| Objective: To observe the therapeutic effect of Modified Chaihu Danggui Powder（MCDP）treating hepatic fibrosis models rat induced by CCl4 and PDGF-BB activated rat hepatic stellate cells（HSC-T6）and explore its molecular biological mechanism.Methods:In vivo experiment: 10 of 60 SPF male SD rats were randomly selected as control group,and the other 50 rats were induced for hepatic fibrosis by CCL4,10 of which were used to verify the modeling.At the end of the 8th week,40 rats Confirmed as hepatic fibrosis were divided into 4 groups with10 rats in each group,which were model group,MCDP high-dose group,MCDP medium-dose group and MCDP low-dose group.The control group and model group were given gavage with an equal dose of normal saline,while the MCDP group was given different doses of MCDP by gavage,once a day,for 4 weeks.During the experiment,the appearance and weight of rats were observed.After the experiment,blood was collected from the abdominal aorta and the liver was removed.The pathological changes in liver tissue were observed through HE and Masson staining.The serum levels of ALT,AST,and ALB were detected.ELISA method was used for detecting HA,LN,COL-I,PⅢNP,α-SMA,TNF-α,IL-6,and IL-1β in serum.Western blot and qRT-PCR method were used to detect the expression of PI3K,AKT,Bcl-2,Bax proteins and mRNAs.In vitro experiment,serum with MCDP was prepared and hepatic stellate cell proliferation model was established.Control group（10% fetal bovine serum）,model group（10% normal rat serum+PDGF-BB）,traditional Chinese medicine group（PDGF-BB+10% MCDP rat serum）,agonist+traditional Chinese medicine group（PDGF-BB+10% MCDP rat serum+HY-101625）,inhibitor+traditional Chinese medicine group（PDGF-BB+10% MCDP rat serum+LY294002）were set.After 24 h of continuous intervention,the morphology of HSC-T6 cells was observed under the microscope.The proliferation activity and apoptosis level of HSC-T6 cells were detected.ELISA was used to detect MMP-2,MMP-9,TIMP-1,COL-Ⅰ,and α-SMA.The expression levels of PI3K,AKT,Bcl-2,and Bax were detected by Western blot and qRT-PCR.Results:In vivo experiments:（1）Fur color and mental state of rats in the model group were worse than those in the control group,and all treatment groups with MCDP were better than those in the model group,and the improvement was most obvious in the high-dose group.（2）In HE and Masson staining,compared with the model group,liver fibrosis,inflammatory cell infiltration and liver cell necrosis were reduced on different degrees in each MCDP treatment group,and the improvement was most obvious in the high-dose group.（3）Compared with blank control group,ALT and AST were increased and ALB were decreased in other groups（P < 0.05）.Among all MCDP treatment groups,ALT and AST were lowest and ALB was highest in high-dose group（P < 0.05）.（4）Compared with blank control group,serum TNF-α,IL-6 and IL-1β in model group were increased,and the difference was statistically significant（P < 0.05）.Compared with model group,TNF-α,IL-6and IL-1β of MCDP treatment groups decreased with the increase of dose（P < 0.05）.（5）Serum HA,LN,COL-I,PⅢNP and α-SMA levels in the model group were the highest.In the MCDP treatment groups,as the dosage increased,the levels of HA,LN,COL-I,PⅢNP,and α-SMA decreased（P<0.05）.（6）（1）WB result: Compared with the control group,the protein levels of PI3K,AKT and Bcl-2 in liver tissues of the other groups increased,and while the proteinn level of Bax decreased（P<0.05）.The levels of PI3K,AKT,and Bcl-2 in the high-dose group were the lowest,while Bax level was the highest（P<0.05）.（2）The results of qRT-PCR: The mRNA of PI3K,AKT,and Bcl-2 in liver tissues of the control group and the MCDP groups increased,while the Bax decreased compared with the control group（P<0.05）.With the increase of dosage,the mRNA of PI3K,AKT,and Bcl-2 decreased,while the Bax level increased（P<0.05）.In vitro experiments:（1）Inverted phase contrast microscopy showed that HSC-T6 growth in the model group was relatively balanced,and the density was higher than that in the control group,with a fuller cell body,relatively rapid growth,and easy clustering.The number of cells in the MCDP group,agonist+MCDP group,and inhibitor+MCDP group decreased,and cell growth was inhibited.They didn’t form clusters and were nearly quiescent phase.（2）The inhibitor+MCDP group had the lowest proliferation activity,and the highest apoptosis rate（P<0.05）.（3）The inhibitor+MCDP group had the highest apoptosis rate（P<0.05）.（4）Among the drug intervention groups,the expression levels of TIMP-1,COL-I and α-SMA in the inhibitor+MCDP group were the lowest,while the expression levels of MMP-2 and MMP-9were the highest（P<0.05）.（5）Among the drug intervention groups,the expression levels of TIMP-1,COL-I and α-SMA in the inhibitor+MCDP group were the lowest,while the expression levels of MMP-2 and MMP-9were the highest（P<0.05）.（6）Compared with the model group,the protein expression levels of PI3K,AKT,and Bcl-2 in each drug intervention group were downregulated,and the protein expression level of Bax was upregulated（P<0.05）.Among them,the protein expression levels of PI3K,AKT,and Bcl-2 in the inhibitor+MCDP group were the lowest,while the protein expression level of Bax was the highest（P<0.05）.（7）Compared with the control group,the mRNA expression levels of PI3K,AKT,and Bcl-2 in each drug intervention group were downregulated,and the mRNA expression level of Bax was upregulated（P<0.05）.Among them,the mRNA expression levels of PI3K,AKT,and Bcl-2 in the inhibitor+MCDP group were the lowest,while the mRNA expression level of Bax was the highest.The mRNA expression levels of PI3K,AKT,and Bcl-2 in the agonist+MCDP group were higher than those in the MCDP group,while the mRNA expression level of Bax was lower than that in the MCDP group（P<0.05）.Conclusion（s）: MCDP can significantly improve hepatic inflammatory injury and fibrosis in rats induced by CCl4.The mechanism may be related to regulation of PI3K/AKT/Bcl-2 signaling pathway,downregulation of inflammatory factors and promotion of collagen degradation.The serum with MCDP can inhibit the proliferation of HSC-T6 activated by PDGF-BB and promote apoptosis.It can promote the degradation of extracellular matrix and reduce the synthesis of extracellular matrix by regulating the balance of MMP-2,MMP-9 and TIMP-1.The anti-hepatic-fibrosis effect of MCDP may be related to the regulation of PI3K/AKT/Bcl-2 pathway,inhibition of hepatic stellate cell activation and promotion of apoptosis. |
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