| Liver fibrosis and cirrhosis are global health problem.About 300 million people suffer from viral hepatitis,non-alcoholic fatty liver disease,alcoholic hepatitis and other chronic liver diseases in China.If not treated in time,liver fibrosis and cirrhosis will progress to liver cancer.There are no effective means to treat or reverse liver fibrosis currently.The number of patients with liver fibrosis is gradually increasing,which urgently requires the development of therapy based on pathogenesis.Triggering receptor expressed on myeloid cells-1(TREM-1)is a member of the immunoglobulin superfamily and has two main forms.A free form that lacks a transmembrane domain is called soluble TREM-1(sTREM-1),a secreted protein molecule.It was found that sTREM-1 expression level was significantly elevated in patients with hepatocellular carcinoma and co-culture of hepatic stellate cells(HSC)and hepatoma carcinoma cells(HCC)also promoted sTREM-1 secretion by activated HSC.Since sTREM-1 lacks a transmembrane domain,we speculated that sTREM-1,as an inflammatory factor,can bind to a membrane protein receptor of HSC to activate its downstream signaling pathway and promote the activation of HSC,thereby promoting liver fibrosis.In this study,recombinant sTREM-1 protein was synthesized to stimulate human HSC line LX-2 and a mouse liver fibrosis model induced by carbon tetrachloride(CCl4)combined with sTREM-1 stimulation was established to observe the effect of sTREM-1 on HSC activation and liver fibrosis in vivo and in vitro.Then the membrane receptors interacting with sTREM-1 were screened by pull-down and mass spectrometry.Finally,the downstream signaling pathways were further searched.This topic mainly includes the following three parts:Part Ⅰ Soluble triggering receptor expressed on myeloid cells-1 can promote liver fibrosis and activate hepatic stellate cellsObjective:To investigate the effect of sTREM-1 on CCl4-induced liver fibrosis and HSC activation.Methods:Firstly,the mouse model of liver fibrosis induced by CCl4 was established and sTREM-1 was injected into tail vein.Hematoxylin-eosin(H&E)staining and Sirius red staining were used to observe the necrosis of liver cells and collagen deposition.Immunohistochemistry staining(IHC)and Western blot analysis were used to detect the expression of(α-smooth muscle actin,α-SMA)and Collagen Ⅰ in liver tissues of mice,meanwhile the serum transaminases of mice were detected.Secondly,LX-2 cells were stimulated with recombinant sTREM-1 protein.The protein expression levels of α-SMA and Collagen Ⅰ were detected by Western blot.The cell migration ability was detected by scratch test.Cell proliferation was detected by cell counting kit-8(CCK-8)assay.Cell cycle changes were detected by flow cytometry.Results:H&E and Sirius red Staining showed that sTREM-1 aggravated liver cell necrosis and collagen deposition in CCl4-induced liver fibrosis mouse model.IHC and Western blot showed that sTREM-1 further promote up-regulation of collagen I and α-SMA expression in CCl4-induced liver fibrosis mice(P<0.05).Meanwhile,serum glutamic-oxalacetic transaminase and alanine transaminase were further increased in mice with CCl4-induced liver fibrosis after sTREM-1 injection(P<0.05).Western blot analysis showed that protein expressions of a-SMA and Collagen Ⅰ were increased significantly after sTREM-1 was applied to stimulate LX-2 cells in a time and concentration dependent manner(P<0.05).Scratch test showed that the scratch healing area of LX-2 cells in sTREM-1 group was significantly larger than that in the control group.CCK-8 assay showed that the cell viability of sTREM-1 group was significantly higher than that of control group(P<0.05).Flow cytometry showed that sTREM-1 decreased the proportion of G1 cells and increased the proportion of S cells(P<0.05).Conclusion:sTREM-1 can aggravate liver cell necrosis,collagen deposition and up-regulation of marker protein of liver fibrosis in mice induced by CCl4.sTREM-1 can promote LX-2 activation,migration and proliferation.Part Ⅱ Soluble triggering receptor expressed on myeloid cells-1 promotes hepatic stellate cell activation and liver fibrosis by binding membrane protein ring directed receptor 2Objective:To screen and verify the membrane protein receptor that interact with sTREM-1.Methods:Firstly,the localization of sTREM-1 protein in LX-2 cells was observed by immunofluorescence method.Secondly,the membrane protein bound to sTREM-1 were screened by pull-down assay combined with mass spectrometry.Finally,immunofluorescence and pull-down assay were used to verify the binding of sTREM-1 to the target receptor.Results:Immunofluorescence assay showed that sTREM-1 was enriched in the cell membrane of LX-2 cells after stimulation.By pull-down combined with mass spectrometry and literature review,the roundabout guidance receptor 2(Robo2)was screened as a possible receptor for sTREM-1.Immunofluorescence confirmed that sTREM-1 and Robo2 proteins co-located in cell membrane.Pull-down results showed that sTREM-1 could bind to Robo2 protein.Conclusion:sTREM-1 binds to cell membrane protein receptor Robo2.Part Ⅲ Soluble triggering receptor expressed on myeloid cells-1 promotes hepatic stellate cell activation by activating Smad2/3 and PI3K/Akt signaling pathwaysObjective:To screen and verify the downstream signaling pathway activated by sTREM-1 and Robo2.Methods:Firstly,LX-2 cells infected with lentivirus vector and stably knocked down Robo2 were screened.Western blot was used to detect key proteins and their phosphorylation levels in PI3K/Akt and Smad2/3 signaling pathway.Secondly,PI3K/Akt and Smad2/3 signaling pathway inhibitors LY294002 and SIS3 were added respectively,and Western blot was used to detect the activation of downstream signaling pathway and HSC.Results:Western blot analysis showed that after sTREM-1 stimulated LX-2 cells with stable knockdown of Robo2,the expressions of α-SMA and Collagen I were significantly decreased(P<0.05),sTREM-1-induced phosphorylation of Smad2,Smad3,PI3K and Akt decreased(P<0.05).SIS3 and LY294002 stimulation significantly reduced sTREM-1-induced collagen I and α-SMA expression(P<0.05).Conclusion:sTREM-1 binding to Robo2 can activate smad2/3 and PI3K/Akt signaling pathways to promote the activation of hepatic stellate cells... |
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