Beneficial Effect And Molecular Mechanism Of Tian Huang Formula On Renal Fibrosis

Objective:Renal fibrosis is an important complication of glycolipid metabolic diseases.It is the main pathological change of various chronic kidney diseases(CKD).It is closely related to the prognosis and outcome of glycolipid metabolic diseases and CKD."Tiao Gan Qi Shu Hua Zhuo" is an important method of traditional Chinese medicine in the treatment of glucose and lipid metabolic diseases.As a representative prescription of "Tiao Gan Qi Shu Hua Zhuo",the TianHuang formula has a good curative effect on glycolipid metabolic diseases,but its treatment of fibrosis injury of the target organ kidney is not clear,which needs further research and exploration.Therefore,based on the research of network pharmacology,in vivo animal experiments,and in vitro cell experiments,this paper discusses the pharmacodynamic material basis of Tianhuang formula in the prevention and treatment of renal fibrosis,explores the protective effect and the molecular mechanism of Tianhuang Recipe on renal fibrosis,to provide a theoretical and data support for the clinical application of Tianhuang formula in the treatment of renal fibrosis.Research methods:1.Network PharmacologyThe main components of the Tianhuang formula were obtained through the TCMSP database,supplemented by the components mined in the literature,and the target genes of the above components were matched.The targets of renal fibrosis were screened by GeneCards and OMIM database.Construct the PPI network between the active components of the Tianhuang formula and the common target of renal fibrosis disease.Input the name of the common target gene on the Metascape database and analyze the richness of functions and pathways,to obtain the possible mechanism and regulatory pathway of the Tianhuang formula in the treatment of renal fibrosis.2.In vivo animal experimentsHealthy male C57BL/6 mice after adaptive feeding were randomly divided into sham operation group(sham),model group(UUO),low,medium,and high dose Tianhuang formula groups(TH-L,TH-M,and THH),and positive drug group(LDX).Each group was divided into three-time nodes:3,7,and 14 days.Except for the sham operation group,the other groups underwent UUO operation to prepare the model of renal fibrosis.Gastric perfusion began on the day of operation.Tianhuang formula group and LDX group were administered by gavage once a day.The sham operation group and UUO group were given equal volumes of water as the control.Serum and renal tissue samples were collected on the 3rd,7th,and 14th days.Creatinine(Scr)and urea nitrogen(BUN)levels in serum were measured by using kits,and HE staining was performed on kidney tissues to evaluate the degree of renal structural and functional injury;Masson staining was used to evaluate the aggregation degree of collagen fibers;The expression levels of fibrosis index proteins and genes in obstructive renal tissue were detected by Western blot and real-time fluorescence quantitative PCR(qRT-PCR).Protein and gene levels of transforming growth factorβ1(TGF-β1),a key factor in renal fibrosis,were detected.the protein expression and phosphorylation levels of p38 and ERK1/2 in obstructive renal tissue were detected,as well as the protein levels of NF-κB and the mRNA expression levels of inflammatory factors downstream of the MAPK pathway,to explore the protective mechanism of the Tianhuang formula on the fibrotic kidney.3.In vitro cell experimentTGF-β1 induces HK-2 cells to establish a fibrotic cell model.CCK-8 experiment determined that the low,medium and high administration concentrations of Tianhuang formula were 25,50,and 100 μg/ml.the cells were collected 24h after administration.The degree of fibrosis was evaluated by measuring the expression levels of fibrosis index proteins and genes in cells.The expression and phosphorylation level of p38,ERK1/2,and mRNA level of TNF-α,and NF-κB proteins were measured,to explore the mechanism of the Tian Huang formula in protecting kidney fibrosis.Results:1.Network pharmacologyThere are 32 main active components in the Tianhuang formula that were screened,targeting 232 potential target genes,Quercetin was the most important.There are 111 core targets for renal fibrosis diseases,among which AKT1,TNF,IL-6,MAPK1,MMP9,JUN,and other target genes may be the core targets of Tianhuang formula in the treatment of renal fibrosis.Through enrichment analysis,30 biological function results related to inflammatory response,apoptosis,and ECM and 20 core pathway results related to inflammatory response,apoptosis,and metabolism were obtained.Based on the visualization of the KEGG pathway diagram,it is inferred that the protective effect of the Tianhuang formula on renal fibrosis may be through the action of Quercetin and other main components on TGF-β1.FN,TNF,and other action targets,based on TGF-β1 and associated MAPK and NF-κB and other signaling pathways regulate inflammation and cell transdifferentiation and play a role in renal protection and anti-fibrosis.2.Pharmacodynamic studyCompared with the UUO group,The treatment group of the Tianhuang formula can maintain the structure and function of renal tissue on the obstructive side;the Tianhuang formula can significantly rreduce collagen proliferation in renal interstitium and downregulate the proteins related to inflammation and fibrosis,α-SMA,COL1,TGF-β1 expression,and related gene FN,α-SMA,TGF-β1,TNF-α mRNA expression level.The effect of the Tianhuang formula on inflammation and fibrosis index protein in HK-2 cell fibrosis was also observed in vitro,the Tianhuang formula has a certain inhibitory effect on the protein level of FN and the mRNA expression of αSMA,FN,and COL1.3.Mechanism researchIn vivo animal experiments,compared with the UUO group,the Tianhuang formula treatment can significantly reduce the phosphorylation levels of ERK1/2 and p38 in the renal tissue.At the same time,we found that the treatment of the Tianhuang formula can significantly inhibit the obstruction of NF-κB protein expression.In vitro,the Tianhuang formula has a certain inhibitory effect on p38 phosphorylation in HK-2 cells induced by TGF-β1,but no obvious regulatory effect on ERK1/2 was observed.It is suggested that the Tianhuang formula can protect renal fibrosis by regulating the MAPK pathway,but it may need the joint participation of other cells and factors.Conclusion:1.Tianhuang formula can effectively protect the morphology and function of the kidney and resist the progress of renal fibrosis.2.The protective effect of the Tianhuang formula on renal fibrosis may be through regulating the TGF-β1 and MAPK pathways,which affect the expression of downstream pro-inflammatory and fibrogenic factors.