Interleukin-26 Promotes The Proliferation And Activation Of Hepatic Stellate Cells To Exacerbate Liver Fibrosis Via The TGF-?1/Smad2 Signaling Pathway

Objective:Liver fibrosis(LF)is an abnormal repair process of liver stimulation to injury,which is characterized by the activation of hepatic stellate cells(HSC)and the deposition of extracellular matrix(ECM).Interleukin(IL)is a type of cytokine with a wide range of functions.IL plays an important role in information transmission,immune cell activation and regulation,T and B lymphocyte activation,proliferation,differentiation,and inflammation.More and more evidences have shown that IL participates in the pathological process of LF by regulating the activation of HSC cells,including IL-1?,IL-6,IL-7 and IL-9.IL-26 belongs to the IL-10 cytokine superfamily and is closely related to the occurrence and development of rheumatoid arthritis and inflammatory bowel disease.However,the biological function of IL-26 in LF remains unclear.The purpose of this study was to examine the effect of IL-26 on LF,to analyze its effects on HSC proliferation and activation,and to identify its possible molecular mechanism.Methods:A HBV-Tg transgenic mouse model was constructed,and 0.25 ?g/g of human IL-26 recombinant protein was injected into the mice via tail vein every day for 2 consecutive weeks.After 8-10 months of feeding,liver tissues were obtained and the condition of LF in liver tissues was observed by hematoxylin-eosin(HE)and Masson staining.ELISA experimnet was used to detect the protein expression levels of TNF-?(tumor necrosis factor-?),IL-6,IL-10,MMP-9(matrix metallopeptidase-9)and ?-SMA(?-smooth muscle actin)in mouse liver tissues.HSC cells were isolated and successfully cultured in vitro.HSC cells were cultured in serum-free medium for 24 h by starvation therapy,and then stimulated with human IL-26 recombinant protein at concentrations of 0,50,100,and 200 pg/ml for 12 h,respectively.Cell proliferation was measured using the Cell Counting Kit-8(CCK-8)assay,cell cycle was detected by flow cytometry,and cell apoptosis was assessed by Annexin V-FITC/PI dual staining.The m RNA expression levels of CASP3(caspase 3),BAX(Bcl-2 associated X protein),TGF-?1(transforming growth factor-?1),and Smad2(SMAD family member 2)in HSC cells were analyzed by real-time quantitative PCR.The protein expression levels of the above genes and phosphorylated Smad2(p-Smad2)protein level were detected by Western blot.The protein expression levels of TNF-?,IL-6,IL-10,MMP-9 and ?-SMA in HSC cells were detected by ELISA.The Student's t test or analysis of variance was applied for statistical analysis.Results:Compared with the control group,injection of human IL-26 recombinant protein promoted LF in HBV-Tg transgenic mice,and significantly increased the protein expression levels of TNF-?,IL-6,and IL-10,MMP-9,and ?-SMA in liver tissues(P<0.05).Treatment with IL-26 significantly increased the proliferation of HSCs in a dose-dependent manner(P<0.05).IL-26 stimulation resulted in an increase of HSC in S phase and a decrease of HSCs in G0/G1 phase in a dose-dependent manner(P<0.05).There was no significant difference in percentages of G2/M phase in the different concentrations of IL-26 stimulation groups(P>0.05).IL-26 treatment significantly decreased the apoptosis of HSCs in a dose-dependent manner(P<0.05).IL-26 stimulation markedly downregulated the m RNA expression levels of CASP3 and BAX in a dose-dependent manner(P<0.05).The protein levels of CASP3,cleaved CASP3,and BAX in HSCs were decreased in a dose-dependent manner after IL-26 treatment(P<0.05).IL-26 significantly induced HSC activation in a dose-dependent manner,and its mechanism was mainly through increasing m RNA levels and protein expression levels of IL-6,IL-10,TNF-a,MMP-9 and ?-SMA(P<0.05).In addition,IL-26 up-regulated the protein expression levels of TGF-?1,Smad2 and p-Smad2 in HSC cells in a dose-dependent manner(P<0.05).Conclusions:Injection of human IL-26 recombinant protein via tail vein promotes HBV-Tg transgenic mouse LF and significantly increases the protein expression levels of TNF-?,IL-6,IL-10,MMP-9 and ?-SMA in liver tissues.IL-26 increases the proliferation and activation of HSC cells in a dose-dependent manner to promote LF.IL-26 promotes the proliferation and activation of hepatic stellate cells by regulating the TGF-?1/Smad2 signaling pathway to promote HBV-Tg transgenic mouse LF.This study reveals an important role of IL-26 in LF and will provide new ideas and potential targets for the treatment of LF.