Based On The Role Of Hepatocytes To Produce ECM1 To Study The Basis Of The Main Effects Of Fuzheng Huayu Recipe Against Liver Fibrosis

Objective:Based on the CCl₄-induced mouse liver fibrosis model,the uniform design method was used to explore the best effect dose ratio and dose-effect relationship of the main active ingredients of Fuzheng Huayu Recipe PA（F01+F02）,and we establish hepatocyte specific ECM1 gene knockout mice in vivo and LO2 hepatocyte injury model in vitro to further clarify the compatibility mechanism of the anti-hepatic fibrosis.Method:1.Uniform design method to screen the optimal effect dose ratio of PA against liver fibrosis in CCl₄ mice:15%CCl₄ was used to prepare a mouse liver fibrosis model.On the first day of the 4th week,the model mice were randomly divided into model control group（CCl₄）,PA1 group,PA2 group,PA3 group,PA4 group,PA5 group,PA6group and PA group,with 10 mice in each group.All drugs group were given the corresponding drug 10m L/kg body weight by gavage,at the same time,the normal control group（Oil）and model control group（CCl₄）mice were given distilled water intragastrically,once a day for 3 weeks.Mice were sacrificed at the 6th weekend to obtain samples.Observe the general conditions of mice,including body weight,liver weight,liver-to-body ratio,spleen weight,spleen-to-body ratio;detect serum ALT,AST activity and ALB content;H&E staining to observe liver tissue inflammation and damage;SR staining to observe liver tissue collagen deposition;determine the content of Hyp in liver tissue by alkaline hydrolysis method.2.The dose-effect experimental study of PA against liver fibrosis in CCl₄ mice:15%CCl₄was used to prepare a mouse liver fibrosis model.On the first day of the 4th week,the model mice were randomly divided into model control group（CCl₄）,1/8PA group,1/4PA group,1/2PA Group,PA group,2PA group and FZHY group,with 10 mice in each group.All drugs group were given the corresponding drug 10m L/kg body weight by gavage,at the same time,the normal control group（Oil）and model control group（CCl₄）mice were given distilled water intragastrically,once a day for 3 weeks.Mice were sacrificed at the6th weekend to obtain samples.Observe the general conditions of mice,including body weight,liver weight,liver-to-body ratio,spleen weight,spleen-to-body ratio;detect serum ALT,AST activity and ALB content;H&E staining to observe liver tissue inflammation and damage;SR staining to observe liver tissue collagen deposition;determine the content of Hyp in liver tissue by alkaline hydrolysis method.3.Research on compatibility effect mechanism of PA against liver fibrosis in CCl₄-ECM1^hep-/-mice:Crispr/Cas9 was used to construct ECM1^hep-/-mice,and WT mice were used as parallel control experiments for ECM1^hep-/-mice.15%CCl₄ was used to prepare a mouse liver fibrosis model.On the first day of the 4th week,the model mice were randomly divided into model control group（CCl₄）,F01 group,F02 group,PA group（F01+F02）,and Positive group（Ps）,with 6 mice in each group.All drugs group were given the corresponding drug 10m L/kg body weight by gavage,at the same time,the normal control group（Oil）and model control group（CCl₄）mice were given distilled water intragastrically,once a day for 3 weeks.Mice were sacrificed at the 6th weekend to obtain samples.Observe the general conditions of mice,including body weight,liver weight,liver-to-body ratio,spleen weight,spleen-to-body ratio;detect serum ALT,AST activity and ALB content;H&E staining to observe liver tissue inflammation and damage;SR staining to observe liver tissue collagen deposition;determine the content of Hyp in liver tissue by alkaline hydrolysis