Effect Of Panax Stipuleanatus Saponins On Liver Fibrosis Rats Based On Gut-Liver Axis

Panax stipuleanatus is root and rhizome of Panax stipuleanatus H.T.Tsai et K.M.Feng,a kind of ginseng plant of the acanthaceae,contains oleanane pentacyclic triterpenoid saponins.According to reports,the natural drugs pentacyclic triterpenoids such as ursolic acid and oleanolic acid have a certain protective effect on liver injury and are used for the adjuvant treatment of acute and chronic hepatitis.The previous study of the research group found that Panax stipuleanatus saponins can significantly reduce the acute liver injury induced by carbon tetrachloride（CCl₄）in mice,but the effect of Panax stipuleanatus saponins on liver fibrosis has not been studied.Therefore,in this paper,the rat liver fibrosis induced by carbon tetrachloride is used as an animal model to study the mechanism of Panax stipuleanatus saponins-induced rat liver fibrosis,based on 16S r RNA gene sequence and UHPLC-QTOFMS analysis of changes in rat intestinal flora and its metabolites,The relationship between"Panax stipuleanatus saponins-intestinal flora-liver fibrosis"was initially established to provide new ideas for clinical treatment of liver diseases.The main research contents and conclusions are as follows:1.Protective effect of Panax stipuleanatus saponins on on CCl₄-induced liver fibrosis in rats.1.1 Panax stipuleanatus saponins can significantly increase the weight of the rat and reduce the liver index.Compared with the model group,the PS medium and high dose groups（150,200 mg/kg）can significantly increase the body weight of rats（p<0.05,p<0.01）;Panax stipuleanatus saponins R₂each dose group（1,10,30mg/kg）,PS each dose group（100,150,200 mg/kg）can significantly reduce the liver index of rats（p<0.05,p<0.01,p<0.001）.1.2 Panax stipuleanatus saponins can effectively improve the plasma liver function index of liver fibrosis model rats.Compared with the model group,the R₂medium and high dose groups（10,30 mg/kg）and PS dose groups（100,150,200mg/kg）can significantly reduce the activity of Alanine aminotransferase（ALT）and Aspartate aminotransferase（AST）（p<0.05,p<0.01,p<0.001）;comparable to the activity of the silymarin（SC）group in the positive group.1.3 Panax stipuleanatus saponins can protect the intestine,reduce the release of LPS from liver cells into the blood,and reduce the content of LPS in plasma.Compared with the model group,the R₂medium and high dose group（10,30 mg/kg）and PS dose groups（100,150,200 mg/kg）can significantly reduce the LPS content（p<0.05,p<0.01,p<0.001）.The inhibitory activity of R₂and PS high-dose group was higher than that of positive group SC.1.4 Panax stipuleanatus saponins can promote the degradation of collagen in rat liver.Compared with the model group,each dose group of R₂（1,10,30 mg/kg）and each dose group of PS（100,150,200 mg/kg）can significantly reduce the contents of Hyaluronidase（HA）、Laminin（LN）、Precollagen III（PCIII）、Collagen type IV（IV-C）（p<0.05,p<0.01,P<0.001）,of which the high-dose PS group activity was due to the positive group SC.R₂high-dose group（30 mg/kg）and PS each dose group（100,150,200 mg/kg）can significantly reduce the content of Hydroxyproline（Hy P）（p<0.05,p<0.01,p<0.001）.1.5 Panax stipuleanatus saponins can inhibit the expression of pro-inflammatory factors.Compared with the model group,each dose group of R₂（10,30 mg/kg）and each dose group of PS（100,150,200 mg/kg）can significantly reduce the content of Tumor necrosis factor（TNF-α）and Interleukin-1β（IL-1β）（p<0.05,p<0.01,p<0.001）。