The Mechanism Of Buyang Huanwu Decoction On Renal Interstitial Fibrosis Based On The Method Of "Buying Qi And Activating Blood"

Purpose:1.Observe the protective effect of Buyang Huanwu Decoction on renal interstitium in rats with unilateral ureteral obstruction(UUO);2.The transcriptome sequencing technique is used to detect the differentially expressed genes in rats,and the significantly enriched differentially expressed genes and signal pathways are screened.3.Western blotting and Pearl time PCR are used to verify the signal pathway and we explore the possible mechanism of Buyang Huanwu Decoction in the treatment of renal interstitial fibrosis(RIF).Method:1.Pharmacodynamic study of Buyang Huanwu Decoction on UUO ratsFifty SPF healthy male SD rats are randomly divided into sham operation group,model group,Buyang Huanwu Decoction low dose group,Buyang Huanwu Decoction medium dose group and Buyang Huanwu Decoction high dose group.From the first day after operation,each dose of treatment group is given by gavage.Rats in sham operation group and model group are given normal saline by gavage.On the 14 th day after operation,blood samples are collected from the inferior vena cava and abdominal aorta one day before operation,and serum biochemical indexes are detected.The levels of Scr and AST are detected by biochemical method;the pathological changes of renal tissue are observed by HE and Masson staining;the expression of FN and α-SMA protein are detected by immunohistochemistry.2.Transcriptomic study of Buyang Huanwu Decoction on UUO ratsThe kidney tissues of UUO rats in each group,sham group,UUO group and high-dose Buyang Huanwu Decoction group are taken out to extract total RNA.Through the quality control and filtering of total RNA,the sequencing library is constructed,the reference genome is compared,the transcripts are assembled and merged,the sample expression quantity is analyzed,and the differentially expressed genes are screened.Finally,the obtained differentially expressed genes are analyzed by Gene Ontology(GO)and Kyoto Encyclopedia of genes and genes Genomes(KEGG)enrichment analysis.3.Effect of Buyang Huanwu Decoction on inflammatory response mediated by NF-κ B signaling pathwayFirst,the protein and m RNA levels of FN,col Ⅰ and col Ⅲ are detected by WB and PCR.Then,The screened signal pathways are verified by WB and PCR according to the main biological functions and signal pathways are screened by GO and KEGG enrichment analysis.Finally,the results were discussed and analyzed.Result:1.Pharmacodynamic study of Buyang Huanwu Decoction on UUO rats(1)Observation on the general state and renal morphology of rats in each group Compared with the sham operation group,the rats in the model group are more depressed,less food intake,less activity and loose body hair.Compared with the model group,the mental state of rats in each drug intervention group is improved in varying degrees,food intake and hair color are improved to a certain extent.Compared with the sham group,the volume of the obstructed kidney in the UUO group is significantly larger than that of the sham group,with rough texture,uneven surface,ecchymosis on the renal tissue surface,a large amount of turbid fluid in the kidney,and no abnormality in the unobstructed kidney.The morphological changes of kidney in each intervention group are similar to those in the UUO group,alleviated to varying degrees.(2)Biochemical detection and pathological changesThe results of biochemical test show that the level of Scr in the UUO group is significantly higher than that in the sham group(P < 0.01).Compared with the UUO group,the Scr levels of low-dose and medium dose Buyang Huanwu Decoction groups has a downward trend,but there is no statistical significance(P > 0.05).Compared with the UUO group,the Scr level of highdose Buyang Huanwu Decoction group is significantly lower(P < 0.01).Compared with the sham group,the SCR level in the UUO group increases slightly,but there is no significant difference between the two groups(P > 0.05).Compared with the model group,the serum AST level of each drug intervention group is slightly increased,and the difference is not statistically significant(P > 0.05).The results of HE show that in the model group,the structure of renal tubule is seriously damaged,the lumen is enlarged,the interstitial is atrophied and collapsed,accompanied by fibrous tissue hyperplasia.There are a lot of inflammatory cells in the stroma.Compared with the model group,the pathological changes of renal tissue in each drug intervention group are alleviated in varying degrees.Masson staining show that a large number of blue collagen fibers are deposited in the renal interstitium in the model group.The degree of collagen fiber accumulation in renal interstitium of rats in each drug intervention group decreases in varying degrees.Compared with the model group,the positive area of blue collagen fibers in the lowdose and high-dose groups is significantly decreased(P < 0.05),and the positive area of blue collagen fibers in the high-dose group is significantly decreased(P < 0.01).(3)Effect of Buyang Huanwu Decoction on UUO induced ECM accumulation in renal interstitium and EMT process in renal tubular epithelial cells The results of immunohistochemistry show that FN and α-SMA protein are only distributed in a small amount in the renal interstitium in the sham operation group,while FN and α-SMA protein are widely distributed in the renal tubulointerstitium in the model group.Compared with the model group,the expression of FN and α-SMA protein in all drug intervention groups are improved in varying degrees,and the effect of high-dose BYHW group is the most obvious(P <0.01).2.Transcriptome study of Buyang Huanwu Decoction on UUO rats(1)The original reads number of the kidney tissue sequencing library of the three groups is all about 97%,and the base proportion of Q20 is all about 98%.By comparing with the reference genome sequence of SD rats,the overall alignment rate is more than 70%,and the multiple alignment rate is less than 10%.