method.ELISA method to detect liver tissue TGF-β1 activity and 4-HNE content;colorimetric method to determine liver tissue iron and GSH content;TBA method to determine liver tissue MDA content;immunohistochemical staining to detect liver tissue Col-Ⅰ,α-SMA,FTH1,TFRC and GPX4 expression;q PCR to detect liver tissueα-SMA,Col-Ⅰ,FTH1,FTL,ACSL4,PTGS2 and GPX4 m RNA expression;Western blot to detect liver tissueα-SMA,Col-Ⅰ,p Smad3/Smad3,TFRC and x CT protein expression.4.Study on the effect mechanism of F01 against CCl₄-induced LO2 cells damage:LO2cells were seeded in 96-well plates,6-well plates or 60mm culture dishes for 24 hours.Except for the control group,the remaining groups are stimulated with 0.4%CCl₄ for 6hours,and then the CCl₄injury solution was removed.The control group and model group were incubated with 0.1%DMSO,and each drug group was incubated with 0.2μM F01,2μM F01,20μM F01,10μg/m LECM1 or 1μM Fer-1,separately.The samples were collected after 24h.CCK8 method to detect cell viability;DCFH-DA fluorescent probe method to detect ROS activity level in cells;q PCR to detect ACSL4 and PTGS2 m RNA expression in cells;Western blot to detect ECM1,x CT and GPX4 protein expression in cells.5.Study on the effect mechanism of F01 against Erastin-induced LO2 cells ferroptosis:LO2 cells were seeded in 96-well plates,6-well plates or 60mm culture dishes for 24hours.Except for the control group,the remaining groups are stimulated with 50μM Erastin,at the same time,each drug group was stimulated with 0.1μM F01,1μM F01,10μM F01 or 10μg/m L ECM1,separately.The samples were collected after 24h.CCK8method to detect cell viability;DCFH-DA fluorescent probe method to detect ROS activity level in cells;q PCR to detect ACSL4 and PTGS2 m RNA expression in cells;Western blot to detect ECM1,x CT and GPX4 protein expression in cells.Result:1.Except for the PA1 and PA2 group,the other drug groups can significantly reduce the serum ALT（P<0.001）and AST（PA3,PA6,P<0.05;PA4,PA5,PA,P<0.01）activities,and increase the serum ALB content（PA2,P<0.05;PA4,PA6,PA,P<0.01）of the model control group mice.PA4,PA5,PA6 and PA can significantly alleviate inflammation damage and collagen deposition in liver tissues,and reduce liver tissue Hyp content（PA4,PA,P<0.01;PA5,PA6,P<0.001）and SPA（PA,P<0.05;PA6,P<0.01;PA5,P<0.001）in the model control group mice,among which the effect of PA5 is particularly significant.2.Except for the 2PA group,the other medication groups can significantly reduce the serum ALT（1/8PA,P<0.05;1/4PA,PA,P<0.01;1/2PA,FZHY,P<0.001）and AST（1/8PA,1/2PA,PA,P<0.01;PA,P<0.01;1/4PA,FZHY,P<0.001）activity,inflammatory injury of liver tissue,and liver tissue Hyp content（1/8PA,P<0.05;PA,FZHY,P<0.01;1/4PA,1/2PA,P<0.001）.Each medication group can significantly reduce liver tissue collagen deposition and SPA（2PA,P<0.05;1/8PA,1/4PA,1/2PA,PA,FZHY,P<0.001）.1/4PA has the most significant effect in reducing liver tissue collagen deposition and Hyp content,and has the same effect as the FZHY.3.In WT mice,compared with the model group,in each drug group mice,the serum ALT（PA,Ps,P<0.05;F01,F02,P<0.01）and AST（Ps,P<0.05;F01,F02,PA,P<0.01）activity were significantly reduced;the liver inflammation damage and collagen deposition were significantly reduced,and the Hyp content and SPA in liver tissue were significantly reduced.In ECM1^hep-/-mice,compared with the model group,the serum ALT activity of mice in the F02,PA and Ps groups was significantly reduced（F02,Ps,P<0.05;PA,P<0.01）,and the AST activity in the F02 and Ps groups was significantly reduced（F02,P<0.05;Ps,P<0.01）,there was no significant improvement in the F01 group;the liver inflammation