| Objective:To explore the material basis and related mechanism of Fluid-Increasing Decoction for Purgation in anti-hepatic Fibrosis.Firstly,we extract polysaccharide from single herb(Ophiopogon japonicus and Scrophularia ningpoensis)and compound herbs(Ophiopogon japonicus,Scrophularia ningpoensis,Rehmannia glutinosa and Rheum officinale)from the tradtional Chinese prescription-Fluid-Increasing Decoction for Purgation to observe the anti-fibrosis effect of Fluid-Increasing Decoction for Purgation on liver cirrhosis mice.The effects on biochemical indicators,immune factors,endotoxin(LPS)and intestinal microecology were tested to explore the mechanism of polysaccharides from Fluid-Increasing Decoction for Purgation in anti-hepatic fibrosis and regulating intestinal microecology.At the same time,investigate the physicochemical properties,structural characteristics and anti-pancreatic cancer activity of the polysaccharides and its acetylated derivatives from O.japonicus.This project lays a theoretical and experimental basis for quality control and innovative drug research based on polysaccharide of Fluid-Increasing Decoction for Purgation.Research methods:1.Single herb and compound herbs polysaccharides of Fluid-Increasing Decoction for Purgation were obtained by water extraction and then ethanol precipitation method.40%carbon tetrachloride(CCl4)combined with multi-factors was used for 8 weeks to induce liver cirrhosis mice model.From the sixth week on,the mice were treated with crude poysaccharide of O.japonicus(0.5 g-kg-1),S.ningpoen.sis(0.5 g·kg-1)compound herbs I(0.5 g·kg-1)and compound herbs II(0.8 g·kg-1)lasting for three weeks.HE staining and Masson’s staining was made to observe liver inflammation and liver fibrosis level.Serum biochemical indicators such as alanine aminotransferase(ALT),aspartate aminotransferase(AST),total protein(TP)and albumin(ALB)were detected by automatic biochemical analyzer.Enzyme-linked immunosorbent assay was used to detect serum concentration of immune factors,such as interleukin-1(IL-1),interleukin-6(IL-6)and tumor necrosis factor-a(TNF-a),and endotoxin in mice.llumina MiSeq sequencing was used to detect intestinal microbial diversity in mice.2.The crude polysaccharides of O.japonicus were isolated by enzyme-water extraction and then ethanol precipitation method.The homogenous polysaccharides were separated from an anion exchange chromatography.The purity and molecular weight were determined by High Performance Liquid Chromatography(HPLC),and the physicochemical properties and structure of the polysaccharide were determined by Infra-red spectrum,Nuclear Magnetic Resonance spectrum,monosaccharides composition and methylation,ect.The acetylated polysaccharides were prepared by pyridine-acetic anhydride method,the degree of acetyl substitution was determined by hydroxylamine colorimetry method,and the acetyl substitution sites were determined by Nuclear Magnetic Resonance spectroscopy.MTT assay was uesd to detect the anti-pancreatic cancer cells proliferation activity of the acetylated polysaccharide.Changes of nuclear morphology were observed by fluorescence microscopy and the expressions of caspase-3,p53,p21,Bax,and FASL were detected by Western blotting.Results:1.After 8 weeks treanment of CCl4 combined with multifactorial effects,the hepatic tissue was stained with HE and masson to comfirm that the mice liver cirrhosis model was successfully established.After 3 weeks of different polysaccharide treatments,mices of treatment groups had the higher weight and were more active than that of the model group,the hepatic inflammation and the liver fibrosis were also lower than that of the model group.The compound herbs II group had the lower liver fibrosis level than the compound herbs I group(p<0.05),while the O.japonicus group had the lowest liver fibrosis level(p<0.01).The hepatic index of the model group was significantly lower than control group(p<0.01),the hepatic index of O.japonicus,S.ningpoensis and compound herbs II groups were higher than model group(p<0.05),and the O.japonicus group was the closest to the control group.Serum biochemical test results showed that the ALT and AST in model group were significantly increased(p<0.01),while the seunn ALT content of O.japonicus,S.ningpoensis and compound herbs II groups decreased significantly(p<0.01)and the AST concertration of each polysaccharide treatment group were also lower than model group(p<0.05).The total protein and albumin level in model group were significantly lower than the control group(p<0.05),but they had not increase significantly