Effect Of Artemether On Adriamycin Renal Tubule Injury

Chronic kidney disease(CKD)is still a global public health problem,which can lead to systemic multi-system complications and eventually to end-stage kidney disease.It is very important to intervene in CKD and delay renal failure as soon as possible.Renal tubules are an important part of the kidney and highly sensitive to external stimuli.For a long time,it has been considered that renal tubules are the target of injury.However,more and more studies have shown that renal tubules can actively participate in and aggravate renal injury.At present,there are few protective drugs for renal tubular injury.Therefore,it is of great significance to study the mechanism of renal tubular injury and develop effective renal tubular protective drugs for the treatment of CKD.Objective1.To explore the syndrome characteristics and medication rule of Artemisia annua in the treatment of chronic kidney disease based on data mining;2.The possible mechanism of artemisinin and its derivatives in the treatment of adriamycin nephropathy was analyzed by network pharmacology,bioinformatics and molecular docking techniques.3.In vivo experiments were conducted to verify the possible protective mechanism of artemether against doxorubicin renal tubule injury;4.To observe the protective effect of artemether on doxorubicin-stimulated renal tubular epithelial cells in vitro.Methods1.Study on the medication rule of Artemisia annua in the treatment of chronic kidney diseaseThrough the cloud platform of ancient and modern medical records,the medical records of ancient and modern artemisia annua for the treatment of chronic kidney disease collected by the database were searched.According to the retrieval strategy and inclusion criteria,the medical records of artemisia annua for the treatment of chronic kidney disease were preliminarily screened.After standardized processing of the data of treating diseases,syndrome distribution and prescription composition in the medical records,The platform data mining function was used to conduct statistical analysis on the prescription indications and diseases,the frequency of TCM use,and the attributes of TCM.Association rule analysis and cluster analysis were conducted for high-frequency TCM.Finally,the complex network analysis of core prescriptions was conducted.2.Network pharmacology,bioinformatics analysis and molecular docking of artemisinin and its derivatives in the treatment of adriamycin nephropathyAdriamycin nephropathy was selected as the research object.Artemisinin and its main derivatives,artemether,dihydroartemisinin,artesunate and artemether,were studied.First,doxorubicin-related nephropathy targets were obtained by searching the disease database.The targets of artemisinin,artemether,dihydroartemisinin,artesunate and artemether were screened by drug database.The common target was obtained from the intersection of disease target and drug-related target,and the core gene was screened.GO functional enrichment analysis and KEGG pathway enrichment analysis were performed on the drug-disease common target to obtain the main biological process and related signal pathways of common target enrichment.The core targets related to renal tubule phenotype and mitochondria were obtained,and the selected targets were verified by molecular docking with artemether,an artemisinin derivative.3.Animal experimentsIn this study,adriamycin nephropathy mice were used as renal tubule injury model,and the mice were randomly divided into Control group(Control group),adriamycin nephropathy model group(AN group)and artemether treatment group(AN+Art group).Control group and AN group were fed with standard diet,and AN+Art group were fed with artemether diet(0.3g/kg),for 2 weeks.After 2 weeks of administration,urine of mice in each group was collected for 24 hours.Total urinary albumin and creatinine were detected and their ratios were calculated.NGAL content in urine was detected to evaluate renal tubular injury.At the same time,the renal tissue samples of mice in each group were collected,PAS staining was performed and renal tubular injury index was evaluated.Combined with the results of pharmacological studies on the network,the positive expressions of Vimentin,ZO-1 Bcl-xL and Bax in renal tissue were observed by IHC method.RT-PCR was used to detect the relative expression levels of kim-1,FN and ND1 mRNA in renal tissue,and Amplex UltraRed was used to detect H2O2 content in renal tissue homogenate.Protein levels of Vimentin,ZO-1,Pe-cadherin,Bax,Bcl-xL,ERRα,NRF1,HSP60,OPA1,VDAC,PDH,PDK1 and PDP1 in renal tissues were detected by Western Blot.The above comprehensive evaluation of artemether on adriamycin renal tubule injury and mitochondrial effects.4.Cell experimentsIn order to further confirm the protective effect of artemether on renal tubules,Normal rat kidney epithelial cells(NRK-52E)were taken as the research object.After adriamycin stimulation,artemether was given different concentrations(0μM,50μM,100μM,150μM)for 24h.Western Blot was used to detect the protein expression levels of ZO-1,p-E-cadherin,Cleaved PARP and Bcl-xL in cells.To investigate the effects of artemether on the phenotype and mitochondria related proteins of doxorubicin-induced renal tubular epithelial cells.Results1.Study on the medication rule of Artemisia