| Rationales: Huangqi is a tonic that has the effects of nourishing qi and raising yang,diuresis and swelling,and solid surface antiperspirant.Its active components are mainly astragalus polysaccharides,flavonoids and saponins,which have many pharmacological effects such as regulating immune function,anti-tumor,regulating metabolism and antiaging.In view of its effect of diuresis and detumescence,Huangqi has played a good role in the intervention of nephrotic syndrome,renal edema and chronic glomerulonephritis.By replicating adriamycin nephrotic rat model,the research group compared the pharmacodynamic difference between Fangji Huangqi Decoction and the negative prescription of Fangqi Huangqi Decoction,which took second-grades Huangqi from Shanxi Hunyuan as the monarch medicine.The results showed that the negative prescription of Fangqi Huangqi Decoction weakened significantly.After administration of aqueous extraction of Huangqi,it was found that the traditional medicinal efficacy indexes such as proteinuria,body weight,and organ index all changed to varying degrees,thereby confirming that the effect of aqueous extraction of Huangqi on adriamycin nephropathy was accurate.So,what is the mechanism of aqueous extraction of Huangqi intervening adriamycin nephropathy rats? Does the aqueous extraction of Huangqi of different commodity specifications have different effects on adriamycin nephropathy? What is the basis of effective substance? At present,it has not been systematically studied from the molecular and chemical level.The purpose of this study is to study the mechanism and the substance basis of aqueous extraction of Huangqi in the treatment of adriamycin nephropathy,and to provide the basis for the clinical application of Huangqi in nephrotic syndrome.Objective: In this study,the aqueous extraction of Huangqi with different product specifications and grades were used as research objects.Adriamycin-induced MPC5 cell injury model and adriamycin-induced nephropathy rat model were used.The mechanism and the substance basis of aqueous extraction of Huangqi intervention in adriamycin nephrosis were elucidated by the methods of transcriptomics,cell biology,chemical analysis,multivariate statistical analysis and network pharmacology,which laying a foundation for the further development of Huangqi-related drugs and providing a research basis for guiding the clinical rational use of Huangqi.Methods: 1.Taking the second-grade Huangqi from Shanxi Hunyuan as the research object,the serum biochemical indexes of blank group,adriamycin nephropathy model group and aqueous extraction of Huangqi group were detected,and the renal tissues of each group were stained with he and observed with transmission electron microscope to evaluate the effect of aqueous extraction of Huangqi on adriamycin nephropathy rats.Through the transcriptome sequencing of kidney tissue,the key target genes and key pathways of aqueous extraction of Huangqi intervention in adriamycin nephropathy were screened,and the accuracy of key target genes and key pathways was verified by cell biology and a variety of molecular biology techniques.2.Taking the aqueous extracts of different commodity grades of Huangqi as the research object,adriamycin-induced MPC5 cell injury model was used to screen out the aqueous extracts of Huangqi which had a greater protection effect.The chemical composition analysis was performed,and then the multivariate statistical analysis method was used to clarify the chemical composition categories and their quantity-ratio relationships that caused differences in protection.3.The key effective components of the aqueous extracts of Huangqi in adriamycin nephropathy rats were screened by the method of transcriptome and network pharmacology,and the activity of the effective components was verified by the mpc5 cell injury model induced by adriamycin.Results: 1.The serum biochemical test results showed that the levels of serum urea nitrogen,triacylglycerol and total cholesterol increased,and the levels of albumin and total protein decreased in rats in the model group.HE staining and transmission electron microscopy results of renal tissue showed that renal tubular epithelial cells in the model group atrophied,the lumen became larger,the glomerular podocyte foot process was fused,partly shed,the basement membrane thickened,and the endothelial cell contour Blurred,the tubular space of renal tubular epithelial cells was reduced and the fusion of foot processes was reduced after administration.Only local fusion,complete foot processes were present,and the outline of endothelial cells was clear.Kidney disease rats have better efficacy.Renal tissue transcriptome sequencing results indicate that the key target proteins and pathways of astragalus aqueous extract for renal protection may be PI3K-Akt signaling pathway(including Col6a6,Nr4a1,Sgk1,Gng7)and related genes(includingCpt1b,Pcsk9,Abca1,Ascm5);The expression levels of key target genes detected by fluorescence quantitative PCR