Objective To investigate the effects of CD24 on CCl4-induced murine liver fibrosis and to analyze the possible molecular mechanism.Methods Wild type (WT) and CD24 knockout (CD24-/-) C57BL/6 mice were treated with CCl4 through intraperitoneal injection.Levels of ALT in serum samples were detected and liver tissues were stained with hematoxylin and eosin (HE) to assess liver tissue injury.Sirius Red staining was used to observe liver fibrosis.Real-time PCR was performed to detect the expression of α-SMA (α-smooth muscle actin), Col1a1 (Collagen, typeⅠ, alpha 1), TGF-β1 (transforming growth factor-β1) and CD24 at mRNA level in liver tissues.Western blot was performed to analyze the expression of α-SMA and Col1a1 at protein level.Flow cytometry analysis was used to detect the macrophages in liver tissues.ELISA was used to detect the expression of TGF-β1 in the culture supernatants of M1 and M2 macrophages.Results The expression of CD24 at both mRNA and protein levels were up-regulated in mice with CCl4-induced liver fibrosis.HE staining showed that liver inflammation in CD24-/-mice was more severe than that in WT mice after treated with CCl4.Sirius Red staining of paraffin-embadded liver sections revealed that compared with WT mice, CD24-/-mice presented with more severe liver fibrosis.Moreover, α-SMA and Col1a1, indicators of liver fibrosis, in CD24-/-mice were significantly higher than those in WT mice.Flow cytometry analysis showed that murine hepatic macrophages significantly increased in CD24-/-mice than in WT mice following CCl4 treatment.Real-time PCR analysis also confirmed that significantly enhanced expression of TGF-β1 at mRNA level in liver tissues was observed in CD24-/-mice than in WT mice.TGF-β1 secreted in the culture supernatant of M2 macrophages derived from CD24-/-mice group was more than that of the WT mice group.No significant difference in TGF-β1 secretion in culture supernatant of M1 macrophages was observed between the two groups.Conclusion Taken together, these data suggest that CD24 plays an important role in attenuating CCl4-induced chronic inflammation and hepatic fibrosis in mice.The mechanism of CD24 in alleviating liver fibrosis might be through regulating intrahepatic macrophages, inhibiting the secretion of TGF-β1 by M2 macrophages and suppressing the activation of hepatic stellate cells.

Objective To investigate the regulatory effects of CD24 on Ly6Chi macrophages in liv-er and its influences on bile duct ligation ( BDL)-induced hepatic fibrosis in mice. Methods Liver fibrosis was induced in wild-type ( WT) and CD24-/- mice by surgical ligation of the biliary duct. Levels of alanine amino transferase ( ALT) in serum were detected and liver sections were stained with haematoxylin and eosin ( H&E) staining to assess the severity of liver injury. Sirius Red staining was used to observe the degree of liver fibrosis. Real-time PCR was performed for detecting the expression of hepatic fibrosis-related markers and TGF-β1 at mRNA level. The percentage of macrophages and the number of TGF-β1-producing macro-phages were measured by flow cytometry. Results BDL-induced liver fibrosis was exacerbated in CD24-/-mice than in WT mice as demonstrated by more serious hyperplasia in bile duct, more inflammatory infiltra-tion at the portal area and higher levels of ALT in serum. Results of Sirius red staining also showed that the liver fibrosis was more severe in CD24-/- mice than in WT mice. Moreover, α-SMA and collagen typeⅠalpha 1 (Col1a1) were significantly upregulated in CD24-/- mice. Flow cytometry analysis revealed that CD24 was highly expressed by hepatic macrophages in BDL-induced WT mice, and the percentages of hepat-ic macrophages were significantly elevated in CD24-/-mice compared with those in WT mice. Further analy-sis revealed that the percentages of Ly6Chi hepatic macrophages in CD24-/- mice were higher than those in WT mice, but there was no significant difference in the percentages of Ly6Clo macrophages. The expression of TGF-β1 at mRNA level was increased in CD24-/-mice as compared with that in WT mice after BDL. Mo-reover, intracellular staining showed that Ly6Chi hepatic macrophages in CD24-/- mice secreted more TGF-β1 than the macrophages in WT mice. Conclusions CD24 might attenuate the BDL-induced liver fibrosis in mice via regulating the percentage of hepatic Ly6Chi macrophages and the secretion of TGF-β1.

Objective: This study was devoted to identifying natural thrombin inhibitors from traditional Chinese medicine (TCM) and evaluating its biological activity in vitro and binding characteristics. Methods: A combination strategy containing molecular docking, thrombin inhibition assay, surface plasmon resonance (SPR) and molecular dynamics simulation were applied to verify the study result. Results: Gallic acid was confirmed as a direct thrombin inhibitor with IC