ and F₁F₀-ATPase can bind each other directly,and the optimal molecular conformation is selected.The total energy of CDOCKER docking system is-24.5262,the system is stable,and the binding energy of CDOCKER is-44.2139.There is little interaction between receptor and ligand in the docking process,and the two bind well.The results of microassay showed that compared with Control group,the activity of F₁F₀-ATPase in F₁F₀-ATPase specific inhibitor Oligomycin group,OPZ group,OPZ+ADM group and Oligomycin+ADM group decreased significantly.The results showed that OPZ could inhibit the activity of F₁F₀-ATPase to some extent.The results of colorimetry showed that compared with ADM group,the content of ATP in OPZ+ADM group and Oligomycin+ADM group was significantly lower.The results of DCFH-DA showed that compared with ADM group,the release of ROS in OPZ+ADM group and Oligomycin+ADM group was significantly higher.The results of JC-1 showed that compared with ADM group,the MMP of OPZ+ADM group and Oligomycin+ADM group was significantly lower.The results of Annexin V/PI double staining showed that compared with ADM group,the apoptosis rate of OPZ+ADM group and Oligomycin+ADM group was significantly higher.5.NAC interferes with the effects of omeprazole and adriamycin on ROS/NF-κB apoptosis pathway and mitochondrial function in NRK-52E cells.The results of flow cytometry showed that the apoptosis rate of OPZ+ADM group was significantly higher than that of Control group,while the apoptosis rate of NAC+OPZ+ADM group decreased significantly than that of OPZ+ADM group.WB showed that the expression of apoptosis-related Bax,Bcl-2,Cyt C and cleaved caspase8were consistent with the results of flow cytometry.Compared with Control group,the expression level of p-P65 in OPZ+ADM group was significantly increased.Compared with OPZ+ADM group,the expression of p-P65 in NAC+OPZ+ADM group decreased significantly.The results of fluorescence probe method showed compared with Control group,that the release of reactive oxygen species in OPZ+ADM group and NAC+OPZ+ADM group was significantly higher,while the release of ROS in NAC+OPZ+ADM group was significantly lower than that in OPZ+ADM group.The MMP of OPZ+ADM group and NAC+OPZ+ADM group was significantly lower than that of Control group,and the MMP of NAC+OPZ+ADM group was significantly higher than that of OPZ+ADM group.CONCLUSION1.Omeprazole aggravated the nephrotoxicity of adriamycin in Wistar rats.2.Omeprazole can increase kidney cell apoptosis through F₁F₀-ATPase-mediated mitochondrial oxidative stress pathway,and then aggravate the nephrotoxicity induced by adriamycin in rats.