Study On Mechanism Of Astragalus Mongholicus And Angelica Sinensis Prescription On The Treatment Of Diabetic Renal Fibrosis Based On TGF-β/Smads Pathway

Objectives: In this study,by collecting and arranging the clinical prescriptions,we analyzed the prescription rules of Prof.Zhao using the method of invigorating qi and promoting blood circulation to treat diabetic kidney disease(DKD).The high frequency matched pair of medicine of astragalus mongholicus and angelica sinensis(AM-AS)was selected,and the core targets and key pathways in the treatment of diabetic renal fibrosis were predicted by network pharmacology combined with molecular docking method.Through the construction of animal model,we evaluated the efficacy of AM-AS prescription on the treatment of diabetic renal fibrosis,and explored the molecular mechanism of AM-AS prescription acting on TGF-β/Smads pathway in diabetic kidney disease fibrosis,in order to provide experimental basis for clinical application.Methods:1.Study on the medication rules of Prof.Zhao Quanlin using the method of invigorating qi and promoting blood circulation to treat DKD: Collect effective prescriptions for DKD from September 2019 to December 2020 in the outpatient department and inpatient ward of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine,and enter them into an Excel table after screening to establish a database.By using SPSS Statistics 19.0 and SPSS Modeler 18.0,the frequency of prescriptions,the nature,flavor and meridian tropism of herbs,and the clustering and association rules were analyzed,and the prescription rules of Prof.Zhao in the clinical treatment of DKD were explored and the core herbal combinations were found.2.Study on the complex network of AM-AS prescription in the treatment of diabetic renal fibrosis: The high-frequency herbal combination-AM-AS prescription obtained in the first part was selected for network pharmacology analysis to topology and predict the core targets and key pathways of the herbal combinations in treating diabetic renal fibrosis.By molecular docking of the core targets in the TGF-β/Smads pathway and the main active components of AM-AS prescription,the binding sites and binding conditions were predicted and simulated,and the molecular mechanism of TGF-β/Smads pathway in diabetic renal fibrosis and the intervention effect of AM-AS prescription was preliminarily analyzed.3.Study on the efficacy and mechanism of AM-AS prescription in the treatment of diabetic renal fibrosis: The rat model of DKD was constructed to observe the effect of continuous increase in blood glucose on the general condition of rats,clinically relevant indicators of renal function,and pathological changes in renal tissue fibrosis.TGF-β1 and Smad7 in the TGF-β/Smads pathway were obtained in the second part.We observed their m RNA and protein expression levels in the kidney tissue of DKD rats and the intervention effect of AM-AS prescription to explore molecular mechanism of AM-AS prescription in treating diabetic renal fibrosis based on TGF-β/Smads pathway.Results:1.After the analysis of 222 effective prescriptions prescribed by Prof.Zhao in the clinical treatment of DKD,we found that and 91 traditional Chinese medicines were used more than 9 times,with a cumulative medication frequency of 2944 times.Among them,angelica sinensis was used most commonly,and the herbs for tonifying deficiency appeared most frequently.The analysis of four properties and five tastes is dominated by sweet,bitter and pungent,warm and mild,which are mostly attributed to the spleen,liver and lung meridians.Clustering and association rules analysis showed that the combination occuring most frequently was astragalus mongholicus and angelica sinensis,and the prescriptions used most commonly were Danggui Buxue decoction,Buyang Huanwu decoction,Jisheng Shenqi decoction,Huanglian Wendan decoction,Xuefu Zhuyu decoction,etc.By analysing Prof.Zhao’s ideas of clinical differentiation and treatment of DKD,the commonly used treatment is mainly invigorating qi and promoting blood circulation,supplemented by warming and regulating qi,nourishing pancreas and benefiting yin,warming and tonifying kidney-yang,clearing away heat and detoxifying,releasing turbidity and relieving bowels,etc.2.Based on the network pharmacology analysis of AM-AS prescription in the treatment of diabetic renal fibrosis,408 common targets were obtained,corresponding to 116 active components,which includes quercetin,isorhamnetin,stigmasterol,β-sitosterol,7-O-methylisomucronulatol,3,9-di-O-methylnissolin,kaempferol,etc.The core targets were found by protein-protein interaction network analysis,such as JUN,MAPK1,IL-6,JUN,IL-10,TGF-β1,Smad7,etc.AM-AS prescription may act on the core common targets through these active components and plays a role in treating diabetic renal fibrosis.This process may regulate 1272 biological processes,such as drug response,oxidative stress,negative regulation of apoptosis,inflammation,etc.,136 signaling pathways including PI3 KAkt signaling pathway,TNF signaling pathway,NF-κB signaling pathway,MAPK signaling pathway,and TGF-β signaling pathway.The main targets of TGF-β signaling pathway,such as TGF-β1,TGF-β2,TGF-β3,Smad2,Smad3,Smad4 and Smad7,were docked with the main active components of AM-AS prescription.The results showed that the above 7 targets could be combined with 20 main active components of AM-AS prescription,suggesting the possibility of AM-AS prescription in treating diabetic renal fibrosis through TGF-β/Smads pathway.3.The experimental results showed that compared with the DKD group,the dapagliflozin group and each AM-AS prescription treatment groups had improved effects on regulating fasting plasma glucose(FPG),glycated serum protein(GSP),serum insulin(INS)levels and renal function.Among the traditional Chinese medicine treatment groups,AM-AS 3:2 group had better curative effect in improving FPG,GSP,INS,blood urea nitrogen(BUN)、β2-microglobulin(β 2-MG)and microalbuminuria(MAU),and next to the positive medicine treatment group.AM-AS 5:1 group and AM-AS 1:1 group had lower serum creatinine(Scr)levels than AM-AS 3:2 group.AM-AS prescription can inhibit the formation of collagen fibers and the infiltration of granulocytes in renal tissue,and when the ratio of AM:AS is 3:2,AM-AS prescription has the best effect on delaying the pathological changes such as vacuolation,nuclear pyknosis and glycogen deposition of renal tubular epithelial cells.RT-PCR and immunohistochemical detection of protein expression showed that the m RNA and protein expression levels of TGF-β1 have significantly increased in DKD rats,and the m RNA and protein levels of Smad7 have significantly decreased.AM-AS prescription can reduce the level of TGF-β1 in renal tissue,and to some extent antagonize the inhibitory effect of continuous high glucose state on the expression of renal protection factor Smad7.Conclusions: As one of the combinations of qi-benefiting and bloodactivating prescription,AM-AS has certain therapeutic and interventional effects on diabetic renal fibrosis.The mechanism may be related to regulating the expression of TGF-β1 and Smad7 in renal tissue of DKD rats through TGF-β/Smads pathway,inhibiting collagen fiber production,glycogen deposition and granulocyte infiltration,delaying the abnormal changes of renal tubular epithelial cell structure,regulating blood glucose and insulin levels,protecting renal tissue and improving renal function.