The high-dose group of R₂（30 mg/kg）and SP（200 mg/kg）can significantly reduce the content of Interleukin-6（IL-6）（p<0.05,p<0.01）,and the activity of the high-dose group of R2 and PS is equivalent to that of the positive group SC.1.6 Panax stipuleanatus saponins can improve the body’s ability to resist oxidation and remove free radicals.Compared with the blank group,the Malondialdehyde（MDA）content of the model group increased significantly（p<0.001）,and the Superoxide dismutase（SOD）enzyme activity and Catalase from micrococcus lysodeikticus（CAT）content decreased significantly（p<0.001）.Compared with the model group,each dose group of R₂（1,10,30 mg/kg）and each dose group of PS（100,150,200 mg/kg）can significantly increase the activity of SOD enzyme in the liver tissue of rats and reduce MDA and CAT content(（p<0.05,p<0.01,p<0.001）,where the activity of the PS high-dose group was due to the positive group SC.1.7 Histopathological results show that Panax stipuleanatus saponins can effectively improve the infiltration,necrosis and fibrosis of liver cell inflammatory cells caused by CCl₄and reduce inflammation.Hematoxylin and eosin（HE）staining results showed that hepatic cell necrosis,collagen fiber hyperplasia,inflammatory cell infiltration,and strip-shaped collagen fiber hyperplasia in the R2 high-dose group,the PS medium and high-dose group were significantly reduced compared with the model group.Sirius scarlet staining showed that hepatic lobular structure,inflammatory cell infiltration,and strip-shaped collagen fiber hyperplasia in R₂high-dose group,PS medium and high-dose group were significantly reduced compared with the model group.1.8 Panax stipuleanatus saponins exerts anti-liver fibrosis effect by inhibiting LPS/TLR4/NF-κBsignaling pathways.Western blotting（WB）experimental results showed that compared with the model group,the expression of TLR4,My D88,p-NF-k B,NF-k B,α-SMA protein in the liver tissues of rats in the Pingbian Notoginsenoside R₂treatment group Significantly reduced;after Pingbian Panax notoginseng saponins treatment,the expression of TLR4,My D88,p-NF-k B,NF-k B,α-SMA and TGF-β1 protein in rat liver tissues was significantly reduced,indicating that these proteins were involved In the pathological process of liver fibrosis,the anti-liver fibrosis effect of Pingbian Notoginsenoside may be related to the LPS/TLR4/NF-κB signaling pathway.2.The effect of Panax stipuleanatus saponins on the changes of fecal intestinal microflora in liver fibrosis rats.2.1 The OTU petal chart shows that the total number of OTUs in each group is282,and there is no significant difference in the number of valid sequencing information.2.2 Alpha diversity and Beta diversity analysis found that after CCl₄caused liver fibrosis,the intestinal flora was disordered and the diversity of intestinal flora decreased;compared with the model group,Panax stipuleanatus saponins significantly regulated the flora in rat feces Diversity,diversity of intestinal flora is proportional to the dose administered.2.3 The first three bacterial groups in rat feces are Firmicute、Bacteroidetes、Proteobacteria.After modeling,Firmicute increases and Bacteroidetes decreases,that is,probiotics decrease and pathogenic bacteria increase,R₂（30 mg/kg）and PS（200 mg/kg）The dose can significantly improve this situation.3.The effect of Panax stipuleanatus saponins on fecal metabolites in liver fibrosis rats.3.1 By comparing the principal component（PCA）of the model group and the R₂high-dose group（30 mg/kg）,the model group and the PS high-dose group（200mg/kg）,it can be seen that the two groups of samples are all within the 95%confidence interval,and There is a certain separation trend,indicating that R₂and PS have an effect on the metabolic network of rat feces.Through principal coordinate analysis（OPLS-DA）analysis,it can be seen that there are significant differences in metabolites between the R₂high-dose group and the PS high-dose group compared with the model group.Compared with the model group,the R₂high-dose group significantly reduced 4 differential metabolites;the PS high-dose group had 9 significantly reduced differential metabolites.3.2 Enrichment analysis was performed using the differential metabolite KEGG to find the key pathway with the highest correlation with metabolite differences.The anti-liver fibrosis effect of R₂is mainly related to the Linoleic acid metabolism pathway,and the differential metabolite is Linoleic acid;The anti-hepatic fibrosis effect of PS is mainly related to the Pentose and glucuronate interconversions pathway between pentose and glucuronate.The differential metabolite is D-ribose 5-phosphate.