TLR3 Ligand Induced The Senescence Of Hepatic Stellate Cell And The Mechanism Of Activated NK Cell Immune Clearance

BackgroundLiver fibrosis is a common scarring reaction of different chronic liver injuries,such as viral hepatitis(hepatitis B and C),alcoholic or non-alcoholic steatohepatitis,autoimmune hepatitis and cholestatic liver disease.The activation of hepatic stellate cells is the key to liver fibrosis development.After liver injury,hepatic stellate cells activate and differentiate into myofibroblasts,which are the main source of extracellular matrix(ECM)in the process of liver fibrosis.Liver fibrosis is a precursor to liver cirrhosis and liver cancer.At present,liver fibrosis is still a global health problem due to the lack of effective treatments.The elimination of activated hepatic stellate cells can limit the progression of liver fibrosis.In addition,senescent hepatic stellate cells are more sensitive to natural killer(NK)cells cytotoxicity,thereby inhibiting the progression of liver fibrosis.It has been reported that polyinosinic acid cytidylic acid(poly I:C)and Interleukin-18(IL-18)can synergistically activate NK cells,which have an inhibitory effect on hepatic stellate cells.However,it remains unclear whether poly I:C has an effect on hepatic stellate cells in addition to activating NK cells.In addition,it was reported in other study that poly I:C up-regulated the expression of Toll-like receptor 3(TLR3)in human umbilical cord blood mesenchymal stem cells(UCB-MSCs)to induce UCB-MSCs senescence.Therefore,we hypothesized that poly I:C can induce the senescence of hepatic stellate cells,which is more sensitive to NK cells cytotoxicity to promote the regression of liver fibrosis.Methods1.In order to verify the effect of poly I:C on TLR3 expression of hepatic stellate cells,we treated primary hepatic stellate cells with poly I:C in vitro,and detected the expression of TLR3 by q PCR and flow cytometry.2.In order to test whether the senescence of hepatic stellate cells was induced by poly I:C,we treated primary hepatic stellate cells and LX2 cells with poly I:C in vitro,and detected the expression of senescence indicators p16,p21 and HMGA1 by western blot and q PCR.At the same time,senescence-associated(SA)β-galactosidase staining was used to detect cell senescence.3.In order to investigate the expression of NKG2 D ligand induced by poly I:C,we treated primary hepatic stellate cells and LX2 cells with poly I:C in vitro,and detected the expression of MICA/MICB and ULBP2 by q PCR and flow cytometry.4.In order to study the killing ability of NK cells after poly I:C treatment of hepatic stellate cells,we used flow cytometry to detect the expression of CD107 a and apoptosis.Results1.Poly I:C up-regulated the expression of TLR3 in hepatic stellate cells.2.Poly I:C up-regulated the expression of senescence indicators such as p16,p21 and HMGA1.The number of SA-β-gal positive cells also increased in hepatic stellate cells.3.Poly I:C up-regulated the expression of NKG2 D ligand MICA/MICB and ULBP2 in hepatic stellate cells.4.Poly I:C treated hepatic stellate cells up-regulated CD107 a expression in NK cells and apoptosis in HSCs.ConclusionsPoly I:C induces cellular senescence of HSCs by upregulating the expression of TLR3 in HSCs,and triggers NK cell immunosurveillance,suggesting that the role of poly I:C on HSCs senescence may promote fibrosis regression.