annua in the treatment of chronic kidney diseaseIn this part of the study,artemisia annua was finally included in 31 prescriptions for the treatment of chronic kidney disease,and the disease distribution was mainly lupus nephritis and chronic glomerulonephritis.The top 6 syndromes in frequency were Yin deficiency and blood heat syndrome,kidney deficiency and blood stasis syndrome,damp-heat stasis syndrome,liver-kidney YinQi deficiency syndrome,spleen-kidney deficiency syndrome and damp-heat accumulation syndrome.31 first prescription involved 179 TCM,in addition to the artemisinin drug use frequency of the top 10 drugs for poria cocos,salvia miltiorrhiza,liquorice,atractylodes,radix astragali,radix rehmanniae,cortex moutan,rhizoma alismatis,coix seed,high frequency and more drugs to the spleen yiqi,water moisture penetration,ziyin clear heat,promoting blood circulation to remove blood stasis drugs is given priority to,is cold sex,grape and more bitter medicine,return to the liver,spleen,lung and kidney meridian.Drug association rule and cluster analysis showed that artemisia annae was commonly used with nourishing Yin,clearing heat,invigorating dampness and strengthening spleen in the treatment of chronic kidney disease.Complex network analysis showed that the core prescriptions of artemisia annua in the treatment of chronic kidney disease were Haoqin Qingdan Decoction,Shenling Baizhu Powder and Artemisia annua biejia Decoction,which were added and subtracted based on the three classic prescriptions.2.Network pharmacology,bioinformatics analysis and molecular dockingA total of 235 intersection targets of artemisinin,artemether,dihydroartemisinin,artesunate,artemether and doxorubicin nephropathy were identified.GO enrichment analysis and KEGG enrichment analysis of intersection targets showed that artemether treatment of adriamycin nephropathy is multi-pathway and multi-target.Among them,47 targets were enriched in mitochondria,indicating that mitochondria play an important role in the treatment of adriamycin nephropathy by artemisinin and its derivatives.Protein interaction network analysis(PPI)and core target screening showed that the top 70 core targets included mitochondrial structure and function related proteins Bcl-xL and Bax,and renal tubular phenotypic related proteins FN,etc.Bcl-xl,Bax,FN and artemether were used for molecular docking verification.The results showed that the binding energies of Bax,Bcl-xL,FN and artemether were all less than-5 kcal/mol,and they all interacted with hydrogen bonds,indicating that the key target had good binding activity with artemether.3.Animal experiments(1)Effects of artemether on urinary protein/creatinine ratio in adriamycin nephropathy miceAt 2 weeks,the ratio of urinary protein to creatinine in AN group was significantly higher than that in Control group(P<0.01).Compared with AN group,urinary protein/creatinine ratio in AN+Art group was significantly decreased(P<0.05).(2)Protective effect of artemether on renal tubule injury in adriamycin nephropathyPAS staining results of renal tissue sections showed that at 2 weeks,a large number of protein tubules were observed in the renal tissues of mice in the AN group,accompanied by atrophy and swelling of some renal tubules.Compared with the Control group,the renal tubule injury index was significantly higher(P<0.01).The above pathological changes were significantly improved in AN+Art group,and the renal tubule injury index was significantly lower than that in AN group(P<0.01).Urine biochemical and RT-PCR results showed that urinary NGAL excretion and the expression of Kim-1 mRNA in renal tissues in the AN group were significantly increased compared with the Control group(P<0.01).Compared with AN group,NGAL excretion in urine and Kim-1 mRNA expression in kidney tissue in AN+Art group were significantly decreased(P<0.01,P<0.05).(3)Effects of artemether on renal tubule phenotype in adriamycin nephropathyWestern blot results showed that the expression of p-E-cadherin and ZO-1 in the kidney tissue of AN group was significantly lower than that of Control group(P<0.01,P<0.05).PE-cadherin and ZO-1 in AN+Art group were significantly increased compared with AN group(P<0.05,P<0.01).The expression of Vimentin in the AN group was significantly increased compared with the Control group(P<0.05),and the expression of Vimentin in the AN+Art group was significantly decreased compared with the AN group(P<0.01).IHC results were consistent with Western blot results.Compared with the Control group,the positive expression of Vimentin in the tubule interstitium of the AN group was significantly increased,while the positive expression of ZO-1 in the tubule was significantly decreased.Compared with the AN group,the positive expression of Vimentin in renal tubule interstitium was decreased and the positive expression of ZO-1 in renal tubule was increased in the AN+Art group.Rt-pcr results showed that FN mRNA expression was significantly increased in the AN group compared with the Control group(P<0.01).Compared with AN group,FN mRNA expression in AN+Art group was significantly decreased(P<0.01).