Study Of The Protective Effect And Mechanism Of DBWP On Liver Fibrosis Induced By CCl₄ In Mice Based On Systems Pharmacology

Objective To analyze and predict the targets of Decoction of Baihe,Wuyao and Pugongying（DBWP）by network pharmacology methods and further verify the protective effect and mechanisms of DBWP on liver fibrosis induced by CCl₄ in mice.Methods Firstly,network pharmacology was used to analyze the effective chemical components of DBWP,and its related targets and signal pathways were screened.Sixty 8-week old SPF male Kunming mice were selected and were randomly divided into normal group（CNTR）,model group（Model）,Ganlixin positive control group（Positive）,DBWP low-dose group（LD）and high-dose group（HD）.The mice in the Positive group were given Ganlixin at the dose of 0.1 mg/10 g,and those mice in LD and HD DBWP groups were given of 0.09 g and 0.27 g crude drug/10 g respectively,once a day for 6 weeks.The mice in the CNTR group were similarly given of the same volume of DDH₂O.Except for CNTR,the mice in all other groups were intraperitoneally injected with 20%CCl₄（solution of olive oil）at dose of 0.1 m L/10 g body-weight twice a week for 6 consecutive weeks.While the mice in CNTR group were administered in the same way with the same dose of olive oil.At the end of experiment,after fasted for 12 hours,the mice in all groups were weighed and anesthetized using isoflurane,then the serum and liver samples were collected for further research.The automatic biochemical detector was used to detect the serum contents of alanine aminotransferase（ALT）and aspartate aminotransferase（AST）to assess the liver function of mice;The liver pathological changes were observed by HE staining;The deposition and distribution of collagen fibers in liver was examined by Masson staining;The contents of superoxide dismutase（SOD）and malondialdehyde（MDA）in liver homogenate was used to evaluate the liver tissue antioxidant capacity;Protein expression levels of Collagen1,Collagen3,smooth actin（α-SMA）,transcription growth factor（TGF-β1）,Smad2/3,p-Smad2/3,and nitric oxide synthase（i NOS）were detected through Western-Blot;The matrix metalloproteinases（including MMP2,MMP9,MMP12）,tissue metalloproteinase inhibitor（TIMP1）,manganese superoxide dismutase（Mn-SOD）,i NOS,interleukins（IL-1β,IL-11）,tumor necrosis factor（TNF-α）and other factors at m RNA level expression were evaluated by quantitative PCR（Q-PCR）.As a result,the protective mechanisms of DBWP on CCl₄ induced chronic liver injury and liver fibrosis in mic were revealed.Results 1 Network pharmacology analysis showed that:（1）DBWP could play a hepatoprotective effect through six active ingredients;（2）The key targets of DBWP were related to IL6,IL-1β,MMP2 and MMP9,etc;（3）The function of DBWP is related to ECM,inflammation and oxidation process;（4）The hepatoprotective effect of DBWP involved in inflammation,apoptosis and immune pathways;2 The results in vivo experiments showed that:Compared with the CNTR,the liver phenotype changes in the Model mice included:（1）Increasing serum ALT and AST concents;The liver occurs severe lesions and severe fibrosis;（2）Increased expression of ECMs（such as Collagen1and 3）,and up-regulatedα-SMA which is the marker of HSCs activation;（3）Raised expression of TGF-β1,Smad2/3,P-Smad2/3 which inferred that TGF-β1→Smad signal pathway was activated;（4）Increased expression of MMPs and TIMP1,which inhibited the degradation of MMPs on ECM;（5）The expression of MDA and i NOS increased,iwhile Mn-SOD and SOD decreased,which implied that decreased liver capability to scavenge free radicals;（6）Increased IL-6,IL-1β,IL-11 and TNF-αm-RNA expression which demonstrated that the mouse liver occurred inflammation.Compared with the Model group,the LD and HD of DBWP groups underwent the following changes:（1）Reduced serum ALT and AST concentration and improved the liver pathological changes,and reduced the collagen fibers deposition;（2）Inhibited the activation of HSCs,and down-regulated the expression of Collagen1,Collagen3,andα-SMA;（3）TGF-β1→Smad signal pathway was blocked,and the development of fibrosis was also inhibited;（4）Up-regulated of MMPs expression,while inhibited of TIMP1 which manifested that enhanced degradation ability of MMPs on ECMs and reduced deposition of ECMs;（5）Reduced expression of IL-6,IL-1β,IL-11 and TNF-αwhich implied that the liver inflammation was inhibited;（6）Down-regulated expression of MDA and i NOS,while increased expression of Mn-SOD and SOD,which suggested that the liver antioxidant capability was enhanced,thus protected the liver cell membrane from being damaged.Conclusion Combined with network pharmacology analysis,DBWP had good protective effect on liver fibrosis induced by CCl₄,which mechanisms are involving the activation of HSC,blocking the TGF-β1/Smad2/3 signaling pathway,the generation and degradation of ECM,anti-inflammatory and anti-oxidation effects.Figure 15;Table 5;References 165...