Mechanism Of Gandoufumu Decoction On Wilson’s Disease Liver Fibrosis Model TX Mice And HepG2 Cell Model Of Copper Loading In Vitro Based On Autophagy By PI3K/AKT/mTOR Signal Pathway

Experiment I:Effect of Gandoufumu Decoction on TX Mice Based on Autophagy by PI3K/Akt/mTOR Signal Pathway in VivoObjective:In this section,toxic milk mice,an animal model of liver fibrosis of Wilson’s disease,are used as research objects.Gandoufumu decoction,an effective traditional Chinese medicine compound,is used as an intervention method.The point is to explore the mechanism of Gandoufumu decoction in treating Wilson’s disease liver fibrosis,focusing on regulating autophagy and inhibiting liver fibrosis by PI3K/Akt/mTOR signal pathwayMethods:Fifty toxic milk mice were randomly divided into model group,Gandoufumu decoction high,medium,low dose group and penicillamine group.Ten wild type mice of the same genetic background were used as control group.Based on 9 times the daily dosage of a 70kg adult,the Gandoufumu decoction group was administered gavage at 31.2,15.6,and 7.8g crude drug/kg in the high,medium,and low dose groups.The penicillamine group was formulated into a suspension with distilled water at a concentration of 0.1 g/kg.The mice were administered orally with a volume of 0.2 mL/10 g of the body weight of the mice,and the model group and the normal control group were administered the same volume of normal saline once a day for 4 weeks.Determination of serum HA,LN,PC-Ⅲ,C-Ⅳ,ALT,AST,TP,ALB,TBIL,superoxide dismutase,glutathione peroxidase,malondialdehyde,hydroxyl by ELISA;and superoxide dismutase,glutathione peroxidase,malondialdehyde and hydroxyproline of liver tissue;HE,Masson and Sirius red collagen staining to observe the pathological changes of liver fibrosis Transmission electron microscope was used to observe the ultrastructure of liver tissue;RT-qPCR was used to observe the expression of PI3K,AKT,mTOR,Beclin-1,LC3-I,LC3-Ⅱ,and p62 mRNA in liver tissue.Results:1.Effect of Gandoufumu decoction on Serum Biomarkers of Hepatic Fibrosis in toxic milk Mice(1)ALT,AST,TP,ALB,TBIL:①Compared with the control group,the activity of ALT and AST in the model group increased,the content of TP and ALB decreased,and the content of TBIL increased(P<0.01).②Compared with the model group,the activity of ALT and AST in Gandoufumu decoction high,middle-dose group and penicillamine group decreased,the content of TP and ALB increased,and the content of TBIL decreased(P<0.05,P<0.01);the content of TBIL in Gandoufumu decoction low dose group decreased(P<0.05).③Compared with Gandoufumu decoction high-dose group,AST activity of Gandoufumu decoction middle-dose group increased(P<0.05);Gandoufumu decoction low-dose group ALT and AST activity increased,TP content decreased(P<0.01).④Compared with the penicillamine group,ALT and AST activity were increased and TP content was decreased in the low-dose Gandoufumu decoction group(P<0.01)(2)HA,LN,PC-Ⅲ,C-Ⅳ and hydroxyproline:①Compared with the control group,the content of HA,LN,PC-Ⅲ,C-Ⅳ,and hydroxyproline in the model group increased(P<0.01).②Compared with the model group,the HA,LN,PC-Ⅲ,C-Ⅳ,and hydroxyproline contents of Gandoufumu decoction middle-dose group and penicillamine group were decreased(P<0.05,P<0.01);Gandoufumu decoction high-dose group HA,LN,PC-Ⅲ.The content of hydroxyproline decreased(P<0.01);HA content of Gandoufumu decoction low-dose group decreased(P<0.05).③Compared with the high-dose Gandoufumu decoction group,the contents of HA,LN,PC-Ⅲ,C-Ⅳ and hydroxyproline increased in the low-dose Gandoufumu decoction group(P<0.05,P<0.01).④Compared with the penicillamine group,the content of HA,LN,PC-Ⅲ,C-Ⅳ,and hydroxyproline in the low-dose Gandoufumu decoction group were increased(P<0.05,P<0.01)(3)Serum superoxide dismutase,glutathione peroxidase,malondialdehyde Compared with the control group,the serum superoxide dismutase and glutathione peroxidase activity of the model group decreased,and the malondialdehyde content increased(P<0.01).