in treatment groups and were even lower in the positive control group.Compared with the control group,the concertration of serum IL-1 and IL-6 in model group were increased significantly(p<0.05).while in the O.japonicus group and compound herbs II group,the seurm IL-1 level decreased significantly(p<0.05).and high-dose compound herbs group was lower than the low-dose compound herbs group(p<0.01).The seurm IL-6 level of compound herbs II group decreased significantly(p<0.05).The content of TNF-a in the model group was higher than the normal group,but in the other treatment groups,it did not show significant difference to the control group except for the S.ningpoensis group.The serum endotoxin concentration of model group was higher than the control group(p<0.05),the treatment of of O.japonicus and S.ningpoensis polysaccharides significantly reduced the endotoxin in seunn(p<0.05).The compound herbs II group had the lower content of LPS than compound herbs I group(p<0.01).The structure of the gut microbes in the model group was changed,like the abundance of bacteriodetes and actinobacteria was reduced(p<0.01),while the abundance of firmicutes increased significantly(p<0.01);For the O.japonicus polysaccharide group,the quantity of bacteriodetes and actinobacteria microflora were increased(p<0.01),and the abundance of fir mi cutes decreased(p<0.05),compare to model group;In the figwort polysaccharide group,the content of firmicutes decreased significantly(p<0.05);the actinobacteria abundance of compound herbs II group was increased(p<0.05),and compound I had the higher content of actinobacteria than other groups(p<0.01).2.A homogeneous polysaccharide ROH05 was isolated and purified from the roots O.japonicus,the average molecular weight was 16.7 kDa.According to the structural analysis,ROH05 is a galactan with the main chain of 1,4-β-D-galactose branched with terminal-β-D-galactose at C-6,The acetylation was prepared to get the acetylated polysaccharide ROH05A,the degree of acetyl substitution was 0.23,and the acetyl substitution occurred in the C-2,C-3 and C-6 positions of 1,4-β-D-galactose and C-6 of terminal galactose.ROH05A could significantly inhibit the proliferation of pancreatic cancer cells(BxPC-3 and PANC-1)with little cytotoxicity to human normal liver cells(LO2).The RO.H05A-treated BxPC-3 cell was observed under fluorescent microscope,the characteristic nuclear fragmentation and crenulation of apoptotic were detected.ROH05A could time-dependently upregulated the expression of caspase-3 in protein level,while the expression of anti-tumor genes p53,p21,Bax and FasL was also increased.Conclusions:1.Successfully induced a mice cirrhosis model.The intervention result of O.japonicus,Radix Scrophulariae,low-dose compound.and high-dose compound polysaccharides showed that Fluid-Increasing Decoction for Purgation could resist fibrosis,decrease the levels of ALT,AST and other biochemical indicators,reduce serum levels of IL-1,IL-6 and other immune factors,lower the serum content of endotoxins in cirrhotic mice.The polysaccharides of Fluid-Increasing Decoction for Purgation could regulate intestinal flora imbalance,treatment of O.japonicus and high-dose compound polysaccharides were the better.The results indicated that the anti-fibrosis effect of Fluid-Increasing Decoction for Purgation could be related to regulating the microecology of the intestine,anti-hepatic injury and improving immune function.2.A homo-galactan ROH05 was extracted from O.japonicus which had the main chain of 1,4-β-D-galactose.The acetylated derivatives of ROH05 exhibited anti-pancreatic cancer prolifer-ation activity by inducing apoptosis.Significance:The eff-ects of polysaccharides from Fluid-Increasing Decoction for Purgation on liver cirrhosis in the perspective of active polysaccharide intestinal microecology were discussed,which provided a theoretical and experimental basis for the modern preparation development of Fluid-Increasing Decoction for Purgation and the research of anti-cirrhosis drugs.The result turned out that polysaccharide of O.japonicus showed good anti-fibrosis effect,then a homo-galatan was purified from O.japonicus,and its acetylated derivatives showed pancreatic cancer effect in vitro,and the mechanism was related to the induction of apoptosis.The structural characteristics and anti-pancreatic cancer effects of the acetylated polysaccharide from 0.japonicus provided theory evidence for the reasearch of the relation between structural and biological activity of polysacch-arides and the development of anti-pancreatic cancer drugs. |
