| Part1Extraction and Determination of the Effective Fractions of Xiongshao DecoctionObjective To create the method for the extraction of Xiongshao decoction, and the method for the extraction and determination of three effective parts of the total extract, namely crude polysaccharides, total alkaloids and total glucosides.Methods Xiongshao decoction was extracted by decoction. In order to separate the crude polysaccharides from the total extract, alcohol precipitation combined with the Sevage method was applied to remove the protein. Water-saturated n-butanol extraction was applied to separate total glucosides from the total extract, and total alkaloids were extracted from the total extract through strong acid cation exchange resin. Visible spectrophotometry was applied to determine the content of total polysaccharides in the crude polysaccharides extract and the content of total ginsenoside in the total glycosides extract of Xiongshao decoction, and high performance liquid chromatography method was applied to determine the content of paeoniflorin in the total glycosides extract of Xiongshao decoction, and acid-base titration method was applied to determine the content of total alkaloids in the total alkaloids extract of Xiongshao decoction.Results The total polysaccharides content of the crude polysaccharides extract is55.74%, and the average recovery of total polysaccharides was 98.23%with RSD0.54%; the total ginsenoside content of the glucosides extract was63.54%, and the average recovery of total ginsenoside was96.34%with RSD0.61%; the paeoniflorin content of the glucosides extract was10.01%, and the average recovery of paeoniflorin was99.67%with RSD1.38%; the total alkaloids content of the total alkaloids extract was79.42%, and the average recovery of total alkaloids was101.12%with RSD3.37%.Conclusion The method for the extraction of Xiongshao decoction is simple and feasible. The methods for the determination of the crude polysaccharide extract, the total alkaloids extract and the total glucosides extract are highly sensitive and reproducible, and can be used for the quality control of the effective parts of Xiongshao decoction.Part2Study of the Effects and Mechanisms of Xiongshao Decoction and its Effective Parts on Hepatic FibrosisObjective The HF rat model was induced by dimethylnitrosamine (DMN), and then the effects of Xiongshao decoction and its effective parts on HF were observed, and their possible mechanisms were investigated. In this way, we want to initially clarify the anti-HF material basis of Xiongshao decoction and lay a solid theoretical foundation for its development and application.Methods86male Wistar rats were randomly divided into the normal group and the modeling group.74rats of the modeling group were intraperitoneally injected with1%DMN by the dose of10mg/kg body weight every other day to establish HF model, three times a week for4weeks. The rats of normal group were intraperitoneally injected with equivalent normal saline. During the modeling,4rats died of illness and4rats were put to death for the determination of the model preparation. After the modeling, the remaining66rats of model group were randomly divided into six groups (n=11):the model group, Fuzheng-Huayu group (FZHY group), Xiongshao group, the polysaccharide group, the total alkaloids group and the total glycosides group. After the modeling, the0.105g/ml FZHY solution, the1.610g/ml Xiongshao decoction, the35.420mg/ml crude polysaccharides extract solution, the0.196mg/ml total alkaloids extract solution and the25.725mg/ml total glycosides extract solution, were respectively administered to the corresponding treatment group by gavage once a day for4weeks. The rats of normal group and model group were given equivalent normal saline by gavage once per day for4weeks. The general situations of rats were observed during the experiment. The seven groups were all administered for4weeks, and after that we killed all the rats and observed the appearance and pathological changes of their livers with HE staining and masson staining. Liver weight index and spleen weight index were calculated; serum parameters of liver function ALT, AST, and TIBL and ALB were determined by automatic biochemical analyzer; serum markers of hepatic fibrosis HA, LN, PC Ⅲ and CIV were detected by radioimmunoassay; the serum SOD activity, GSH-PX activity, as well as the serum contents of MDA, MMP-13, TIMP-1and hydroxyproline were respectively measured by xanthine oxidase, DTNB reduction, TBA, ELISA and alkaline hydrolysis. The expressions of CTGF, PDGF-B, Col-Ⅰ and α-SMA were detected by the immunohistochemical assay, and the