(4)Effects of artemether on mitochondrial structure and function homeostasis of renal tissue in mice with adriamycin nephropathy①Mitochondrial related expression of Bcl-xL and BaxWestern blot results showed that compared with Control group,Bax expression in AN group was significantly increased(P<0.01)and Bcl-xL expression was significantly decreased(P<0.05).Compared with AN group,Bax in AN+Art group was significantly decreased(P<0.05)and Bcl-xL was significantly increased(P<0.01).IHC results showed that Bax and Bcl-xL proteins were mainly expressed in renal tubules.Compared with the control group,the positive expression of Bax and Bcl-xL in renal tubules was increased and decreased in the AN group.Compared with the AN group,the positive expression of Bax and Bcl-xL in renal tubules was decreased and increased in the AN+Art group.②Expression of mitochondrial pyruvate metabolism-related proteins:Western blot results showed that compared with Control group,PDH protein expression in AN group was significantly down-regulated(P<0.05),and PDP1/PDK1 expression was significantly increased(P<0.01).Compared with AN group,PDH protein expression in AN+Art group was up-regulated,but the difference was not statistically significant(P>0.05),while PDP1/PDK1 was significantly decreased(P<0.01).③Expression of OPA1 and VDAC proteins in mitochondrial inner and outer membrane:Western blot results showed that compared with the Control group,the expression of OPA1 and VDAC protein in the AN group was significantly decreased(P<0.01,P<0.01).Compared with AN group,the protein expressions of OPA1 and VDAC in AN+Art group were significantly increased(P<0.05).④mtDNA copy number of kidney tissue:RT-PCR results showed that mtDNA copy number in AN group was significantly decreased compared with Control group(P<0.01).Compared with AN group,the mtDNA copy number in AN+Art group was significantly increased(P<0.01).⑤H2O2 content of renal tissue homogenate:Compared with Control group,H2O2 content in renal tissue homogenate in AN group was significantly decreased(P<0.05);Compared with AN group,H2O2 content in AN+Art group was significantly increased(P<0.05).⑥Expression of mitochondrial biosynthesis-related transcription factors ERRα,NRF1 and HSP60:Western blot results showed that compared with Control group,the expressions of ERRα,NRF1 and HSP60 in AN group were significantly decreased(P<0.01,P<0.01,P<0.05).Compared with the AN group,ERRα and NRF1 in the AN+Art group were significantly increased(P<0.01),while HSP60 was slightly increased but the difference was not statistically significant(P>0.05).4.Cell experiments(1)Effects of artemether on the phenotype of NRK-52E cells stimulated by adriamycinWestern blot results showed that the expression of ZO-1 and p-E-cadherin protein in NRK-52E cells significantly decreased after adriamycin stimulation(P<0.05,P<0.01).The protein expressions of ZO-1 and p-E-cadherin were up-regulated by artemether co-incubation,and ZO-1 was significantly up-regulated by artemether 150μM(P<0.05),and p-E-cadherin was significantly up-regulated by artemether 50μM and 100μM(P<0.05,P<0.01).(2)The effect of artemether on adriamycin stimulated NRK-52E cells Cleaved PARP and Bcl-xL proteinWesternblot showed that Cleaved PARP expression was significantly increased(P<0.05),and Bcl-xL expression was significantly decreased(P<0.05)in NRK-52E after adriamycin stimulation.Artemether could down-regulate Cleaved PARP protein at 150μM(P<0.05),and up-regulate Bcl-xL expression in NRK-52E cells at 100μM(P<0.05).Conclusion1.Through data mining,it was found that artemisia annica prescriptions for the treatment of chronic kidney disease mainly treated deficiency heat syndrome,damp-heat syndrome and blood stasis syndrome;Artemisia annae is mostly used with nourishing Yin and clearing heat,invigorating dampness and strengthening spleen,playing the function of clearing heat and clearing dampness and penetrating evil.Most of the core prescriptions are artemisia qinqingdan decoction and Artemisia annua decoction.This part of the study preliminarily summarized the rule of artemisia annua treating kidney disease,and laid a foundation for the further study of artemisia annua and its related drugs.2.Through network pharmacology and molecular docking technology,it was verified that artemisinin and its derivatives are multi-pathway and multi-target in the treatment of adriamycin nephropathy,among which mitochondria related targets may play an important regulatory role,providing theoretical support for the treatment of chronic kidney disease by artemisinin and its derivatives.3.In vivo studies have confirmed that artemether can reduce urinary NGAL level and kim-1 mRNA expression in mice with adriamycin nephropathy,alleviate pathological damage of renal tubules,and restore renal tubule phenotypic changes.The protective effect of artemether on renal tubules may be realized by increasing mtDNA copy number,regulating mitochondrial pyruvate metabolism,increasing mitochondrial biosynthesis,and maintaining the structural integrity of mitochondrial inner and outer membrane,suggesting that the protective mechanism of artemether on renal tubules may be related to the maintenance of mitochondrial structure and function homeostasis.4.It was confirmed again in vitro that artemether can improve the phenotypic changes of adriamycin-induced renal tubular epithelial cells,regulate the expression of Bcl-xL and Cleaved PARP,participate in the regulation of mitochondrial function,and achieve the protective effect of renal tubular.