The Study Of Effect Of Artemether Combined With ACEi On Type 1 Diabetic Nephropathy

ObjectiveDiabetic kidney disease(DKD),one of the common microvascular complications of diabetes mellitus(DM),is the main cause of end stage renal disease(ESRD).At present,the treatment of DKD mainly includes hypoglycemic,hypotensive and lipid-lowering,among which,renin-angiotensin-aldosterone System(RAAS)inhibitor is the first-line therapy of DKD,which can control blood pressure and reduce proteinuria.The effect of reducing proteinuria does not depend on its antihypertensive effect.At present,RAAS inhibitors are the first-line treatment of DKD.However,RAAS inhibitors also have certain side effects in clinical application,such as dry cough,increased serum creatinine and other adverse reactions.In addition,many studies have confirmed sufficient blocking of RAAS,such as combining with angiotensin converting enzyme inhibitors(ACEi)and angiotensin receptor blocker(ARB)failed to achieve additional renal benefit in DKD patients,but may cause adverse events such as hyperkalemia,doubling creatinine and acute kidney injury.With the in-depth study on the pathogenesis of DKD,the role of oxidative stress in DKD has been paid more and more attention.A large number of studies have shown that RAAS inhibitors can enhance the antioxidant effect of DM animal models,which further supports their clinical use in DKD.Despite the treatment of DKD has been improved in recent years,some patients still inevitably develop into ESRD.Therefore,the search for more effective drugs for the treatment of DKD is still in progress.Studies have shown that traditional Chinese medicine and its extracts can not only reduce the proteinuria of DKD patients,but also attenuate kidney damage,and the effect of integrated traditional Chinese and western medicine in treating kidney disease is better than that of traditional Chinese medicine or western medicine alone.Therefore,on the basis of excavating the treasure house of traditional Chinese medicine and relying on western medicine,exploring the feasibility of combining traditional Chinese medicine with ACEi or ARB in the prevention and treatment of DKD may provide new ideas for the clinical treatment of DKD.Artemether(Art)is one of the derivatives of artemisinin,which has antiinflammatory,modulates immunity and improves metabolism effects.In recent years,several studies have confirmed that artemether can reduce urinary albumin excretion rate in DM animal models and improve kidney damage caused by DM,which has renoprotective effects,and its mechanism may be related to the regulation of oxidative stress response.However,there is no relevant study on whether artemether combined with ACEi can attenuate DKD lesions or even bring further renoprotective effects.In this study,the biological functions and signaling pathways of artemether and ACEi in the treatment of DKD were discussed with the help of network pharmacology,providing theoretical basis for animal research.Subsequently,animal experimental were conducted to verify whether artemether combined with ACEi can lessen kidney injury in Type 1 diabetes(T1D)mice,and to explore the potential mechanism.Methods1.To explore the mechanism of artemether and enalapril for the treatment of DKD based on network pharmacology.The targets of artemether and enalapril were obtained by pharmmapper and swiss targetprediction databases respectively.The main targets of DKD were obtained through Genecards,DrugBank,Online Mendelian Inheritance in Man(OMIM)and Therapetuic Target Database(TTD)database.After obtaining common targets of drugs and diseases,the metascape platform was used to analyze the biological process and signaling pathway involved in the treatment of DKD by artemether and ACEi.2.Discussion on the effect and mechanism of artemether combined with ACEi in the treatment of DKD.Streptozotocin was used to intraperitoneally injected into the C57BL/6J male mice to induced DKD model,and the mice were randomly divided into T1D control group(T1D-ctrl),STZ,STZ+artemether(STZ+Art),STZ+enalapril(STZ+ACEi),STZ+artemether+enalapril(STZ+Art+ACEi)group.T1D-ctrl group and STZ group were given conventional diet.STZ+ Art group and STZ+ACEi group were given diets supplemented with 0.8g/kg artemether and 0.4g/kg enalapril,respectively.Mice in the STZ+Art+ACEi group were given diets supplemented with 0.8g/kg artemether