expression of TGF-β1mRNA by FQ-PCR, and the protein expressions of Smad3and Smad7by western blotting analysis.Results1.Fuzheng-Huayu capsule, Xiongshao decoction, crude polysaccharides and total glucosides extracted from Xiongshao decoction can all improve the general situation of HF rats, but total alkaloids extracted from Xiongshao decoction does not have this effect.2. Liver weight index and spleen weight index:liver weight index of the model group was significantly lower than that of the normal group, its spleen weight index was significantly higher than that of the normal group (P<0.01); compared with the model group, there was no significant differences between the total alkaloids group and the model group, whereas liver weight indices of other treatment groups were significantly increased, and their spleen weight indices were significantly decreased (P<0.01); there was no significant difference between FZHY group and Xiongshao group, and compared with these two groups, liver weight indices of the effective parts groups were all significantly reduced, and their spleen weight indices were all significantly improved.3. Indicators of liver function:the levels of serum ALT, AST and TBIL of the normal group were significantly higher than those of the normal group, and the level of its serum ALB was significantly lower than that of the normal group (P<0.01); compared with the model group, there was no significant differences between the total alkaloids group and the model group, but the levels of serum ALT, AST and TBIL of other treatment groups were significantly decreased, and their serum ALB level were significantly elevated (P<0.05or P<0.01); compared with Xiongshao group, the levels of serum ALT, AST and TBIL of all the effective parts groups were significantly increased (P>0.01).4. HF markers:the levels of serum HA, LN, PCⅢ and CIV of the model group were significantly higher than those of the normal group (P<0.01); compared with the model group, there was no significant difference between the total alkaloids group and the model group, however the levels of serum HA, LN, PCⅢ and CIV of other treatment groups were significantly reduced (P<0.05or P<0.01); compared with Xiongshao group, there was no significant difference between the polysaccharide group, the total glycosides group and Xiongshao group in the level of serum CIV, and the levels of the rest indices of all the effective parts groups were significantly elevated (P<0.05or P<0.01).5. Serum SOD, GSH-PX and MDA:the levels of serum SOD and GSH-PX of the model group were significantly lower than those of the normal group, and the level of its serum MDA was significantly higher than that of the normal group(P<0.01); compared with the model group, there was no significant difference between the polysaccharide group, the total glycosides group and the model group, whereas the levels of serum SOD and GSH-PX of other treatment groups were significantly increased (P<0.01), and the levels of their serum MDA were significantly reduced (P<0.05or P<0.01); compared with Xiongshao group, the levels of serum SOD and GSH-PX of all the effective parts groups were significantly decreased, and the level of their serum MDA were significantly elevated (P<0.05or P<0.01).6. Serum MMP-13and TIMP-1:the level of serum MMP-13of the model group was significantly lower than that of the normal group, and the level of its serum TIMP-1was significantly higher than that of the normal group (P<0.01); compared with the model group, there was no significant difference in serum TIMP-1between the polysaccharide group and the model group, as well as no significant difference between the total alkaloids group and the model group, while the levels of serum MMP-13of other treatment groups were significantly improved (P<0.05or P<0.01), and the level of serum TIMP-1of other treatment groups were significantly reduced (P<0.01); compared with Xiongshao group, the levels of serum MMP-13of the polysaccharide group and the total alkaloids group were significantly decreased, and the levels of their serum TIMP-1were significantly elevated (P<0.05or P<0.01).7. Hydroxyproline in liver tissue and HF degree:the content of hyp and semi-quantitative score of HF of the model group were significantly higher than those of the normal group (P<0.01); compared with the model group, there was no significant difference between the total alkaloids group and the model group, while the contents of hyp and semi-quantitative scores of HF of other treatment groups were significantly reduced (P<0.01); there was no significant difference between FZHY group and Xiongshao group, and the contents