Mechanism Of Wuwei Xiaoduyin On NF-κB Signaling Pathway In IgA Nephropathy Rats

Part one Anti-inflammatory mechanism of Wuwei Xiaoduyin on IgA nephropathy rats based on NF-κB inflammatory signaling pathwayObjective:To observe the effect of Wuwei Xiaoduyin on nuclear factor-κB（NF-κB）signaling pathway in IgA nephropathy（IgAN）rats,and explore the mechanism of its treatment of IgA nephropathy.Methods:40 SD rats were randomly divided into control group,model group,benazepril group(10 mg·kg·d^-1)and Wuwei Xiaoduyin group(2.75g·kg·d^-1),with 10 rats in each group.The IgA nephropathy rat model was established by intragastric administration of bovine serum albumin（BSA）,subcutaneous injection of carbon tetrachloride（CCl₄）and tail vein injection of lipopolysaccharide（LPS）for 9 weeks.The rats in each group were given corresponding doses of drugs by gavage,while the control group and model group were given the same amount of normal saline for 28 days.The levels of24-hour urinary protein（UTP）,serum creatinine（SCR）,urea nitrogen（BUN）and serum albumin（ALB）were detected.The levels of tumor necrosis factor-α（TNF-α）,interleukin-1β（IL-1β）and interleukin-6（IL-6）in serum were detected by enzyme-linked immunosorbent assay（ELISA）,hematoxylin eosin staining（HE）,immunofluorescence and transmission electron microscopy were used to observe the renal pathological changes,the expression of IL-6,IκB kinaseβ（IKKβ）,phosphorylated IκB kinaseβ（p-Ikkβ）,NF-κB inhibitor proteinα（IκBα）,phosphorylated NF-κB inhibitor proteinα（p-IκBα）,NF-κB p65 and phosphorylated NF-κB p65 were detected by immunohistochemistry（IHC）,real-time PCR（RT-PCR）and Western blot.Results:Compared with the control group,the level of UTP in the model group was significantly increased（P<0.01）,mesangial cells proliferated,mesangial matrix increased,mesangial thickening,electron dense matter deposition increased,and a large amount of IgA was deposited in the mesangial area and showed irregular particle and mass distribution;the levels of TNF-α,IL-1β,IL-6 in serum were significantly increased（P<0.01）;the levels of IL-6,IKKβ,p-IKKβin renal tissue were significantly increased（P<0.01）The expression of NF-κB p65,p-p65,IκBα,p-IκBαwere significantly increased（P<0.01）.Compared with the model group,the levels of UTP were significantly decreased（P<0.05,P<0.01）,and the renal tissue injury was alleviated;the levels of TNF-α,IL-1β,IL-6 in serum were significantly decreased（P<0.05,P<0.01）;the expressions of IL-6,IKKβ,p-IKKβ,IκBα,p-IκBα,NF-κB p65,p-p65 in renal tissue were significantly decreased（P<0.05,P<0.01）.There was no significant difference in serum creatinine,urea nitrogen and serum albumin among the groups（P>0.05）.Conclusion:1 The IgAN rat model was established by the combined method of BSA+CCl₄+LPS.At the end of 9 weeks,the rats showed significant increase in urinary protein and renal pathological damage,which showed that the IgAN rat model was successfully replicated.2 Wuwei Xiaoduyin has a benign intervention effect on renal pathological injury of IgAN rats,which can reduce urinary protein and renal pathological injury of IgAN rats.3 NF-κB is involved in the development of proteinuria and kidney injury in IgAN rats.4 Wuwei Xiaoduyin can obviously inhibit high expression of NF-κB signaling pathway related proteins in renal tissue,it can also inhibit the excessive secretion of related inflammatory factors in serum and renal tissue,and then play a role in inhibiting the inflammatory reaction.Part two Effect of Wuwei Xiaoduyin containing serum on cell proliferation,extracellular matrix and NF-κB signaling pathway of rat mesangial cellsObjective: To observe the effect of Wuwei Xiaoduyin containing serum on the proliferation of rat mesangial cells,extracellular matrix and NF-κB signaling pathway induced by lipopolysaccharide.Methods: rat mesangial cell lines were cultured in vitro and intervened with control group,lipopolysaccharide group,benazepril group,Wuwei Xiaoduyin low dose group and Wuwei Xiaoduyin high dose group for 24 hours;Cell proliferation was measured by MTT.The expression of IL-1β,ICAM-1,FN and LN in the supernatant of mesangial cells was detected by ELISA.IL-1β,FN,NF-κB p65 m RNA expression was detected by RT-PCR.The expression of p-IKKβ,p-IκBα,NF-κB p65 in mesangial cells was detected by Western blot.Results: compared with the control group,the expression of IL-1β,ICAM-1,FN,LN,p-IKKβ,p-IκBα,NF-κB p65 in LPS group was significantly increased（P<0.01）.Compared with LPS group,the expression of IL-1β,ICAM-1,FN,LN,p-IKKβ,p-IκBα,NF-κB p65 in the treatment group was significantly decreased（P<0.05,P <0.01）.Conclusion: 1 The inflammation model of rat mesangial cells was established by lipopolysaccharide stimulation of rat mesangial cells.After modeling,mesangial cells showed obvious cell proliferation and inflammatory damage,which showed that the inflammation model of mesangial cells was successfully replicated.2 Wuwei xiaoduyin medicated serum has obvious intervention effect on inflammatory injury of rat mesangial cells induced by lipopolysaccharide,which can reduce the proliferation of mesangial cells,the excessive release of inflammatory cytokines and the excessive secretion of extracellular matrix.3 Wuwei xiaoduyin medicated serum can obviously inhibit the overexpression of NF-κB signaling pathway related proteins inhibited the phosphorylation of the activation of NF-κB in mesangial cells may be one of the main mechanisms of the prescription in reducing proteinuria and renal pathological injury.Conclusion: 1 The IgAN rat model was established by the combined method of BSA +CCl4 +LPS.At the end of 9 weeks,the rats showed significant increase in urinary protein and renal pathological damage,indicating that the IgAN rat model was successfully replicated.The inflammatory model of mesangial cells was established by lipopolysaccharide stimulation of rat glomerular mesangial cells.After modeling,mesangial cells showed obvious cell proliferation and inflammatory damage,indicating that the inflammatory model of mesangial cells was successfully replicated.2 Wuwei Xiaoduyin has a benign intervention effect on renal pathological injury of IgAN rats.It can reduce the urinary protein of IgAN rats,alleviate the renal pathological injury,and inhibit the excessive release of inflammatory factors.3 Wuwei Xiaoduyin can improve the inflammatory injury of rat mesangial cells induced by lipopolysaccharide,reduce the proliferation of mesangial cells,reduce the excessive release of inflammatory cytokines,and reduce the excessive secretion of extracellular matrix.4 According to in vivo and in vitro experiments,Wuwei Xiaoduyin can significantly inhibit the overexpression of NF-κB signaling pathway related proteins and inhibit the expression of NF-κB.This may be one of the main mechanisms of reducing proteinuria and renal pathological damage.