| ObjectiveAn increasing number of studies have shown that abnormal lipid metabolism in renal tubular epithelial cell plays a key role in the development of renal fibrosis and CKD progression.The aim of this study is to investigate whether the protective effect of Bupi Yishen formula on renal function is achieved by regulating renal tubular lipid metabolism,in order to interpret the mechanism of Bupi Yishen formula in the treatment of CKDMethodsIn this study,we used adenine-induced rats as a model of chronic kidney disease(CKD)to observe the progression of CKD and the intervention of Bupi Yishen formula.The experiment was divided into 4 groups:normal control group(CTL group),adenine-induced chronic kidney disease rats(CKD group),low-dose group of Bupi Yishen formula(BYF-L group),and high-dose group of Bupi Yishen formula(BYF-H group).The rats in each group were given adenine-free saline,adenine 200 mg/kg,and the low and high doses of Bupi Yishen formula in water decoction by gavage for 4 weeks.After 4 weeks,serum specimens,24h urine specimens and kidney tissue specimens were collected.The serum and urine specimens were collected and tested for biochemical parameters related to renal functions.Histopathological staining(including HE,PAS,Masson,Oil red O staining)and subsequent molecular biology experiments were performed on the kidney tissue specimens for verification,respectively.In vitro cellular experiments were also validated by TGF-β1induced HK-2 cell model.The cell experiments were divided into 4 groups,normal control group(CTL group),TGF-β1-induced HK-2 cell model group(TGFβ1 group),low-dose intervention group(TGFβ1+BYF-L group)of Bupi Yishen formula,and high-dose intervention group(TGβ1+BYF-H group)of Bupi Yishen formula.Diffeerential gene enrichment analysis between groups was performed by transcriptome sequencing(mRNASeq)technology for normal group(CTL group),adenine-induced chronic kidney disease rat model group(CKD group)and low-dose treatment group of Bupi Yishen formula(BYF group).The key enrichment pathways were validated by in vivo and vitro experimenls.Combined with the transcriptomic analysis results and in vivo and vitro validation results,the specific mechanism of Bupi Yishen formula was fully elucidated.Results1.In vivo animal experiments of physiological and biochemical indexes:physiological indexes included body weight and urine volume;biochemical indexes included blood creatinine,blood urea and 24h urine protein quantification:compared with the CTL group,the adenine induced CKD rats showed a significantly reduced trend of body weight gain(P<0.05)and significantly increased urine volume(P<0.05).Blood creatinine,blood urea and 24b urine protein quantification was significantly higher(P<0.05).After BYF treatment.the trend of weight gain was significantly increased(P<0.05),and urine volume,blood creatinine.blood urea and 24h urine protein quantification was significantly decreased(P<0.05).By HE,PAS and Masson staining experiments,the results showed that a large number of inflammatoty cell infiltration,renal tubular dilatation,renal structural destruction and collagen matrix deposition were clearly visible in pathological sections of CKD rats in the model group.After BYF treatment,the inflammatory cell infiltration was significantly reduced.the structural damage of renal tubules was reduced,and the deposition of collagen matrix in the renal interstitium was significantly reduced.2.BYF inhibits renal fibrosis and tubular cell apoptosis in CKD rats.Results showed that compared with the CTL group.the mRNA expression levels of fibrosis-related indicators,α-sma,Tg-β1,Fn-1,Col3α1,were significantly higher in the adenine-induced CKD rat model group(P<0.05);the mRNA expression levels of the pro-apoptotic indicator Bax were significantly higher,and Bcl-2,an anti-apoptotic indicator,were significantly decreased(P<0.05).After BYF treatment,the mRNA expression levels of fibrosis indexes were all downregulated,and the differences among α-sma,Fn-1,Co13α1 groups were not significant.The mRNA expression levels of pro-apoptotic index Bax were significantly down-regulated,and the mRNA expression levels of anti-apoptotic index Bcl-2 were significantly up-regulated(P<0.05).WB results were consistent with qPCR results,and the expression levels of Caspase3 and BAX proteins were significantly increased in the model group(P<0.05),and the expression levels of BCL-2 protein were significantly down-regulated(P<0.05).FN-1 and COL3α1 protein expression levels were significantly upregulated(P<0.05).3.BYF ameliorates TGF-β1-induced fibrogenesis and apoptosis in HK-2 cells.Results showed that compared with the CTL group,in the TGF-β1-induced HK-2 cell model,the mRNA expression levels of fibrosis-related indicators,TGF-β1,α-SM4,FN-1 and COL3α1,were significantly higher in the TGFβ1 group(P<0.05),the mRNA of the pro-apoptotic indicator BAX expression levels were significantly increased.and the mRNA expression levels of anti-apoptotic indicator BCL-2 were significantly decreased(P<0.05).After BYF treatment.the mRNA expression levels of fibrosis-related indicators were significantly reduced(P<0.05).The mRNA expression levels of BAX,a pro-apoptotic indicator,were significantly down-regulated,and the mRNA expression levels of BCL-2 an anti-apoptotic indicator.were significantly up-regulated(P<0.05).WB results were consistent with qPCR results.and Caspase-3 protein levels