of hyp and semi-quantitative scores of all the effective parts groups were all significantly higher than those of the two groups (P<0.05or P<0.01).8. Immunohistochemistry:the expressions of CTGF, PDGF-B, Col-I and a-SMA in liver of the model group were significantly higher than those of the normal group (P<0.01); compared with the model group, there was no significant difference between the total alkaloids group and the model group, while the expressions of CTGF, PDGF-B, Col-Ⅰ and α-SMA of other treatment groups were significantly decreased (P<0.05or P<0.01); there was no significant difference between FZHY group and Xiongshao group, compared with the two groups, there was no significant difference in the expression of a-SMA between the polysaccharide group, FZHY group and Xiongshao group, while the expressions of the rest indices of all the effective parts groups were significantly elevated (P<0.05or P<0.01).9. Expressions of TGF-β1mRNA, Smad3and Smad7proteins:the expressions of TGF-β1mRNA and Smad3protein in liver of the model group were significantly higher than those of the normal group, and the expression of its Smad7protein was significantly lower than that of the normal group (P<0.01); compared with the model group, there was no significant difference in the expression of Smad7protein between the total glycosides group and the model group, as well as no significant difference between the total alkaloids group and the model group, while the expressions of TGF-β1mRNA and Smad3protein of other treatment groups were significantly reduced, and the expressions of their Smad7protein were significantly increased (P<0.05or P<0.01); compared with Xiongshao group, the expression of TGF-β1mRNA of the polysaccharide group was significantly decreased (P<0.05), and the expressions of Smad3protein of the total alkaloids group and the total glycosides group were significantly elevated (P<0.01).Conclusion Crude polysaccharides and total glucosides that are extracted from Xiongshao decoction can improve or reverse the pathological changes of hepatic fibrosis induced by DMN, while total alkaloids extracted from Xiongshao decoction does not have the effect. The therapeutic material basis of Xiongshao decoction is as follows:(1) the function of anti-lipid oxidative damage of Xiongshao decoction is mediated by total alkaloids;(2) the effects of Xiongshao decoction that promote MMP-13generation and inhibit TIMP-1generation so as to promote the degradation of type I collagen, are mediated by total glycosides, and the role of crude polysaccharides is secondary;(3) the effects of Xiongshao decoction that down-regulate the expressions of TGF-β1, α-SMA and Smad3and up-regulate Smad7expression, are mediated by crude polysaccharides, and the role of total glycosides is secondary;(4) the effect of Xiongshao decoction that inhibits the expressions of the fibrogenic cytokine CTGF and PDGF-B is mediated by crude polysaccharides and total glycosides.Part3Study of the Effects of Xiongshao Decoction and its Effective Parts on the proliferation of human hepatic stellate cells LX-2Objective The effects of the drug-containing serum of Xiongshao decoction and its effective parts on the proliferation of LX-2were observed and were used to explore the effect of the therapeutic material basis of Xiongshao decoction on HSC proliferation.Methods The effects of the drug-containing serum of Xiongshao decoction and its effective parts on the proliferation of LX-2were detected by MTT.Results Compared with the normal groups with the20%concentration and the10%concentration, there was no significant difference between the total alkaloids group and the normal group, while other treatment groups can all inhibit the proliferation of LX-2cell (P<0.01); compared with Xiongshao decoction with the20%concentration, the inhibition rates of the total alkaloids group and the total glycosides group with the20%concentration on the proliferation of LX-2cell were significantly reduced (P<0.05or.P<0.01); compared with Xiongshao decoction with the10%concentration, the inhibition rates of all the effective parts groups with the10%concentration on the proliferation of LX-2cell were significantly reduced (P<0.05or P<0.01).Conclusion Crude polysaccharides and total glucosides that are extracted from Xiongshao decoction can inhibit the proliferation of HSC, while total alkaloids extracted from Xiongshao decoction does not have the effect. The effect of Xiongshao decoction that inhibits the proliferation of HSC is mediated by crude polysaccharides, and the role of total glucosides is secondary. |
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