Protective Effects And Mechanisms Of Omeprazole On Cisplatin-induced Kidney Injury

Cisplatin(CP)is widely used in the treatment of various solid tumors through preventing DNA replication and affecting the synthesis of protein.Cisplatin mainly accumulates in the kidney tissue to induce serious nephrotoxicity,which may lead to the occurrence of acute renal injury or acute renal failure.No drugs could effectively protect cisplatin-induced kidney injury.Hydration therapy is adopted in clinical treatment to reduce the toxicity of cisplatin,however,there are still about 40 % of patients who suffer kidney damage after receiving that.Therefore,how to reduce the cisplatin-induced nephrotoxicity is a key problem,which needs to be solved urgently.Omeprazole(OME)is often used to alleviate the gastrointestinal reactions in patients receiving chemotherapy as one of the classical proton pump inhibitors(PPIs).Studies showed that omeprazole could reduce the nephrotoxicity induced by cisplatin in chemotherapy patients,but the mechanism is not yet clear.So in this study,we used the renal tubular epithelial HK-2 cell and SD rats to validate the protective effects and explore the mechanism of omeprazole on cisplatin-induced kidney injury.This study is mainly divided into the following parts:1.To verify the protective effect of omeprazole on cisplatin-induced kidney injury in vivo and in vitro.In this study,SD rats and renal tubular epithelial HK-2 cells were used to verify the protective effect of omeprazole on cisplatin-induced nephrotoxicity.SD rats were randomly divided into several groups: the control group(normal saline);cisplatin group(15 mg/kg·d);cisplatin+omeprazole group(cisplatin 15mg/kg·d + omeprazole 1.8mg/kg·d or 3.6mg/kg·d)and omeprazole group(3.6mg/kg·d).Intraperitoneal injection of omeprazole or normal saline was carried out on 1-5 d,and cisplatin was injected on the 5th day.All rats were weighed and killed on day 7,blood and kidneys were stored at-80 °C.In vitro study,cells were divided into several groups and incubated for 24 h: control group,cisplatin group(2 nmol/m L),cisplatin + omeprazole group(2 nmol/m L cisplatin + 4 ?g/m L or 40 ? g/m L)and omeprazole group(40 ? g/m L).Cell viability was determined by MTT assay and flow cytometry assay.Optical microscope and fluorescence microscope were used to observe the cell growth and nuclear damage after JC-1/DAPI staining.The expressions of apoptosis-related proteins as Bax,Bcl-2 and Caspase 3 were detected by Western Blot.Renal function was determined by creatinine and urea nitrogen assay.Renal injury was evaluated by renal index and HE staining.The results showed that cisplatin could impair the renal function and the kidney structure,and induce oxidative stress injury.However,co-treated with cisplatin and omeprazole could effectively alleviate the acute renal injury caused by cisplatin in SD rats.After co-treatment with omeprazole,the levels of creatinine and urea nitrogen were declined obviously as compared with cisplatin group.MTT results showed that 40 ? g/m L omeprazole significantly improved the viability of HK-2 cells,and the results were confirmed by Annexin V-FITC/PI double staining assay.Western Blot results showed that cisplatin+omeprazole group could reduce the expression of Bax,increase the level of Bcl-2,and inhibit the activation of Caspase-3 protein,which were obviously different from those of cisplatin group(P<0.05).In addition,cisplatin+omeprazole group effectively ameliorated cell morphology and nuclear damage,and decreased the mitochondrial membrane potential in HK-2 cells.40 ? g/m L omeprazole could achieve better protection.These data suggested that omeprazole had a protective effect on cisplatin-induced renal injury in vivo and in vitro.2.Protective mechanism of omeprazole against cisplatin-induced nephrotoxicity.The mechanism of cisplatin nephrotoxicity is involving multiple molecular signaling pathways,including oxidative stress,inflammatory response and drug accumulation.This study explored the protective mechanism of omeprazole against cisplatin-induced nephrotoxicity.Based on the previous research,SOD,GSH and MDA assay kit were used to evaluate the degree of oxidative stress injury in cells and kidney tissues.Intracellular ROS accumulation was detected by ROS assay kit.HPLC-MS/MS method was established to detect the content of cisplatin in cell lysate and kidney tissues.Western Blot,immunohistochemistry and cellular immunofluorescence were used to detect the expression of OCT2 and P-gp.Immunofluorescence and Western Blot were used to study the expression of inflammatory cytokines such as TNF-?,IL-6 and IL-1? and proteins in TLR4/NF-?B signaling pathway.LPS,the excitant of TLR4,was used to verify the blocking effect of omeprazole.The results showed that cisplatin induced the accumulation of ROS,decreased the activity of GSH and SOD,and increased the level of MDA in cells and kidney tissues.After co-administration of cisplatin