| Objective:To explore the mechanism of paeonol against alcohol liver disease(ALD)inflammation injury based on the intestinal fungal flora and its metabolite b-glucanmediated Dectin-1/IL-1b signal pathway.Methods: In this study,90 male C57BL/6 mice were randomly divided into normal group,ALD model group,positive drug group,paeonol high,medium and low dose groups.Lieber-De Carli alcohol liquid feed free diet was used to establish murine model of alcohol liver disease.The levels of TG,TC,ALT,AST,?-GT were detected by serum biochemistry,and liver HE staining and oil red O staining were used to verify the model.Intestinal fungal flora of mice was detected by ITS high-throughput sequencing,and the changes of intestinal fungal species were analyzed.The pathological changes of ileum were observed by HE staining,and the expression of ZO-1,Claudin-1 protein and gene were detected by immunohistochemistry,Western blotting,and qRT-PCR.The serum levels of b-glucan,caspase-1,IL-1b and the liver tissue homogenate levels of NLRP3 were detected by ELISA.The expression of liver caspase-1 and NLRP3 was detected by immunohistochemistry.The expression of liver macrophage F4/80 and IL-1b was detected by immunofluorescence.The expression of Dectin-1,Syk,NLRP3,ASC,procaspase-1,caspase-1,pro-IL-1b and IL-1b protein and gene were detected by Western blotting and qRT-PCR.In addition,we also established b-glucan-induced macrophage inflammatory model in vitro to further verify the mechanism of paeonol against ALD inflammation injury.We further validated the anti-ALD mechanism of paeonol by an in vitro b-glucan-induced macrophage inflammation model.The cell activity was measured by MTT assay.The morphological changes of cells were observed by inverted microscope.The expression of macrophage surface marker F4/80 was evaluated by flow cytometry.The expression of macrophage NLRP3 was detected by immunofluorescence.The expression of Dectin-1,Syk,NLRP3,ASC,pro-caspase-1,and caspase-1 protein and gene in macrophages were detected by Western blotting,and qRT-PCR.The cell supernatant levels of IL-1? and IL-18 were detected by ELISA assay.Results: The HE staining and oil red O staining showed that a large number of fat droplets appeared in the liver of the ALD model group.The serum levels of TG,TC,ALT,AST,and ?-GT were significantly increased after alcohol feeding compared with the control group.After paeonol treatment,the liver fat droplets were reduced,and the serum levels of TG,TC,ALT,AST,and ?-GT were significantly decreased compared with the ALD model group.The ITS sequencing results showed that the intestinal fungal species and abundance in the ALD model group were significantly increased compared with those of the control group.The intestinal fungal species and abundance were significantly reduced after paeonol treatment compared with the ALD model group,especially,at levels of Saccharomycetales,Debaryomycetaceae,Candida,and fungi?sp.The ileum HE staining showed that the intestinal mucosa of ALD model group was seriously damaged,and the damaged intestinal mucosa was improved after paeonol treatment.The immunohistochemistry,Western blotting and qRT-PCR results showed that the expressions of ZO-1?Claudin-1 protein and gene in ileum of ALD model group were significantly decreased compared with the control group,and the expression were significantly increased after paeonol treatment compared with the ALD model group.The ELISA results showed that the serum levels of b-glucan,caspase-1,IL-1b,and the liver tissue homogenate level of NLRP3 in the ALD model group were significantly increased compared with those in the control group,and the expression were significantly decreased after paeonol treatment compared with the ALD model group.The immunohistochemical results showed that the expression of liver caspase-1 and NLRP3 protein in the ALD model group were significantly increased compared with the control group,and the expression were significantly decreased after paeonol treatment compared with the ALD model group.The immunofluorescence results showed that the expression of liver macrophages F4/80 and IL-1b in the ALD model group were significantly increased compared with the control group,and the expression were significantly decreased after paeonol treatment compared with the ALD model group.The Western blotting and qRTPCR results showed that the levels of Dectin-1,Syk,NLRP3,ASC,pro-caspase-1,caspase-1,pro-IL-1b,IL-1b protein and gene in the ALD model group were significantly increased compared with the control group,and the expression were significantly decreased after paeonol treatment compared with the ALD model group.In vitro cell experiments,the MTT results showed that the cell activity of the b-glucan-induced group was significantly decreased compared with the control group,and the cell activity was significantly decreased after paeonol treatment compared with the b-glucan-induced group.The morphological results showed that the macrophages in the b-glucan-induced group had obvious cell differentiation,while the paeonol-treated group had less cell differentiation.The flow cytometry and immunofluorescence results showed that the expression of macrophages F4/80 and NLRP3 in the b-glucan-induced group were significantly increased compared with the control group,and the expression were significantly decreased after paeonol treatment compared with the b-glucan-induced group.The Western blotting and qRT-PCR results showed that the expressions of macrophages Dectin-1,Syk,NLRP3,ASC,pro-caspase-1,caspase-1 protein and gene in the b-glucan-induced group were significantly increased compared with the control group,and the expression were significantly decreased after paeonol treatment compared with the b-glucan-induced group.The ELISA results showed that the levels of IL-1b and IL-18 in the b-glucan-induced group were significantly increased compared with the control group,and the levels were significantly decreased after paeonol treatment compared with the b-glucan-induced group.Conclusion: In vivo experiments has shown that paeonol could inhibit the b-glucanmediated Dectin-1/IL-1b inflammation signal pathway by reducing the abundance of intestinal Candida albicans,and ultimately play a role in anti-alcoholic liver disease inflammatory injury.In vitro experiments further verified that paeonol played a role in anti-alcoholic liver disease inflammatory injury by regulating key proteins of Dectin-1/IL-1b signaling pathway related to intestinal fungal flora. |
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