Study On Kaempferol Ameliorates Liver Fibrosis By Targeting Macrophage Polarization

Objective:Liver fibrosis is a general pathological process of various chronic hepatic diseases,which is characterized by the activation of hepatic stellate cells（HSCs）and excessive accumulation of collagen.The previous studies have found that the important roles of macrophages with different functions and states in liver fibrosis.Literature survey found that astragalus membranaceus and paeony appeared frequently in several anti-liver fibrosis Chinese herbal formulas,which together contained various flavonoids.Among them,Kaempferol（KA）has been extensively studied in the treatment of liver diseases,including liver fibrosis.The purpose of this study is to investigate the effect of kaempferol on macrophage polarization during liver fibrosis and to determine whether kaempferol playing an anti-fibrosis role by mediating the polarization of macrophages.Methods:Male C57BL/6 mice of appropriate age were selected and randomly divided into the four groups:Oil group,CCl₄ group,KA-Low-dose group（50mg/kg）and KA-High-dose group（100mg/kg）.Mice were intraperitoneally injected with CCl₄ twice a week to establish liver fibrosis model.During the same period,mice in kaempferol treatment group were intraperitoneally injected with the corresponding dose of kaempferol（once a day）,and Oil group and CCl₄group were given the same amount of solvent.Serum and liver tissues of mice were collected 6 weeks later.Recording liver weight ratio,liver function was measured by examination of serum alanine aminotransferase（ALT）activity.The paraffin-embedded liver tissues of each group were used to detect the difference of liver pathological morphology by H&E and Sirius Red staining.The protein and gene levels ofα-SMA,Collagen I,i NOS,TNF-α,CD206 and Arg-1 in liver were detected.Immunohistochemistry（IHC）and immunofluorescence（IF）were used to observe the degree of liver fibrosis and the change of polarization of macrophages.THP-1 cells and LX-2 cells were selected as experimental objects in vitro.LPS+IFN-γand IL-4+IL-13 were used as inducers to construct macrophage polarization models,respectively.The THP-1 and LX-2 cells were treated with kaempferol at 5/10/20μM doses to observe the pharmacodynamic effects of kaempferol on THP-1 polarization and LX-2activation.Results:1.Compared with Oil group,the liver surfaces of mice in CCl₄group were rough and full of folds,liver/weight ratio and serum ALT contents were significantly increased.However,the liver surfaces of mice in the low and high doses of kaempferol tended to be smooth,the increase of liver/weight ratio and ALT were inhibited in both KA groups.2.According to the results of liver HE,Sirius Red and immunohistochemical staining,liver parenchymal cell necrosis,F4/80⁺macrophage infiltration and collagen fiber accumulation were observed in the portal area of CCl₄ group.Kaempferol at both low and high doses can reduce the expression and accumulation of collagen and the recruitment of F4/80⁺macrophages.3.Kaempferol inhibited the m RNA and protein expression ofα-SMA and Collagen I in liver of mice with hepatic fibrosis at both low and high doses.4.Kaempferol significantly inhibited the expression of i NOS,TNF-α,CD206 and Arg-1 in M1 and M2 macrophages of fibrotic mice.5.At the cellular level,compared with M1 and M2 model groups,kaempferol administration group could down-regulate the expression levels of i NOS,TNF-αmarkers of M1 polarization and CD206 and Arg-1,markers of M2polarization.6.Kaempferol treatment not only directly inhibited the activation of LX-2,but also blocked the activation of LX-2 cells in conditioned medium of M2 macrophages.Conclusions:Kaempferol can effectively improve the liver injury and the liver fibrosis in CCl₄-induced mice.Kaempferol could disturb the activation of HSCs by suppressing the polarization of M2 macrophage to achieve anti-fibrosis effect.