| IgA nephropathy,as one of the most common primary glomerular diseases around the world,has significant regional differences in incidence,with a high incidence in Asia especially.The clinical manifestations of the disease are mainly proteinuria of varying degrees,which may be accompanied by recurrent gross hematuria or microscopic hematuria.Renal immunopathology was characterized by IgA or igAdominated immunoglobulin deposition in the mesangial region,mesangial cell proliferation and mesangial matrix increase,with or without capillary loop deposition.About 30%to 40%of patients progress to end-stage renal disease within 20 years,which is the main primary glomerular disease that causes chronic renal failure,placing a huge burden on both patients and society.Although hormone and supportive therapy can reduce proteinuria in patients with IgA nephropathy to a certain extent,they have not been shown to slow the progression of renal function,and both have adverse events.Therefore,it is necessary to find the combination of traditional Chinese medicine therapy,which has important scientific value and social significance to reduce proteinuria in patients while delaying the progression of renal function and reducing the occurrence of adverse events.As the disadvantages of single-target drug research gradually emerged and the research of multi-target drugs gradually emerging,the term"network pharmacology" was put forward and widely used in scientific research,making new breakthroughs in the pathogenesis of diseases and bringing effective research methods to the research and development of new drugs.The application of network pharmacology to study Traditional Chinese medicine and its compounds has also become one of the current many research hotspots.Based on the results of previous studies on network pharmacology related methods,this study selected IL-17 signaling pathway to further explore the mechanism of action of flavored Huangqi Chifeng Decoction on IgA nephropathy.Objective:Based on THE IL-17 signaling pathway,this study investigated the effect of Modified Huangqi Chifeng Decoction on proteinuria in rats with IgA nephropathy and its improvement effect on renal pathological injury,and explored the molecular mechanism of this formula in the treatment of IgA nephropathy.Methods:The rat model of IgA nephropathy was constructed by the method of bovine serum albumin+carbon tetrachloride+lipopolysaccharide.After the successful production of the model,dividing the rats into model group,telmisartan group,Modified Huangqi Chifeng Decoction high-dose,medium-dose and low-dose groups with 10 rats in each group according to the random number table randomly.The groups were treated with telmisartan and Modified Huangqi Chifeng Decoction for 8 weeks,the length of experiment is 21 weeks totally.Urine of the rats was collected every 3 weeks for 24h,and the relevant 24h urinary protein was detected.At the end of the experiment,blood samples were collected from abdominal aorta,and serum samples were collected after centrifugation.The relevant blood biochemical levels of rats were detected by automatic biochemical analyzer,and IL-6 and IL-17A levels were determined by enzyma-linked immunosorbent assay(ELISA).After the abdominal aorta blood sampling,the rats’ bilateral kidneys were dissected immediately.HE,Masson and PAS staining methods were used to stain the kidney sections of rats,and then the pathological changes of glomerulus in each group were observed under light microscope.IgA fluorescence deposition in mesangial area of glomerulus in each group was observed under immunofluorescence microscope and its intensity was evaluated.The pathological changes of glomerulus ultrastructure in each group were observed under electron microscope.The expression of HSP90,MMP9,NF-κB P65 and P-NFκB P65 in the IL-17 signaling pathway were detected by immunohistochemical method.The expressions of HSP90,MMP9,NF-κB P65 and P-NF-κB P65 were detected by Western Blot.Results;1.Quantification of 24h urine protein in rats:① Modeling stage:since the 9th week,the quantification of 24h urine protein in rats in modeling group was significantly increased compared with control group,and the difference was statistically significant(P<0.05).② Drug intervention stage:at 21 weeks,compared with the model group,the 24h urinary protein quantification of telmisartan group and TCM intervention groups was decreased,the difference was statistically significant(P<0.05);Compared with telmisartan group,there was no significant difference in the decrease degree of urine protein quantification at 24h in the intervention groups(P>0.05).2.After 8 weeks of drug intervention(at the end of the 21st day of the experiment),blood biochemical indexes were detected,and there were no statistical differences in Scr,ALB,AST and ALT among all groups(P>0.05).Compared with telmisartan group,BUN in all groups had statistical difference(P<0.05),considered that telmisartan may have a certain effect on renal function due to its metabolism in the kidney.3.Observation of renal specimens stained by HE,PAS and Masson under light microscope:Blank group:glomerular morphology was basically normal,intact structure,smooth balloon wall,thin and clear vascular cavity,normal distribution of mesangial cells and mesangial matrix,no hyperplasia,fibrosis,hyaline lesions,smooth balloon wall;The lumen of renal tubules was arranged neatly,and the lining monolayer cubic epithelium had no swelling and necrosis.There was no inflammatory cell infiltration and fibrous tissue proliferation in the stroma.Compensatory expansion model group:glomerular appear,the part shrinkage,part of the capillary swelling,congestion,part of the glomerular capillary loops atresia or stenosis,blood capillary endothelial proliferation and mesangial cell hyperplasia,mesangial matrix deposition,glomerular obvious lobulated or nodular changes,balloon wall Narrows,balloon clearance generally slightly smaller;Renal tubules are arranged in disorder,with dilated lumen,tubule cell swelling and vacuolar degeneration,tubule atrophy occasionally,hyperemia,edema and inflammatory cell infiltration occasionally,and fibrous hyperplasia occasionally.Telmisartan group:glomerular