Protective Effect And Mechanism Of Dendrobium Officinale Polysaccharide On Liver And Intestinal Mucosal Barrier In Rats With CCl₄ Induced Liver Fibrosis

Aims: Dendrobium officinale polysaccharide(DOP)is one of the main effective ingredients of Dendrobium officinale Kimura et Migo.Modern pharmacology shows that DOP has a variety of therapeutic effects,including antioxidant,anti-diabetes,antiinflammatory and other effects.However,the mechanism of its effect on liver fibrosis is not yet clear.Therefore,this article established a CCl4 induced liver fibrosis model to explore the protective effect and mechanism of DOP on liver fibrosis.Methods: DOP was extract by water extraction,alcohol precipitation,repeated freezethaw,microfiltration,ultrafiltration and freeze-drying.A rat model of liver fibrosis was constructed by intraperitoneal injection of CCl4 in vivo,and LPS-induced HSC-T6 cells model,TGF-β-induced HSC-T6 cells model and LPS-induced Caco-2 cells model were also constructed in vitro.The protective effects of DOP against liver fibrosis and its mechanisms were evaluated by histopathological stained sections,serum liver injury and liver fibrosis index assays,immunohistochemistry,horseshoe crab reagents,Western blot,q RT-PCR,immunofluorescence,gene silencing and flow cytometry assays in vitro and in vivo.Results: In this study,the obtained DOP had stable properties and uniform molecular weight,and the serological indexes showed that DOP could significantly reduce the levels of ALT and AST as well as the four liver fibrosis levels in rat serum.In addition,DOP also reduced the expression of α-SMA and Collagen Ⅰ in rat liver,and the above results indicated that DOP has a good anti-fibrotic effect.Next,the mechanism of anti-hepatic fibrosis of DOP was studied in three parts.(1)The result showed that DOP significantly alleviated the increase of LPS induced by CCl4.LPS activated the expression of TLR4/NF-κB in the liver,and DOP down-regulated the expression of TLR4,and NF-κB in the liver,as well as the levels of P-IκBα/IκBα.It was confirmed by LPS-induced HSC-T6 cells model that DOP could alleviate liver inflammation and liver fibrosis through LPS/TLR4/NF-κB signaling pathway.(2)In vitro and in vivo experiments verified that DOP could inhibit SMO/Gli1 signaling pathway transduction,suppress protein expression of three angiogenic markers CD31,CD34 and VWF,as well as downregulate the expression of angiogenesis-related proteins VEGF,VEGFR2 and Angiopoietin 1,so as to alleviate liver fibrosis.(3)The integrity of intestinal barrier function is an important factor in reducing the entry of toxic substances into the liver.On the one hand,DOP could elevate the expression of Bcl-2 and diminish the expression of Bax and Caspase-3 in colon tissue on the other hand,DOP could ascend the protein levels of three tight junction proteins ZO-1,Occludin and Claudin-1 and protect the intestinal mucosal barrier.The ability of DOP to protect the LPS-induced barrier function of Caco-2 epithelial cells was verified by the LPS-induced Caco-2 cell model.Conclusion:DOP could protect the intestinal mucosal barrier in rats and reduce the entry of toxic substances such as LPS into the liver,thereby inhibiting the activation of TLR4/NF-κB signaling pathway.DOP inhibited autocrine and paracrine of TGF-β,inhibited SMO/Gli1 signal transduction,and reduced hepatic angiogenesis,thereby reducing the expression of α-SMA and collagen deposition in the liver,ultimately played a role in alleviating hepatic fibrosis.