are consistent with the expression trend of transcriptome sequencing;the verification results of the key pathways show that astragalus aqueous extract can significantly reduce the adriamycin-induced MPC5 injured cells ROS expression level protects adriamycin-induced apoptosis of MPC5 cells by activating the PI3K-AKT signaling pathway.Results: 1.The results of serum biochemical test showed that the levels of serum urea nitrogen,triglyceride and total cholesterol increased,the levels of albumin and total protein decreased in the model group.After the intervention of aqueous extract of Huangqi,the indexes were all reversed;The results of HE staining and transmission electron microscopy showed that in the model group,the epithelial cells of renal tubules were atrophic,the lumen was enlarged,the podocyte of glomerulus was fused,some of them were exfoliated,the basement membrane was thickened,and the contour of endothelial cells was indistinct.After administration,the lumen space of renal tubule epithelial cells was narrowed,the fusion of podocytes was reduced,only local fusion,complete podocytes existed,and the contour of endothelial cells was clear;The results showed that the aqueous extract of Huangqi had a good effect on adriamycin nephropathy rats.The results of transcriptome sequencing showed that PI3 K Akt signaling pathway(including col6a6,NR4A1,SGK1,gng7)and related genes(including cpt1 b,PCSK9,ABCA1,ascm5)were the key targets and pathways for the aqueous extract of Huangqi to play the role of renal protection;The expression trend of key target genes detected by fluorescence quantitative PCR was consistent with that of transcriptome sequencing;the verification results of key pathways showed that the aqueous extract of Huangqi could significantly reduce the expression level of ROS in MPC5 cells damaged by adriamycin,and protect MPC5 cells apoptosis induced by adriamycin by activating PI3K-AKT signal pathway.2.The protective effect of aqueous extracts of Huangqi on different commercial specifications on adriamycin-induced MPC5 cell damage does not correspond to the grade of medicinal materials.The protective effects of aqueous extracts of different grades of Huangqi in the same place on adriamycin-induced damage to MPC5 cells are: imitation of wild Huangqi,second grade of hunyuan > first grade of hunyuan > special grade of hunyuan > third grade of hunyuan;Transplanting Huangqi,second grade of Yingxian >first grade of Yingxian > third grade of Yingxian,second grade of Longxi > first grade of Longxi > third grade of Longxi.The comparison of aqueous extracts of Huangqi protective effects of different planting methods in the same habitat shows that Hunyuan imitation wild Huangqi is better than Yingxian transplanted Huangqi.The comparison of the protective effects of aqueous extracts of Huangqi in different growing areas on the same planting method showed that transplanting Huangqi in Longxi was better than transplanting Huangqi in Yingxian.The comparison of the protective effects of aqueous extracts of Huangqi in different growing areas and different planting methods showed that Hunyuan imitation wild Huangqi was better than Longxi transplanted Huangqi.Chemical analysis and multivariate statistical analysis of aqueous extracts of Huangqi showed that 9chemical components(Astragaloside IV,Ononin,Calycosin-7-O-glc-6-O-acetate,7,2’-Dihydroxy-3’,4’-dimethoxyisoflavan,Astraisoflavan-7-O-β-D-glucoside,7-hydroxy-6,4’-dimethoxyisoflavan,Formononetin,Isoastragaloside I,Astragaloside I)have protective effects on protecting MPC5 cell damage,and the difference of content and different quantitative composition may be the main reason for the difference of MPC5 cell protective effects.3.Transcriptomics combined with network pharmacology screening of medicinal ingredients of aqueous extracts of Huangqi showed that the cumulative contribution rate of the four compounds was 87.28%.Cell experimental verification results show that the four active ingredients have obvious protective effects on MPC5 cell injury induced by adriamycin,and they can significantly increase the expression of podocin protein in MPC5 cells.The best protective concentration of Astragaloside IV on adriamycin-induced MPC5 cell damage was 6.25 μg/m L,and the best protective concentration of formononetin was3.125 μg/m L.The best protective concentration of calycosin is 6.25 μg/m L,and the optimal protective concentration of quercetin is 3.125 μg/m L.Conclusion: The aqueous extracts of Huangqi protects adriamycin-induced MPC5 cells from apoptosis by reducing the ROS expression level in cells injured by adriamycininduced MPC5 and activating the PI3K-AKT signaling pathway.In addition,the aqueous extracts of Huangqi may interfere with lipid metabolism of AN rats by acting on lipid metabolism-related genes(including Cpt1 b,Pcsk9,Abca1,Ascm5).The key medicinal ingredient for adriamycin nephropathy is Astragaloside IV,calycosin,quercetin,formononetin. |
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