②Compared with the model group,the superoxide dismutase and glutathione peroxidase activity of Gandoufumu decoction high,middle dose group and penicillamine group increased,and the malondialdehyde content decreased(P<0.01);the indicators of Gandoufumu decoction low dose group did not change significantly(P>0.05)③Compared with Gandoufumu decoction high-dose group,superoxide dismutase and glutathione peroxidase activity in Gandoufumu decoction low-dose group decreased and malondialdehyde content increased(P<0.01)④Compared with the penicillamine group,the superoxide dismutase and glutathione peroxidase activity in the low-dose Gandoufumu decoction group were decreased,and the malondialdehyde content was increased(P<0.01)2.Effects of Gandoufumu decoction on liver tissue biomarkers in toxic milk mice(1)Liver tissue superoxide dismutase,glutathione peroxidase,malondialdehyde and hydroxyproline:①Compared with the control group,the activity of superoxide dismutase and glutathione peroxidase in the liver tissue of the model group decreased,and the content of malondialdehyde and hydroxyproline increased(P<0.01)②Compared with the model group,the superoxide dismutase and glutathione peroxidase activity of the high-dose Gandoufumu decoction group and the penicillamine group increased,and the malondialdehyde and hydroxyproline contents decreased(P<0.05,P<0.01);the Gandoufumu decoction middle-dose group hydroxyproline content decreased(P<0.01);Gandoufumu decoction low-dose group had no significant changes in indicators(P>0.05).③ Compared with the Gandoufumu decoction high-dose group,the superoxide dismutase activity of the Gandoufumu decoction low-dose group decreased,and the malondialdehyde and hydroxyproline levels increased(P<0.05,P<0.01).④Compared with the penicillamine group,the activity of superoxide dismutase and glutathione peroxidase in the middle-dose group of Gandoufumu decoction decreased and the content of malondialdehyde increased(P<0.05,P<0.01);the activity of superoxide dismutase and glutathione peroxidase in the low-dose group of Gandoufumu decoction decreased and the content of malondialdehyde and hydroxyproline were increased(P<0.05,P<0.01)(2)Semi-quantitative SSS score of liver tissue pathological fibrosis and Sirius red staining positive collagen area:①The SSS score of liver fibrosis and the percentage of collagen positive staining area in the model group were significantly higher than those in the control group(P<0.01).②Compared with the model group,the SSS score and percentage of collagen positive staining area of Gandoufumu decoction high,middle dose group and penicillamine group were decreased(P<0.05,P<0.01);Gandoufumu decoction low dose group had no significant change(P>0.05).③Compared with Gandoufumu decoction high-dose group,SSS score and percentage of collagen positive staining area in Gandoufumu decoction low-dose group increased(P<0.01).④Compared with the penicillamine group,the SSS score and percentage of collagen positive staining area in the low-dose Gandoufumu decoction group were increased(P<0.01)(3)Observation of the structure of autophagosomes in liver tissues by transmission electron microscopy:Compared with the control group,typical autophagosomes can be seen near the nucleus in the model group.The inner membrane of the phage is degraded by lysosomal enzymes,and a single layer of membrane structure around the autophagosome is also surrounded by some degraded cytosolic components and a small amount of autophagosome-like structure.Compared with the model group,the number of autophagosomes in the high and medium dose Gandoufumu decoction and penicillamine groups was reduced,and the number of autophagosomes in the low-dose Gandoufumu decoction group was not significantly changed.3.Effect of Gandoufumu decoction on PI3K/Akt/mTOR signal pathway and autophagy in toxic milk mice(1)Western blot to observe the expression levels of phospho-PI3K/PI3K,phospho-AKT/AKT,phospho-mTOR/mTOR,Beclin-1/Actin,LC3-Ⅱ/LC3-Ⅰ,p62/Actin in each group of liver tissues:①Compared with the control group,the expression levels of phospho-PI3K/PI3K,phospho-AKT/AKT,phospho-mTOR/mTOR,p62/Actin in the liver tissue of the model group were down-regulated,and the expression levels of Beclin-1/Actin,LC3-Ⅱ/LC3-Ⅰ Increased(P<0.01)②Compared with the model group,the expression levels of phospho-PI3K/PI3K,phospho-AKT/AKT,phospho-mTOR/mTOR,p62/Actin were increased in Gandoufumu decoction high and middle dose groups,Beclin-1/Actin,LC3-Ⅱ/LC3-ⅠThe expression level was down-regulated(P<0.05,P<0.01);the expression levels of phospho-PI3K/PI3K,phospho-AKT/AKT,phospho-mTOR/mTOR in the penicillamine group were up-regulated,Beclin-1/Actin,LC3-Ⅱ/LC3-Ⅰ The expression level of I was down-regulated(P<0.05,P<0.01);the indicators of Gandoufumu decoction low-dose group showed no significant change(P>0.05)③Compared with Gandoufumu decoction high-dose group,Gandoufumu decoction low-dose group phospho-PI3K/PI3K,phospho-AKT/AKT,phospho-mTOR/mTOR,p62/Actin expression levels were down-regulated,Beclin-1/Actin,LC3-Ⅱ/LC3-Ⅰ The expression level was up-regulated(P<0.01);the expression level of p62/Actin in Gandoufumu decoction mid-dose group was down-regulated(P<0.05);the expression levels of phospho-AKT/AKT,phospho-mTOR/mTOR and p62/Actin in the penicillamine group were down-regulated(P<0.01).