Exploring The Effect And Mechanism Of Qingli Huoxue Method In Treating Chronic Kidney Disease Based On "Adriamycin Nephropathy" Model

Background/ObjectiveChronic kidney disease(CKD)is racking up accompanied by the increase of metabolic diseases and the aggravation of the aging social population,which seriously threatens people’s life and health.Therefore,how to effectively prevent and treat CKD has become an urgent problem for clinicians.Drug therapy is the main means to delay the progression of CKD.Although the treatment of CKD has made great improvement with the development of modern medicine,the types of drugs available to protect the kidney and delay the progression of CKD in clinical practice are limited,and most of them are expensive.It is urgent to find more cheap and efficient drugs for the treatment of CKD.Traditional Chinese medicine(TCM)has been draw more and more attention to the treatment of CKD,and the experience of famous old TCM practitioners points out the direction for us to find CKD therapeutic drugs.Professor Sun Wei,a famous expert in traditional Chinese medicine and well-known nephrology in Jiangsu Province,has been involved in clinical practice for more than 40 years and has rich clinical experience in the treatment of CKD,with significant effect.His medication experience is a potential treasure trove for finding drugs against CKD.Based on the clinical experience of Professor Sun Wei in the treatment of CKD,this study evaluates the efficacy by computer data mining method,filters Out the drugs with potential renal protective effects,verifies the effect of potential effective drugs by constructing the pharmacological disease model of CKD,explores the mechanism,and further analyzes the effective active ingredients and mechanisms using network pharmacology method,in order to scientifically and efficiently find the potential CKD therapeutic drugs in traditional Chinese medicine,clarify the mechanism,provide the basis for clinical medication,and provide new ideas for new drug research and development.MethodsAccording to the unified standard,the medical records and data of Professor Sun Wei’s outpatient CKD diagnosis and treatment recorded in real time in the Big Data Platform System of China Nephrology Big Data Application Innovation Alliance were collected and collated,the efficacy before and after treatment was evaluated by computer data mining and statistical methods,the application method of medication was analyzed,and the most commonly used drugs by the tutor in the treatment of CKD were selected as potential effective drugs for basic experimental study.Cell experiments:Adriamycin-induced renal tubular epithelial cell injury model was established by directly stimulating rat renal tubular epithelial cells(NRK-52E)with adriamycin hydrochloride(DOX)in vitro.Different concentrations of aqueous solubles of TCM granules were used for intervention to observe the effects of different TCM on DOX-induced cell damage.Cell viability was measured using the MTT assay.Apoptosis was observed by colorimetric TUNEL staining and protein changes of cleaved Caspase-3 in western blotting.Reactive oxygen species(ROS)/superoxide(O2-)fluorescent probes were used to evaluate intracellular oxidative stress damage.The expression of ERK,JNK,p38 and their phosphorylated proteins was detected by immunoblotting to observe the effect of traditional Chinese medicine on adriamycin-induced MAPK signaling pathway.Animal experiments:Male BALB/c mice were given a single injection of DOX via tail vein to construct an in vivo adriamycin kidney injury model.Urine,serum and tissue samples were collected after two weeks of intragastric treatment with the corresponding different concentrations of aqueous solutions of Chinese herbal granules,respectively.H&E,Masson and PAS staining were performed on renal tissues to observe the pathological changes of renal tissues.The changes of proteinuria,serum albumin,serum creatinine(Scr),serum urea nitrogen(BUN),superoxide dismutase(SOD)activity,malondialdehyde(MDA)and glutathione(GSH)in renal tissue were detected by kit.TCMSP database and literature mining method were used to obtain the monomer active components of traditional Chinese medicines with definite curative effect and similar effect,the network map of"traditional Chinese medicine-active components" was constructed,and the cross-active components between different drugs were found as potential effective traditional Chinese medicine monomers for further basic experimental study.ResultData mining and statistical methods were used to analyze the medical records of the tutor in the treatment of CKD.The results showed that Professor Sun Wei used traditional Chinese medicine to treat CKD with effective improvement rate of 38.5%,stable rate of 28.02%,effective improvement rate of symptom efficacy of 79.96%,and glomerular filtration rate,serum creatinine,urea nitrogen,proteinuria,urine occult blood,etc.were well controlled,confirming that Professor Sun Wei had a significant effect in the treatment of CKD.The regularity of prescription medication was analyzed,and it was found that Professor Sun Wei paid special attention to the role of Qingli Huoxue drugs in delaying the progression of CKD.Therefore,a total of 16 commonly used Qingli Huoxue herbs,such as Shiwei,Polygonum cuspidatum,Yujin,Chuanxiong,Angelica sinensis,Smilax glabra,Dioscorea nipponica,Hedyotis diffusa,Yinchen,Caulis Spatholobi,Jixuecao,Liuyuexue,Corn whisker,Zelan,Guijianyu.Huangsukuihua.The results showed that adriamycin could induce renal injury in vivo and renal tubular epithelial cell injury in vitro.At the same time,zelan,Jixuecao and Guijianyu could significantly reduce adriamycin-induced renal injury in vivo and in vitro.Liuyuexue could significantly reduce adriamycin-induced renal tubular epithelial cell injury in vitro,but the effect was weak in vivo.Zelan,Jixuecao,Guijianyu,and Liuyuexue all had good antioxidant effects,and Zelan,Jixuecao and Guijianyu could also significantly inhibit doxorubicin-induced activation of MAPK/ERK signaling pathway,while Guijianyu had no significant effect on MAPK signaling pathway.Zelan,Jixuecao and Guijianyu have definite curative effect and similar effect.The active components of the three are analyzed by network pharmacology method,and the cross active components are further verified.The results showed that apigenin,a common active component,could significantly reduce doxorubicin-induced renal damage,reduce oxidative stress injury,inhibit the activation of MAPK signaling pathway,and also inhibit renal inflammatory response in vivo and in vitro.Conclusion① Professor Sun Wei has a significant effect on the treatment of CKD with the method of Qingli Huoxue,and his medication experience has important guiding significance in delaying the progression of CKD and improving the quality of life of patients;② Doxorubicin can induce continuous progressive damage changes in the kidney in vivo and in vitro,and is a mature CKD disease model.Among them,oxidative stress injury and activation of MAPK/ERK signaling pathway play a crucial role in the process of adriamycin renal injury;③ The commonly used drugs zelan,Jixuecao and Guijianyu in Qingli Huoxue method can reduce doxorubicin-induced renal injury by inhibiting oxidative stress and MAPK/ERK signaling pathway,suggesting that oxidative stress and MAPK/ERK signaling pathway may be important targets for protecting the kidney in Qingli Huoxue method,and zelan,Jixuecao and Guijianyu may have important application values in the treatment of CKD;④Apigenin,a common active component of zelan,Jixuecao and Guijianyu,can reduce doxorubicin-induced renal injury through anti-oxidation,inhibition of MAPK/ERK and effect,suggesting that Apigenin may be an important active component of Qingli Huoxue method to play a renoprotective role and may have important application prospects in the treatment of CKD.