were significantly increased(P<0.05)and BCL-2 protein levels were significantly decreased(P<0.05)in the TGFβ1 group.BAX was no difference among groups.FN-1 and COL3al protein expression was significantlyincreased(P<0.05).Caspase-3 and BAX were significantly downregulated(P<0.05)-BCL-2 protein expression was significantly increased(P<0.05),and fibrosis index protein expression was significantly downregulated(P<0.05)after BYF treatment.4.Based on mRNA-Seq sequencing technology and bioinformatics analysis,The main regulatory processes of BYF on differentially expressed genes include monocarboxylic acid metabolic process,energy derivation by oxidation of organic compounds,cellular respiration.fatty acid metabolic process.glycoprotein metabolic process,generation of precursor metabolites and energy production,regulation of Wnt signaling pathway,amino-acid betaine metabolic process,canonical Wnt signaling pathway.The KEGG database was enriched for differential genes in the following metabolic pathways:citrate cycle,carbon metabolism,fatty acid degradation,fatty acid metabolism,2Oxocarboxylic acid metabolism,ABC transporters,N-Glycan biosynthesis,Gastric cancer,multiple Longevity regulating pathway-multiple species,Glycine,serine and threonine metabolism,Biosynthesis of unsaturated fatty acids,biosynthesis of unsaturated fatty acids,Hepatocellular carcinoma,Choline metabolism in cancer,Endometrial cancer,mTOR signaling pathway,Vitamin digestion and absorption,Glyoxylate and dicarboxylate metabolism,Peroxisome,Basal cell carcinoma,Colorectal cancer.In combination with GO biological process analysis and KEGG metabolic pathway results,kidney samples from CKD rats were significantly associated with lipid metabolism-related pathways.To verify the changes in the expression of genes involved in lipid metabolism-related pathways in CKD rats,the transcriptional expression levels of most genes related to fatty acid oxidative metabolism were altered.The expression levels of Acadvl,Acadm,Cpt1a,Ppargcla,and Ppara,which are involved in the fatty acid oxidation process,were downregulated in the model group,and the expression trends of these genes were up-regulated after BYF treatment.The abnormal alterations of fatty acid oxidation-related genes were partially reversed after treatment with BYF intervention.The results of transcriptome differential gene enrichment analysis tentatively identified that the process of renal fibrosis in CKD rats was closely associated with abnormal fatty acid metabolism5.In vivo animal studies show that BYF improves renal fatty acid oxidation and renal tubular lipid droplet accumulation.Results showed that the expression of CPT-1α,Pparα,and Ppargcla were significantly downregulated in the CKD model group compared with the CTL group(P<0.05),and the mRNA expression levels of fatty acid oxidation-related indicators were significantly increased after BYF treatment(P<0.05).WB results were consistent with the qPCR results.The proteins expression levels of CPT-1α,PGC-1α were significantly down-regulated in the CKD model group compared with the CTL group(P<0.05).and the expression levels of fatty acid oxidation-related indicator proteins were significantly increased after BYF treatment(P<0.05).no significant difference in ACOX1 protein expression level between groups.The results of oil red O staining showed that a large amount of lipid accumulation in the kidney tissue was observed in the model group,and the lipid accumulation was reduced in the BYF-treated group.6.In vitro cellular assays show that BYF improves renal tubular fatty acid oxidation and lipid droplet accumulation.Results showed that the expression of CPT-1α PPARα,and PGC1a was significantly downregulated in the TGFβ1 group compared with the CTL group(P<0.05),and the mRNA expression levels of fatty acid oxidation-related indicators were significantly increased after BYF treatment(P<0.05).WB results were consistent with the qPCR results.Compared with the CTL group,the expression levels of CPT-1α,PGC-1α and ACOX1 proteins were significantly down-regulated in the TGFβ1 group(P<0.05),and the protein expression levels of fatty acid oxidation-related indicators were significantly increased after BYF treatment(P<0.05).The results of oil red O staining showed that a large amount of intracellular lipid accumulation was observed in the model group,and the lipid accumulation was significantly reduced in the BYF-treated group.Conclusion1.BYF effectively alleviated adenine-induced renal impairment and inhibited renal fibrosis and tubular apoptosis in CKD rats.BYF ameliorated TGF-β1-induced fibrogenesis and apoptosis in HK-2 cells.2.The mRNA-Seq sequencing technology and systematic bioinformatics analysis of kidney tissues suggested that Bupi Yishen formula had significant regulatory effects on lipid metabolism-related pathways in CKD rats,especially closely related to fatty acid oxidation process.3.Further in vivo and vitro experimental studies confirmed that,BYF partially restored the down-regulation of fatty acid oxidation and reduced lipid accumulation.In summary,the renoprotective effect of BYF on CKD may be achieved by restoring the homeostasis of lipid metabolism in renal tubular epithelial cells,inhibiting renal fibrosis markers and reducing renal tubular cell apoptosis,thus achieving anti-renal fibrosis and delaying the progression of CKD. |
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