damage and collagen deposition in the F02,PA and Ps groups were significantly reduced,and the Hyp content in liver tissue（PA,P<0.05;F02,Both Ps,P<0.01）and SPA（PA,P<0.05;F02,P<0.01;Ps,P<0.001）decreased significantly,and there was no significant improvement in the F01 group.In WT mice,the medication group could significantly reduce Col-Ⅰm RNA（Ps,P<0.01;F01,F02,PA,P<0.001）andα-SMA m RNA（F01,P<0.05;F02,PA,P<0.01）and protein（P<0.001）expression;reduced liver TGF-β1 activity and Smad3 phosphorylation level（P<0.05）.In ECM1^hep-/-mice,the above effects of F01 are eliminated,and the effects of F02 and PA were not significantly affected.In WT mice,the medication group can significantly reduce the iron content in the liver tissue of the model control group（F01,F02,PA,P<0.001;Ps,P<0.01）,and increase the liver tissue FTH1（F02,Ps,P<0.05;F01,PA,P<0.01）and FTL（P<0.05）m RNA expression,reduced liver tissue TFRC protein expression（F02,P<0.05;F01,PA,P<0.01）.In ECM1^hep-/-mice,the regulation of F01 on liver iron metabolism was abolished,and the effects of F02 and PA were not significantly affected.In WT mice,the medication group can significantly reduce the liver tissue MDA（F01,F02,P<0.05;Ps,P<0.01;PA,P<0.001）and 4-HNE（F01,P<0.01;PA,P<0.001）content,decrease the ACSL4（F01,F02,P<0.05;PA,P<0.01）and PTGS2（P<0.05）m RNA expression,increase liver x CT protein expression（P<0.05）,GPX4 m RNA（F02,P<0.01;F01,PA,Ps,P<0.001）expression and GSH（F01,Ps,P<0.05;F02,P<0.01;PA,P<0.001）level.In ECM1^hep-/-mice,the inhibitory effect of F01 on liver lipid peroxidation damage was abolished,and the effects of F02 and PA were not significantly affected.4.0.4%CCl₄ can significantly inhibit LO2 cells viability,increase the ROS activity level and the m RNA levels of ACSL4 and PTGS2 in LO2 cells,and reduce the protein expression of ECM1,x CT and GPX4 in LO2 cells.Compared with the model control group,F01 can decrease ROS activity level and ACSL4 and PTGS2 m RNA levels in a dose-dependent manner,and increase ECM1,x CT and GPX4 protein expression in LO2cells.Recombinant human ECM1 protein and Fer-1 can also play an anti-hepatocyte damage effect by improving the above indicators.5.50μM Erastin can significantly inhibit LO2 cells viability,increase the level of ROS activity and the m RNA levels of ACSL4 and PTGS2 in LO2 cells,and reduce the protein expression of ECM1,x CT and GPX4 in LO2 cells.Compared with the model control group,F01 can dose-dependently reduce ROS activity level and ACSL4 and PTGS2m RNA levels,and increase ECM1,x CT and GPX4 protein expression in LO2 cells.Recombinant human ECM1 protein can also play an anti-hepatocyte ferroptosis effect by improving the above indicators.Conclusion:1.The combination of F01 and F02 has a dose-effect relationship for anti-liver fibrosis.When the dose of F01 is between 5.6-44.8 mg/kg and the dose of F02 is between 0.175-1.4 mg/kg,the optimal combination dose ratio of them is 32:1.The best effective dose combination is F01（5.6mg/kg）+F02（0.175mg/kg）.When F01 and F02exceed this range,its anti-liver fibrosis effect is weakened.2.ECM1 can play an anti-hepatocyte damage effect by inhibiting lipid peroxidation and reducing hepatocyte ferroptosis.3.The combination of F01 and F02 may regulate the activity of the x CT/GPX4 signaling pathway by up-regulating the expression of liver ECM1,and inhibit hepatocyte ferroptosis,thereby exerting an anti-liver fibrosis effect.Among them,F01 works by affecting ECM1 produced by hepatocytes,while the effect of F02 is hardly affected by ECM1.4.F01 inhibits hepatocyte lipid peroxidation damage by up-regulating the ECM1/x CT/GPX4 signaling pathway,thereby inhibiting hepatocyte ferroptosis.