and 0.4g/kg enalapril.The treatment lasted for 8 weeks.At the 0,2,4,6,and 8 weeks of the experiment,24 hours urine samples of all the included mice were collected using the mouse metabolic cage,and 24 hours metabolism of the mice,such as food intake,water intake,urine volume and other data were recorded.The body weight,urinary albumin excretion and fasting blood glucose of mice were measured at 0,2,4,6 and 8 weeks in the experiment.At the end of the experiment,glycated hemoglobin(HbA1c)concentration was measured by Ultra2 HbAlc analyzer,Automatic biochemical analyzer was used to detect serum creatinine,urea nitrogen,urine N-acetyl-β-d glucosidase(NAG),urine creatinine and other serum biochemical and urinary biochemical indexes.Blood pressure was monitored by an animal noninvasive sphygmomanometer at 8 weeks after treatment.Urinary H2O2 level was detected by Amplex UltraRed kit.The renal pathological changes were assessed by PAS staining.The area and volume of capillary loops and mesangial matrix in the glomerulus were measured.The area of renal tubules,lumen and wall area of renal tubules were also measured.The changes of glomerular basement membrane and podocyte foot processes were observed by electron microscopy.The glucagon/insulin area ratio was assessed by laser confocal microscopy,the total islet area/pancreas area ratio was assessed by HE staining,and the relative expression levels of catalase,Cu/Zn superoxide dismutase(SOD1),Manganese superoxide dismutase(MnSOD,SOD2)and extracellular superoxide dismutase(SOD3)in renal tissues were detected by real-time fluorescence quantitative PCR.The contribution of catalase,antiMnSOD acetyl-lysine 68 antibody(MnSOD(acetyl K68))and anti-MnSOD acetyl-lysine 122 antibody(MnSOD(acetyl K122))in renal tissues was detected by immunohistochemical.Western blots were used to detect the expression of MnSOD(acetyl K122),MnSOD(acetyl K68),sirt3 and catalase in kidney tissues.Results1.In the part of network pharmacology,artemether regulates the pathways of DKD mainly acting on pathways in cancer,RAS signaling pathway,insulin signaling pathway and PPAR signaling pathway.Its functions mainly include phosphotransferase activity,nuclear receptor activity,insulin receptor binding and antioxidant activity.The pathways of DKD regulated by enalapril mainly act on pathways in cancer,IL-17 signaling pathway,PPAR signaling pathway and arachidonic acid metabolism,whose functions are mainly concentrated in metallopeptidase activity,protein kinase activity,lipid binding,insulin receptor binding and antioxidant activity.2.Animal experiments(1)The combination of artemether and ACEi could reduce the 24-hour urinary albumin excretion,urinary NAG and urinary albumin creatinine ratio in STZ mice;(2)The combination of artemether and ACEi could reduce kidney weight and renal hypertrophy in STZ mice;(3)The combination of artemether and ACEi could inhibit the proliferation of glomerular capillary loops and renal tubule,and the proliferation of mesangial matrix and foot process fusion;(4)The combination of artemether and ACEi could reduce the level of fasting blood glucose,urine glucose and HbAlc in STZ mice,and improve the diabetic metabolic symptoms of polydipsia and polyuria;(5)The combination of artemether and ACEi could increase serum insulin level and decrease the proportion of glucagon/insulin area in islets;(6)The combination of artemether and ACEi could reduce blood pressure and triglyceride level,and increase total protein and serum albumin level of STZ mice;(7)The combination of arternether and ACEi could reduce H2O2 content in urine of STZ mice,and the effect is more obvious than that of artemether or ACEi alone;(8)The combination of artemether and ACEi could increase the protein expression level of catalase in the kidney of STZ mice,decrease the protein expression level of MnSOD(acetyl K68),MnSOD(acetyl K122)and sirt3,and up-regulate the mRNA level of SOD 1.Conclusion1.From the part of network pharmacological,it suggests that artemether or enalapril may treat DKD by regulating antioxidant activity.2.This study confirmed that the combination of artemether and ACEi could reduce proteinuria and delay the progression of DKD in STZ mice,and the combination of artemether and ACEi was more effective than artemether or ACEi alone.Its renoprotective effect might correlate to the enhancement of antioxidant capacity.