Kidney Flaccidity Compound Ameliorates Kidney Fibrosis In UUO Rats By Influencing Macrophage Polarization Via TLR4/NF-?B Signaling Pathway

Objective: In this study,we observed the effects of Chinese medicine Kidney Flaccidity Compound on pathological damage and fibrosis in rats induced with unilateral ureteral obstruction(UUO),which is a means to establish the model of renal inflammation and fibrosis.The purpose of this study was to explore the effects and mechanisms of Kidney Flaccidity Compound on M1/M2 macrophage polarization and inflammatory factors expression,so as to provide scientific basis for the prevention and treatment of chronic kidney disease with Chinese herbal medicine.Methods:After one week of adaptive feeding,twenty-five male SD rats were randomly divided into five groups,namely sham group,UUO model group,UUO group treated with low-dose Kidney Flaccidity Compound,UUO group treated with high-dose Kidney Flaccidity Compound,UUO group treated with irbesartan.There are 5 rats in each group.In this experiment,five rats in each group were respectively pretreated with saline,low-dose and high-dose of Kidney Flaccidity Compound and irbesartan by means of intragastric administration for 3 days before the establishment of the model.After modeling,each group continued to be treated for 7 days.After treatment,obstructed kidney tissues were taken and the morphological changes of kidney were observed by HE staining.To observe the change of renal fibrosis,renal interstitial collagen fibers,Col III expression and a-SMA expression in kidney tissues were detected by Sirius red staining,immunohistochemical staining and Western Blot,respectively.Flow cytometry and RT-PCR were used to detect the expression of M1 macrophage markers i NOS and M2 macrophage markers MRC1 to understand macrophage polarization in renal tissues.The expression of inflammatory mediators TNF-? and IL-1? in kidney tissues were detected by immunohistochemical staining and RT-PCR,and the expression of TLR4/NF-k B pathway related protein in kidney tissues was analyzed by Western Blot.Results: 1.The change of pathology and renal fibrosis: Compared with sham-operated rats,HE staining of kidney tissue in UUO rats showed that renal tubules were dilated and interstitial inflammatory cells were infiltrated.Sirius red staining showed a large amount of red collagen fibers deposited in renal interstitial tissues in UUO rats(P < 0.01).Compared with UUO rats,after treatment with Kidney Flaccidity Compound and Irbesartan,the degree of tubular dilatation and interstitial inflammatory cell infiltration in rats were significantly reduced and the area of red collagen fibers in renal interstitium were significantly reduced(P < 0.01).Similarly,Kidney Flaccidity Compound decreased the expression of Col III(P < 0.01)and ?-SMA(P < 0.01)in renal significantly and the effect of high dose group was more obvious.2.The macrophage polarization: Compared with sham-operated rats,the percentage of M1 macrophages(F4/80+i NOS+)increased significantly in UUO rats(P < 0.01),while the percentage of M2 macrophages(F4/80+CD206+)decreased(P < 0.01),the macrophages polarized to M1 type.However,after treatment with Kidney Flaccidity Compound and Irbesartan,the percentage of M1 macrophages and the expression of i NOS gene(surface marker of M1 macrophages)were decreased significantly(P < 0.01),and the percentage of M2 macrophages and MRC1 gene expression were(surface marker of M2 macrophages)increased significantly(P < 0.01),especially inthe high dose of Kidney Flaccidity Compound group(P < 0.01).3.Expression of inflammatory mediators: Compared with sham-operated rats,the average optical density of TNF-? and IL-1? in kidney tissues of UUO rats increased significantly(P < 0.001),however the expression of inflammatory mediators decreased significantly after treatment with Kidney Flaccidity Compound and Irbesartan(P < 0.001).The results of RT-PCR in detecting gene expression of inflammatory mediators were consistent with the immunohistochemical analysis.4.The change of TLR4/NF-?B signaling pathway : Compared with sham operation rats,the expression levels of proteins : TLR4,Ik B?,P-Ik B?,P65 and P-P65 in TLR4/NF-?B pathway of UUO rats kidney tissues increased significantly(P < 0.005),while the treatment of Kidney Flaccidity compound and Irbesartan decreased the expression levels of TLR4/NF-?B pathway-related proteins significantly(P < 0.005).Conclusion: 1.Chinese medicine Kidney Flaccidity Compound can reduce the degree of pathological injury of renal tissue and the deposition of interstitial collagen fibers to alleviate renal fibrosis and protect renal function in UUO rats.Dose effect exists in the effect of Kidney Flaccidity Compound.2.Kidney Flaccidity Compound can down-regulate the expression of M1 macrophages,up-regulate the expression of M2 macrophages,promote the polarization of pro-inflammatory M1 macrophages to anti-inflammatory M2 macrophages,thereby reducing the expression of inflammatory mediators;3.Kidney Flaccidity Compound can down-regulate the expression of TLR4/NF-?B pathway.Kidney Flaccidity Compound may regulate macrophage polarization via TLR4/NF-?B pathway,thus ameliorating renal fibrosis and delaying the progress of chronic kidney disease.