CD24 Regulates Carbon Tetrachloride-induced Murine Liver Fibrosis And Its Molecular Mechanisms

Object and contents:The subject was performed to investigate the regulation of CD24 on CCl4-induced murine liver fibrosis and the possible molecular mechanisms.The work includes four parts: One.Establish the mouse model of liver fibrosis induced by CCl4 and observe CD24 molecular changes in liver fibrosis tissue.Two.Explore the mouse liver fibrosis after CD24 knockout.Three.Detect CD24 molecular change in the different immune cells in WT mice liver fibrosis and observe CD24 molecules on regulation of macrophages in liver fibrosis.Four.Analyze the possible molecular mechanisms about CD24 molecules on regulation of macrophages subtypes in mouse liver fibrosis.Methods and results:The project is divided into four parts:Part One: Established the mouse model of liver fibrosis induced by CCl4 and observed CD24 molecular changes in liver fibrosis tissue,including the following two parts.1.Established the mouse model of liver fibrosis:WT mice with intraperitoneal injection of CCl4 after 6 weeks,H&E staining and serum ALT level were detected to observe mice liver tissue injury.Sirius red staining and detection of α-SMA,Col1a1 mRNA by Real-time PCR were used to assess liver fibrosis.Results show that WT mice with intraperitoneal injection of CCl4 after 6 weeks,H&E staining showed inflammatory cells infiltration increasing.Serum ALT level increased.Sirius Red staining revealed thick collagen on liver fibrosis tissue.Likewise,α-SMA and Col1a1 mRNA level increased.We built successfully liver fibrosis model in mice.2.Detected CD24 expression in liver fibrosis tissue:The expression of CD24 mRNA in liver tissue was detected by Real-time PCR.The expression of α-SMA and CD24 protein level in liver tissue were detected by Western Blot.Results show that CD24 mRNA expression and protein expression of CD24 were upregulated in CCl4-induced mice liver fibrosis.Part Two: Explore the regulation of CD24 molecules on liver fibrosis in mice,including the following two parts experiment.1.The change of WT and CD24-/-mice liver fibrosis by liver tissue pathology evaluation: Wild type mice(WT)and CD24 knockout mice(CD24-/-)in the background of C57BL/6 were treated with CCl4 by intraperitoneal injection.H&E staining and serum ALT level were detected to observe mice liver tissue injury.Sirius red staining was used to assess collagen fiber.Results show that H&E staining showed that compared to WT mice,CD24-/-mice treated with CCl4 presented more obvious liver inflammation.Sirius Red staining of paraffin-embadded liver sections revealed thicker bands of collagen on CD24-/-mice compared with WT mice.ALT showed the same result.Wild type mice(WT)and CD24 knockout mice(CD24-/-)in the background of C57BL/6 were treated with CCl4 by intraperitoneal injection.The liver injury and liver fibrosis of CD24-/-mouse was more serious.2.The change of WT and CD24-/-mice liver fibrosis by gene expression and protein level assessment: Real-time PCR detected the expression of α-SMA,Col1a1.The expression of α-SMA in liver tissue was detected by Western-blot.Results show that α-SMA and Col1a1 in CD24-/-mice were significantly higher than that in WT mice.Similarly,expression of α-SMA by Western blot detection was higher than that of WT mice.From the gene expression and protein level: the mice liver fibrosis index increased after CD24 gene knockout compared with WT mouse.Part Three: Detect CD24 molecular change in the different immune cells in WT mice liver fibrosis and observe CD24 molecules on regulation of macrophages in liver fibrosis,including the following three parts experiment.1.CD24 molecular changed in the different immune cells by flow cytometry detection in WT mice liver fibrosis.Results show that CD24 molecules on the surface of macrophage increased obviously in WT mice liver fibrosis,compared to other immune cells.2.Flow cytometry analysis was applied to observe changes of intrahepatic macrophages in WT and CD24-/-liver fibrosis.Results show that Flow cytometry analysis showed that after treatment of micewith CCl4,mice hepatic macrophages significantly increased in CD24-/-mice compared with in WT mice.3.We evaluated the degree of liver fibrosis in mice after using liposomal clodronate to deplete macrophage.Results show that after macrophage depletion,Sirius Red staining showed that collagen fibers reduced and expression of Col1a1 by Real-time PCR detection decreased compared with liver fibrosis in mice.Macrophage depletion attenuated liver fibrosis.Part Four: Analyze the possible molecular mechanisms about regulation of CD24 on CCl4-induced murine liver fibrosis,including the following two parts experiment.1.TGF-β1 mRNA in liver tissue was detected by Real-time PCR in the liver fibrosis.Results show that Real-time PCR analysis confirmed significantly higher expression of TGF-β1 in the liver tissue of CD24-/-mice compared with WT mice.2.Detection of expression and secretion of TGF-β 1 in different macrophage subtypes: WT and CD24-/-macrophages was stimulated by LPS and IL-4 to type of M1 and M2 macrophages.Expression of TGF-β1 in M1 and M2 macrophages was detected by ELISA and Real-time PCR.Results show that Secretion of TGF-β1 in M2 macrophages supernatant of CD24-/-exceeded that of the WT.By Contrast,Secretion of TGF-β1 did not change obviously in M1 macrophages supernatant.Real-time PCR analysis also got the same results.Conclusion:Expression of CD24 is upregulated in mouse liver fibrosis.And we believe CD24 knockout increase mouse liver fibrosis following CCl4-treatment.The possible mechanisms regulate intrahepatic macrophages,reducing the secretion of TGF-β1 to down-regulation of activation the Hepatic stellate cells,which contribute to inhibition of liver fibrosis.