Regulation Of Dendrobium Candidum From Yunnan On Hh Signaling Pathway Of Hepatic Fibrosis

OBJECTIVE:Hepatic stellate cells are one of the key cells in the development of hepatic fibrosis.Reversing the process of hepatic fibrosis with traditional Chinese medicine is one of the important research directions in the prevention and treatment of hepatic diseases.In this study,we first used the fresh juice of Dendrobium officinale from Yunnan to intervene the experimental hepatic fibrosis rat model,observed the effect of the drug and its effect on hepatic Hh signaling pathway from the point of view of Pathomorphology and molecular biology,and preliminarily explored the anti-hepatic fibrosis effect of Dendrobium officinale and its mechanism.METHODS:Sixty SD rats of 180-200 g were randomly divided into five groups:normal control group?NG?,model group?MG?,low-dose group?LMG?,medium-dose group?MMG?and high-dose group?HMG?.Rats in MG,LMG,MMG and HMG groups were subcutaneously injected with CCl₄-olive oil solution 5 ml/kg twice a week for 8 weeks to establish experimental liver fibrosis model,while rats in NG group were subcutaneously injected with saline of equal volume per kilogram.After calculating the mortality rate,8rats in each group were randomly selected to continue the experiment.The LMG,MMG and HMG groups were given 1.5 g/kg,4.5 g/kg and 7.5 g/kg fresh juice of Dendrobium candidum by gavage,and the NG and MG were gavaged by normal saline of equal volume.After 8 weeks of drug intervention,rats were sacrificed to observe the general situation and liver morphological changes,and liver tissues were taken for pathological sections.The pathological changes of rat liver tissues were observed by HE staining and Masion staining.The expression of a-SMA and Collagen 1 in liver tissues of rats in each group was detected by IHC method.In addition,some fresh liver tissues were taken for detection of Hh signaling pathway related factors.HC method was used to detect the expression of Shh and Gli1 proteins in each group,and this quantitative RT-PCR method was used to detect the levels of Gli1 and Ptch1.RESULTS:?1?Compared with NG group,the general state of rats in MG group was worse and the liver index was significantly higher?P<0.01?,while the general state of rats in each drug group was significantly improved.Compared with MG group,the liver index had significant difference?P>0.05?.?2?HE and Masion staining showed that compared with NG group,there were a large number of hepatocyte necrosis,disordered arrangement of hepatocyte cord and formation of hepatic lobules in MG group rats;lipid droplets were widely formed in hepatocytes,inflammatory cells infiltrated in portal area,and extensive red-stained collagen fibers were formed around portal area.After drug intervention,the liver injury of rats in LMG,MMG and HMG groups was significantly alleviated,manifested by reduced lipid droplets,reduced inflammatory cell infiltration,reduced pseudolobule formation and fibrosis in portal area.?3?The results of IHC and RT-PCR showed that the expression of alpha-SMA and Collagen I in liver tissue of rats in MG group was significantly higher than that of rats in NG group,while the expression of alpha-SMA and Collagen I in liver tissue of rats in LMG,MMG and HMG groups was significantly lower than that in MG group?P<0.01or P<0.05?,suggesting that the anti-fibrosis effect of Dendrobium officinale might be present in a certain range.A certain dose dependence.?4?IHC assay showed that the expression of Shh and Gli1 protein was low in NG group,but high in MG group?P<0.01?.After treatment,compared with MG group,the expression of Shh and Gli1 protein in MG group decreased gradually,with a significant decrease in HMG group?P<0.05?.In addition,the expression of Ptch1 and Gli1 in MG group was significantly higher than that in NG group?P<0.01?,but the expression of Ptch1 and Gli1 decreased gradually with the increase of dosage,especially in HMG group?P<0.05?.CONCLUSION:Dendrobium officinale fresh juice from Yunnan can not only improve the general state of experimental liver fibrosis model rats,but also reduce the pathological changes and the degree of liver fibrosis by inhibiting the expression of gene and protein of alpha-SMA and Collagen I,the main component of extracellular matrix.At the same time,Hh signaling pathway is further blocked by reducing the production of swimming protein Shh and inhibiting the activation of H signaling pathway,and by inhibiting the activation of downstream target genes Ptch1,Gli1 and Gli1 protein expression.Therefore,Dendrobium officinale from Yunnan may inhibit or reverse the development of hepatic fibrosis by inhibiting the activation of HSCs and the expression of collagen,blocking the Hh signaling pathway of hepatocytes.