Effect And Mechanism Of Dendrobium Officinale Polysaccharide Against Hepatic Fibrosis And Its Ultrasonic Evaluation

Objective:To extract and prepare Dendrobium polysaccharide（DOP）with high polysaccharide content and high solubilization rate of active ingredients,to establish a CCl₄-induced liver fibrosis model in rats,to observe the intervention effect of high and low doses of DOP on hepatic fibrosis（HF）,and to further study and explore its anti The effect of DOP on HF was investigated.We also applied CEUS and ARFI techniques to evaluate the effects of HF and anti-HF drugs,which can provide some theoretical basis and new treatment methods for clinical application.Methods:DOP was extracted by hot water immersion method and DOP concentrate was prepared.All 70 male SD rats were randomly divided into normal group（NG group）,model group（MG group）,colchicine group（CG group）,DOP low-dose group（DL group）and DOP high-dose group（DH group）,in which all four groups except NG group were injected subcutaneously with 40%CCl₄ olive oil mixture,after 6 weeks of modeling.The CG,DL and DH groups were given colchicine and high and low doses of DOP by gavage for 4 weeks,and the serum was collected to detect serum liver function,liver fibrosis indexes and oxidative stress indexes using kits.The liver tissues of rats were stained with HE and Masson stain to observe the histopathological changes.The expression levels of related genes were determined by real-time fluorescence quantitative PCR and the expression levels of related proteins were detected by Western-Blot.At the same time,two techniques,CEUS and ARFI,were applied to observe the changes of liver ultrasound indexes in rats.Results:1.The total polysaccharide content of the extracted DOP was 35.49%,and the main monosaccharides contained were glucose and mannose.2.Effects on general condition of rats:After drug intervention,the general condition of CG,DL and DH group was improved compared to MG group,with DH and CG group gaining weight while DL group gained slightly more weight than MG group.Although the livers of DL group showed improvement compared to MG group,the adhesion condition remained,the appearance of the livers in DH and CG group returned to near normal,and the adhesion condition of the livers in each group improved markedly.Liver index decreased in all drug-interfering groups,including CG,DH,and DL（P<0.01 or P<0.05）.3.Hepatology markers were measured:ALT,AST,and AKP decreased in all drug-treated groups,compared to MG groups,while ALB increased in all groups,and fibroblasts increased in both DH and DL groups,while fibroblasts increased in all groups,with less fibroblasts（P<0.01）.4.Hepatic histopathologic changes:Compared to the MG group,HE staining showed less hepatocyte degeneration and necrosis in each drug group,liver cord structure tended toward NG group,and fibroconnective tissue proliferation in sink area decreased to varying degrees,especially in CG group and DH group.Masson staining showed a decrease in the area of blue coloring and an improvement in the degree of fibrosis in each drug group,compared to CG and DH groups,with no significant increase in fiber spacing or pseudolobule formation.5.Results of liver fibrosis-related indexes:Compared with the MG group,the levels of LN,HA,PC-III and Col-IV in the CG,DH and DL groups,as well as the levels of Col-III in the CG and DH groups,showed a significant decrease（P<0.01）.6.Indexes of oxidative stress:Compared with the MG group,the levels of SOD increased and the levels of MDA decreased in all drug groups.Among them,the level of SOD content increased（P<0.05）and MDA content decreased（P<0.01 or P<0.05）in DH and CG groups.7.Results of collagen expression:RT-q PCR showed that the expression levels ofα-SMA and Col-III m RNA also decreased to different degrees in each drug group compared with MG group,among them,α-SMA and Col-Western blot showed that the relative expression ofα-SMA,Col-III and Col-Ⅰprotein in each drug group was significantly decreased compared with that in the MG group（P<0.01）.8.Changes in related signaling pathway factors:RT-q PCR showed that the relative expression of TGF-β,TIMP-1 andβ-catenin m RNA decreased and that of DKK-1 m RNA increased in each drug group compared with that in the MG group.The relative expression of TGF-β1,TIMP-1 andβ-catenin m RNA in the DH and CG groups decreased（P<0.01or P<0.05）,and the relative expression of DKK-1 m RNA was higher than that in the MG group（P<0.01）.The relative expressions ofβ-catenin and Wnt-1 in DH and CG groups were lower than those in MG group（P<0.05 or P<0.01）,while the expression of Wnt-1 in DL group was not statistically different.9.Changes in liver ultrasound indexes:CEUS examination results showed that the relative expressions ofβ-catenin and Wnt-1 in DH and CG groups were lower than those in MG group（P<0.05 or P<0.01）.The values of HVAT,VAT,and VVT increased in each drug group compared with the MG group,and the increase in HVAT values was more significant in the CG and DH groups（P<0.01）;the ARFI test showed that there was no statistical difference in the Vs values of each drug group before administration compared with the MG group,and the Vs values decreased in each group after administration（P<0.05）,and the decrease was more significant in the CG and DH groups（P<0.01）.Conclusions:1.The DOP extraction and rat HF model were successfully prepared.2.DOP can improve the extent of HF in rats,including improving the general condition,liver index,some indexes of liver function,HF index,and the extent of liver histopathological damage.3.DOP can significantly inhibit oxidative stress and achieve the effect of improving HF.4.DOP can reduce ECM deposition by inhibitingα-SMA and collagen expression,and may achieve anti-HF effects by regulating TGF-βand Wnt/β-catenin signaling pathways.5.The combined application of CEUS and ARFI ultrasound techniques is more favorable for assessing the extent of HF and the effect of anti-HF effects of drugs.