| Objective:To investigate the protective effects of polysaccharide of Prismatomeris tetrandra（PSPT）on hepatic fibrosis（HF）that induced by carbon tetrachloride（CCl4）,and to explore its mechanism.Methods:1.Using the STRING database and the DAVID system’s GO and KEGG functional enrichment analysis,a search for proteins that interact closely with the TGF-β1 gene is conducted.2.The traditional liver fibrosis model was created by administering CCl4 intraperitoneally to animals to cause HF.60 Kunming mice were allocated into six groups at random:Control,Model,CCl4+PSPT low dose group（100 mg kg-1）,CCl4+PSPT medium dose group（250 mg kg-1）,CCl4+PSPT high dose group（500 mg kg-1）,and the positive control group CCl4+silymarin（200 mg kg-1）.To create a mouse model of liver fibrosis,all other experimental groups except for the blank control group received efficient dosages of CCl4.Both the blank control group and the model group received the same amount of clean water via gavage at the same time as the mice in the drug trial group,which received the corresponding dose of medicines every day.The experimental mice were given treatment to get the matching blood and liver tissue samples after 8 weeks of continuous involvement.（1）Using H&E,Masson,and Sirius red stains,the pathological morphology of the mice in each experimental group was examined.（2）An assay kit was used to measure the levels of ALT and AST in the blood of the mice in each experimental group.（3）The expression ofα-SMA in the liver tissue of the mice in each experimental group was examined using immunofluorescence staining.（4）TGF-β1,Smad2,Smad3,and CTGF m RNA expression in the liver tissue of mice in each experimental group was determined using RT-q PCR.（5）Protein expression levels of the essential downstream effector factor CTGF and the key players（TGF-β1、Smad2、Smad3、P-Smad2/3）in the TGF-β1/Smad signaling pathway were determined in each experimental group using immunohistochemistry and immunofluorescence staining.Results:1.Bioinformatics analysis showed that Smad2,Smad3,TGFβRI and TGFβRII were closely interacting proteins of TGF-β1,and functional enrichment analysis showed that they were closely related to TGF-β1/Smad signaling pathway.2.The results of animal experiments:Compared with the blank control group,the serum ALT(**P<0.01)and AST(***P<0.001)were significantly increased in the model group.Masson staining,Sirius red staining and immunohistochemistry showed that a large number of collagen fibers were deposited around the large and small blood vessels in the liver tissue(**P<0.01).RT-q PCR showed that the m RNA expressions of TGF-β1,CTGF,Smad2,and Smad3 were significantly increased(***P<0.001).Immunofluorescence staining showed that the protein expressions ofα-SMA,TGF-β1,CTGF,Smad2 and Smad3 were significantly increased(***P<0.001),p-Smad2/3 were distinctly increased(**P<0.01).Compared with the model group,the serum ALT levels of all PSPT groups and silymarin group were significantly decreased(###P<0.001).AST decreased significantly(##P<0.01).Masson staining,Sirius red staining and immunohistochemistry showed that the degree of liver fibrosis and the expression of TGF-β1 and Smad2 protein in each dose group of PSPT were reduced(##P<0.01;#P<0.05).RT-q PCR showed that the m RNA expressions of TGF-β1,CTGF,Smad2 and Smad3 were significantly down-regulated in each dose group of PSPT and silymarin group(###P<0.001).Immunofluorescence staining showed that the protein expression levels ofα-SMA,CTGF,Smad3 and p-Smad2/3 in each dose group of PSPT were significantly decreased(###P<0.001).Conclusion:1.PSPT has a protective effect on CCl4-induced liver fibrosis in mice.2.PSPT inhibited CCl4-induced HF in mice by down-regulating TGF-β1/Smad signaling pathway. |
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