④Compared with the penicillamine group,the expression levels of phospho-AKT/AKT,phospho-mTOR/mTOR,and p62/Actin were increased in the high-dose Gandoufumu decoction group(P<0.01);phospho-PI3K/PI3K,phospho-AKT/in the low-dose Gandoufumu decoction group The AKT expression level was down-regulated,and Beclin-1/Actin expression level was up-regulated(P<0.05,P<0.01)(2)Immunofluorescence observation of the expression levels of Beclin-1,LC3-Ⅱ,and p62 proteins in the liver tissue of mice in each group:The expressions of Beclin-1,LC3-Ⅱ in the liver tissue of the model group were higher than those of the control group,and the expressions of p62 were lower than those of the control group.The expressions of Beclin-1 and LC3-Ⅱ in Gandoufumu decoction high and middle dose groups and penicillamine group were lower than those in model group,and the expression of p62 was higher than that in control group.There was no significant change in expression between the low-dose group and the model group(3)RT-qRCR:The relative expression levels of PI3K,AKT,and mTOR mRNA in liver tissue of each group did not change significantly(P>0.05).Compared with the control group,the relative expression level of p62 mRNA in the model group was down-regulated(P<0.01),and the relative expression level of Beclin-1,LC3-Ⅰ and LC3-Ⅱ mRNA was up-regulated(P<0.01).②Compared with the model group,the relative expression levels of p62 mRNA in Gandoufumu decoction high and medium dose groups were up-regulated(P<0.01),and the relative expression levels of Beclin-1,LC3-Ⅰ,LC3-Ⅱ mRNA were down-regulated(P<0.05,P<0.01);The relative expression levels of p62 mRNA in the penicillamine group were up-regulated,and the relative expression levels of LC3-Ⅰ and LC3-Ⅱ mRNA were down-regulated(P<0.05,P<0.01);there was no significant change in the indicators of the low-dose Gandoufumu decoction group(P>0.05).③Compared with Gandoufumu decoction high-dose group,the relative expression level of p62 mRNA in Gandoufumu decoction low-dose group was down-regulated(P<0.01),and the relative expression level of Beclin-1,LC3-Ⅰ,LC3-Ⅱ mRNA was up-regulated(P<0.01);Gandoufumu decoction medium-dose group The relative expression levels of Beclin-1 and LC3-ⅡmRNA were up-regulated(P<0.01);the relative expression levels of Beclin-1 mRNA in the penicillamine group were up-regulated(P<0.01).④Compared with the penicillamine group,the relative expression level of Beclin-1 mRNA in Gandoufumu decoction high-dose group was down-regulated(P<0.01);the relative expression level of Beclin-1 mRNA in Gandoufumu decoction middle-dose group was down-regulated,and the relative expression level of LC3-Ⅱ mRNA was up-regulated(P<0.01);the relative expression level of p62 mRNA in Gandoufumu decoction low-dose group was down-regulated,and the relative expression level of LC3-Ⅰ and LC3-Ⅱ mRNA was up-regulated(P<0.01)Conclusions:1.Gandoufumu decoction can effectively reduce the serum liver function ALT and AST activity,increase the content of TP and ALB,reduce the content of HA,LN,PC-Ⅲ and C-Ⅳ of toxic milk mice,and improve the histopathological changes of liver fibrosis,which has a good anti liver fibrosis effect2.Gandoufumu decoction can increase the activity of superoxide dismutase and glutathione peroxidase,reduce the content of malondialdehyde and hydroxyproline in serum and liver tissue of toxic milk mice,reduce the autophagy activity of liver tissue,and inhibit the PI3K/AKT/mTOR signal pathwayExperiment Ⅱ:Effect of GandouFumu Decoction on Autophagy in CuCl2-induced HepG2 Cell Based on PI3K/AKT/mTOR Signal PathwayObjectives:This part takes the copper-loaded HepG2 cells as the research object,based on the PI3K/AKT/mTOR signal pathway to explore the effect of Gandoufumu decoction-containing serum on autophagy of copper-loaded HepG2 cells.Methods:CuCl2(200 μmol/L)was used to establish a copper-loaded HepG2 cell model HepG2 cells were divided into a control group(HepG2 cells+normal rat serum),a model group(HepG2 cells+CuCl2+normal rat serum),and a Gandoufumu decoctiongroup(HepG2 cells).