(2)Statistical results of pairwise comparison of different genes Sham＿VS＿UUO differential analysis show that there are 4268 differentially expressed genes,including 2018 up-regulated genes and 2251 down regulated genes＿VS＿UUO differential analysis show that there are 626 differentially expressed genes,including 273 up-regulated genes and 353 down regulated genes.The up-regulated gene and down regulated gene in the two groups are crossed,which is the potential target gene of Buyang Huanwu Decoction in the treatment of UUO rats.A total of 169 target genes are identified.(3)Go and KEGG enrichment analysis of target genes The biological processes of significant enrichment mainly include immune response,inflammatory response,etc.;the cellular components of significant enrichment include extracellular space,extracellular domain,nucleosome,etc.;the molecular functions of significant enrichment include protein domain specific binding,chemokine activity,etc.The pathways of target gene enrichment include c-cytokine-cytokine receptor interaction,insulin signaling pathway,NF-κ B signaling pathway,TNF signaling pathway,and the regulation of inflammatory mediators on tryptophan channel.3.Effect of Buyang Huanwu Decoction on inflammatory response mediated by NF-κ B signaling pathwayCompared with the sham operation group,the results of WB show that the protein expressions of FN,col Ⅰ and col Ⅲ in the model group are significantly increased(P < 0.01).Compared with the model group,the protein expression of FN in the low-dose and medium dose Buyang Huanwu Decoction treatment groups is decreased,but the difference is not statistically significant(P > 0.05);the protein content of FN in the high-dose Buyang Huanwu Decoction treatment group is significantly decreased(P < 0.05).The expression of Col I in low-dose treatment group is significantly lower than that in medium dose and high-dose treatment groups(P < 0.01).The content of col Ⅲ protein in renal interstitium of rats in each drug intervention group is significantly decreased(P < 0.01).Compared with the sham group,the results of PCR show that the m RNA expression levels of FN,col Ⅰ and col Ⅲ in the model group are significantly increased(P < 0.01).Compared with the model group,the m RNA expression levels of FN,col Ⅰ and col Ⅲ in the drug intervention groups are significantly decreased(P < 0.01),and the effect of Buyang Huanwu Decoction high dose group is the most obvious.WB test results show that the expression of p65,p-p65,P-I kappa,IL-6 and TNF-a in kidney tissue of UUO rats are significantly higher than that in sham group(P < 0.01),while the expression of I kappa B α is slightly decreased,but the difference is not statistically significant(P > 0.05).Compared with the model group,the content of p65 protein in the low dose treatment group of Buyang Huanwu decoction is significantly reduced(P < 0.05).The protein content of pp65 in the middle and high dose treatment groups is significantly reduced(P < 0.01),while the pp65 protein content in the low-dose treatment group is lower,but the difference is not statistically significant(P > 0.05),and the p-p65 protein content in the middle dose treatment group is significantly reduced(P < 0.05).The p-p65 protein content in the high dose treatment group is significantly reduced(P < 0.01),and the P-I kappa B α protein content in the low and high dose treatment group is significantly reduced(P < 0.05).The level of I kappa B α in the low and medium dose treatment groups is significantly increased(P < 0.05),while that of the high dose treatment group is higher,but the difference is not statistically significant(P > 0.05);the TNF-α in the drug treatment group is significantly lower(P < 0.01).The content of IL-6 in the low and medium dose treatment group is lower than that in the high dose group(P < 0.05).The results of PCR show that the expression level of I kappa B α is significantly lower(P < 0.05)and that of p65 m RNA is higher than that in sham group(P < 0.05),but the difference is not statistically significant(P > 0.05),and the expression level of IL-6 and TNF α is significantly increased(P < 0.05).Compared with the model group,the expression level of I kappa B α in the low-dose treatment group is significantly increased(P < 0.05),while the expression level of I kappa B α in the low and medium dose treatment groups increased to different degrees,but the difference is not statistically significant(P > 0.05).The p65 m RNA in each drug intervention group is not significantly lower,but the difference is not statistically significant(P > 0.05).The expression of IL-6 and TNF α m RNA in the drug intervention group was significantly reduced(P< 0.05).Conclusion1.The intervention of Buyang Huanwu Decoction on renal interstitial fibrosis in UUO rats involves multiple pathways,multiple targets and multiple processes.2.Buyang Huanwu Decoction can improve the general condition of UUO rats,improve serum biochemical indexes and reduce renal pathological damage.3.Buyang Huanwu Decoction can inhibit the process of renal interstitial fibrosis,and itsmechanism is related to the inhibition of inflammatory response induced by NF-κ B.