and omeprazole,the activities of GSH and SOD were significantly increased;the level of MDA was significantly decreased;and the content of intracellular ROS accumulation was significantly reduced,which compared with the cisplatin group.Omeprazole could effectively alleviate the kidney injury and cell apoptosis induced by cisplatin via attenuating oxidative stress,reducing ROS accumulation and increasing the activities of antioxidant enzyme.In this study,HPLC-MS/MS method for cisplatin detection in biological samples was established,and methodology was examined for linear range,average recovery,precision,exclusivity and stability.HPLC-MS/MS method was used to detect the cisplatin content in HK-2 cells and kidney tissues after drug treatment.It was found that the cisplatin content of cisplatin+omeprazole group was significantly lower than that of cisplatin group.The results of Western Blot and immunohistochemistry assay confirmed that omeprazole could regulate the expression of OCT2 and P-gp,affecting cisplatin uptake and efflux.The above data indicated that omeprazole could alleviate cisplatin-induced nephrotoxicity in vivo and in vitro,through regulating the expression of OCT2 and P-gp transporter to reduce cisplatin accumulation.In this study,cisplatin could up-regulate the expression of TLR4,then activate NF-?B transferring from cytoplasm to nucleus,and eventually increase the expression of inflammatory cytokines such as TNF-?,IL-6 and IL-1?.However,omeprazole could block the activation of TLR4 and NF-?B to reduce the level of inflammatory cytokines.Above results indicate that omeprazole could alleviate cisplatin-induced renal injury and renal tubular epithelial cells apoptosis through reducing oxidative stress injury,blocking the activation of the TLR4/NF-?B signaling pathway and reducing cisplatin accumulation.3.The effect of omeprazole on the anti-tumor effect of cisplatin.In order to further clarify the interaction between omeprazole and cisplatin,tumor-bearing mice and human liver cancer Hep G2 cells were used to explore the effect of omeprazole on the anti-tumor effect of cisplatin.When the average of tumor volume was bigger than 80 mm3,the mice were randomly divided into several groups,including the control group(normal saline,ip.,q.d);cisplatin group(2 mg/kg·d of cisplatin,ip.,q.3d);cisplatin + omeprazole group(2 mg/kg·d of cisplatin,ip.,q.3d,and omeprazole 5 mg/kg·d,ip.,q.d);and omeprazole group(omeprazole 5 mg/kg·d,ip.,q.d).The changes of tumor volume and body weight were recorded daily.All the mice were sacrificed on the 12 th day;tumor tissue,kidney and blood were stored at-80 °C.Western Blot was used to detect the expression of P53.Serum creatinine assay kit,urea nitrogen assay kit,and HE staining were used to analyze the damage degree and renal pathological structure in tumor-bearing mice.MTT and flow cytometry assay were used to analyze the effect of omeprazole on cisplatin induced apoptosis of Hep G2 cells.Tunel and DAPI staining were used to observe the damage induced by omeprazole combined with cisplatin.The results showed that omeprazole could enhance the inhibitory effect of cisplatin on tumor growth in tumor-bearing mice,decreasing the tumor volume and weight.HE and Tunel staining exhibited that there was a large area of cellular necrosis in the cisplatin group,while the tumor necrosis and disintegration was more severe after co-treated with omeprazole.The number of apoptotic cells in the field was significantly higher than that of cisplatin group and control group.MTT and flow cytometry assay confirmed the conclusion that omeprazole enhanced the cytotoxicity of cisplatin in Hep G2 cells.Tunel and DAPI staining showed that the number of apoptotic cells in the combination group was significantly higher than that of other groups.In addition,the expression of P53 was markedly increased after co-treated with cisplatin and omeprazole,as compared with cisplatin group.The above results showed that omeprazole could enhance the anti-tumor effect of cisplatin in H22 tumor-bearing mice and Hep G2 cells.In this study the renal function of cisplatin group was decreased and inflammation occurred in the kidney tissue,while the injury could be alleviated by omeprazole indicating that omeprazole could not only enhance the anti-tumor effect of cisplatin,but also reduce the kidney injury during the treatment.In conclusion,omeprazole could reduce cisplatin-induced nephrotoxicity in vivo and in vitro through regulating oxidative stress,inflammatory response and drug accumulation.In addition,omeprazole could enhance the anti-tumor effect of cisplatin.This study confirmed that omeprazole could protect cisplatin-induced kidney injury,and explored the protective mechanisms.This study has the certain theories innovation and actual leading meanings for the clinical application of cisplatin and omeprazole,providing new targets and mechanisms for the study of cisplatin-induced renal injury protection.