mild dilatation,few glomerular vascular hyperemia and swelling,no obvious proliferation of mesangial matrix,slight narrowing of balloon space,rare adhesion of balloon wall;A small number of renal tubule epithelial cells were swollen and denatured,arranged in order,and there was very little inflammatory cell infiltration in the stroma.Compared with the model group,the lesions in the high,medium and low dose groups of Traditional Chinese medicine were significantly reduced,including mild hyperemia of the vascular sphere,a small amount of matrix deposition in the mesangial region of the glomerular,mild thickening of the basement membrane,and no balloon adhesion.Tubule epithelial cells were not swollen,vacuolar degeneration existed,and there was less inflammatory cell infiltration in the stroma.The improvement was significant in high and medium dose,but weak in low dose.4.IgA deposition was observed under immunofluorescence:no obvious IgA fluorescence deposition was found in the glomerular mesangial area of rats in the blank group,and granular or lump-like IgA fluorescence deposition with different fluorescence intensity was observed in the glomerular mesangial area of rats in the model group and all drug intervention groups.The intensity of IgA fluorescence deposition was different among all groups,and the difference was statistically significant(P<0.05).Compared with blank group,IgA fluorescence deposition was significantly enhanced in model group(H=-46.5,P’=0.000).Compared with model group,the intensity of IgA fluorescence deposition in kidney of telmisartan group,Chinese medicine with high and medium dose intervention was decreased(P’<0.05);Compared with telmisartan group,there was no statistical difference in IgA fluorescence deposition intensity in each dose of TCM intervention group(P’>0.05).Compared with the high-dose group,there was no significant difference in IgA deposition intensity between the medium-dose group and the low-dose group(P’>0.05).5.The ultrastructure of renal tissue was observed under electron microscope.In the blank group,there were no obvious abnormalities in the mesangial area,the foot processes were clear and arranged in a tightly ordered manner,and the basement membrane was intact and uniform in thickness.The mesangial region of the model group was scattered with a large area of high-density electron density,and the foot processes were arranged in disorder,with a large number of fusion and part of detachment,and the basement membrane was different in thickness.In telmisartan group and Chinese medicine high-dose group,a small amount of high density electron density was scattered in the mesangial area,the local fusion of foot process was rare,and the thickness of basement membrane was uniform.In the medium and low dose groups of traditional Chinese medicine,a small amount of high density electron density was scattered in the mesangial area,accompanied by a small amount of fusion of some foot processes,small foci exfoliation and different thickness of basement membrane,which was more serious in low dose groups.6.ELISA results showed that the expression levels of IL-6 and IL-17A in serum of model group were significantly higher than that of blank group,and the difference was statistically significant(P<0.05).Compared with model group,after the intervention of telmisartan and Modified Huangqi Chifeng Decoction,the rat serum IL6,IL-17A expression levels were lower,telmisartan group and high dose group compared with model group,difference has statistical significance(P<0.05),Chinese medicine low dose group compared with model group,do not have statistically significant(P>0.05).7.Western Blot analysis of renal tissue protein showed that compared with blank group,there was no significant difference in NF-κB P65 protein expression in model group,telmisartan group and TCM intervention groups(P>0.05).Compared with blank group,the expression levels of P-NF-κB P65,MMP9 and HSP90 protein in renal tissues of model group were increased(P<0.05).Compared with model group,the protein expression levels of P-NF-κB P65,MMP9 and HSP90 in renal tissues of rats treated with telmisartan and modified Modified Huangqi Chifeng Decoction were significantly decreased(P<0.05).Compared with telmisartan group,there was no significant difference in the expression of P-NF-κB P65,MMP9 and HSP90 protein in the highdose group(P<0.05),but there was significant difference in the expression of P-NF-κB P65,MMP9 and HSP90 protein in the medium-dose and low-dose groups(P>0.05).8.Compared with blank group,there was no significant difference in NF-κB P65 protein expression in model group,telmisartan group,Chinese medicine high-dose,medium-dose and low-dose rats(P>0.05).Compared with blank group,the protein expression levels of P-NF-κB P65,MMP9 and HSP90 in renal tissues of model group were increased(P<0.05).Compared with model group,the protein expression levels of P-NF-κB P65,MMP9 and HSP90 in renal tissues of telmisartan group,high,medium and low dose groups were significantly decreased(P<0.05).Compared with telmisartan group,the protein expression levels of P-NF-κB P65,MMP9 and HSP90 in renal tissues of rats in high dose Chinese medicine group had no statistical difference(P<0.05).There were significant differences in the protein expression levels of P-NF-κB P65,MMP9 and HSP90 in kidney tissues between the medium-dose and low-dose groups(P>0.05).Conclusion:1.The expression levels of IL-6 and IL-17A in serum of inflammatory molecules related to IL-17 signaling pathway were increased in IgA nephropathy rats.2.The expression levels of p-NF-κB P65,MMP9 and HSP90 in IL-17 signaling pathway were increased in IgA nephropathy rats.3.Modified Huangqi Chifeng Decoction can down-regulate the expression levels of IL-17 signaling pathway related molecules IL-6,IL-17A,p-NF-κB P65,MMP9 and HSP90,thereby inhibiting renal inflammatory response and renal fibrosis.4.The protective effect of modified Huangqi Chifeng Decoction on the kidney of rats with IgA nephropathy may be related to the down-regulation of IL-17 signaling pathway and the inhibition of renal inflammatory response and renal fibrosis. |
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