+CuCl2+Gandoufumu decoctionserum),inhibitor group(HepG2 cells+NVP-BEZ235+normal rat serum),Gandoufumu decoction+NVP-BEZ235 group(HepG2 cells+NVP-BEZ235+Gandoufumu decoctiondrug serum).ELISA method was used to determine cell superoxide dismutase,glutathione peroxidase activity and malondialdehyde content;Flow cytometry to observe cellular ROS fluorescence intensity;Immunofluorescence observation of LC3-II protein expression;Western blot was used to observe cells phospho-PI3K/PI3K,phospho-AKT/AKT,phospho-mTOR/mTOR,Beclin-1/Actin,LC3-II/LC3-I,P62/Actin expression;RT-qPCR observation of cell PI3K,AKT,mTOR,Beclin-1,LC3-Ⅰ,LC3-Ⅱ,p62 mRNA expressionResults:1.Cell superoxide dismutase,glutathione peroxidase activity and malondialdehyde content:①Compared with the relative,the superoxide dismutase and glutathione peroxidase activity of the model group cells decreased,and the malondialdehyde content increased(P<0.01).②Compared with the model group,the 10%Gandoufumu decoction drug-containing serum group superoxide dismutase,glutathione peroxidase activity increased,malondialdehyde content decreased(P<0.01);NVP-BEZ235 group glutathione peroxidase activity decreased,malondialdehyde content increased(P<0.05).③Compared with the 10%Gandoufumu decoctiondrug-containing serum group,the superoxide dismutase,glutathione peroxidase activity of the 10%Gandoufumu decoction+NVP-BEZ235 group decreased,and the malondialdehyde content increased(P<0.01)2.Flow cytometry:①Compared with the control group,the ROS content of the model group increased(P<0.01).②Compared with the model group,the ROS content of the 10%Gandoufumu decoctiondrug-containing serum group decreased(P<0.01).③Compared with the 10%Gandoufumu decoctiondrug-containing serum group,the ROS content of the 10%Gandoufumu decoction+NVP-BEZ235 group increased(P<0.05)3.Immunofluorescence:LC3-Ⅱ 4immunofluorescence was red fluorescence,compared with the control group,the model group LC3-Ⅱ expression was up-regulated.Compared with the model group,the expression of LC3-Ⅱ was down-regulated in the 10%Gandoufumu decoction group.Compared with the 10%Gandoufumu decoction group,the expressions of LC3-Ⅱ were up-regulated in the 10%Gandoufumu decoction+NVP-BEZ235 group4.Western Blot:Compared with the control group,the expression levels of phospho-PI3K/PI3K,phospho-AKT/AKT,phospho-mTOR/mTOR,p62/Actin in the model group were down-regulated,and the expression levels of Beclin-1/Actin,LC3-Ⅱ/LC3-Ⅰ were up-regulated(P<0.01).②Compared with the model group,the expression levels of phospho-PI3K/PI3K,phospho-AKT/AKT,phospho-mTOR/mTOR,p62/Actin were increased in the 10%Gandoufumu decoction group,and the expression levels of Beclin-1/Actin,LC3-Ⅱ/LC3-Ⅰ down-regulated(P<0.05,P<0.01);NVP-BEZ235 group phospho-PI3K/PI3K,phospho-mTOR/mTOR expression levels were down-regulated,Beclin-1/Actin,LC3-Ⅱ/LC3-Ⅰ expression levels were up-regulated(P<0.05,P<0.01).③Compared with the 10%Gandoufumu decoction group,the expression levels of phospho-AKT/AKT and phospho-mTOR/mTOR in the 10%Gandoufumu decoction+NVP-BEZ235 group were down-regulated,and the expression levels of Beclin-1/Actin and LC3-Ⅱ/LC3-Ⅰ were up-regulated(P>0.05,P<0.01)5.RT-qPCR:①The relative expression of PI3K,AKT and mTOR mRNA in each group did not change significantly(P>0.05).Compared with the control group,the relative expression of p62 mRNA in the model group was down-regulated,and the relative expression of Beclin-1,LC3-Ⅰ,LC3-Ⅱ mRNA Increased(P<0.01)②Compared with the model group,the relative expression level of p62 mRNA in the 10%Gandoufumu decoction group was up-regulated,and the expression level of Beclin-1,LC3-Ⅰ and LC3-Ⅱ mRNA was down-regulated(P<0.01);the relative expression level of Beclin-1 mRNA in the NVP-BEZ235 group was up-regulated(P<0.05).③Compared with the 10%Gandoufumu decoction group,the relative expression levels of Beclin-1 and LC3-Ⅱ mRNA in the 10%Gandoufumu decoction+NVP-BEZ235 group were increased(P<0.01)Conclusions:1.Gandoufumu decoction can increase the superoxide dismutase and glutathione peroxidase activity,reduce ROS and malondialdehyde content,inhibit autophagy activity,and up-regulate the phosphorylation level of PI3K/AKT/mTOR signal pathway of copper-loaded HepG2 cells2.PI3K/AKT/mTOR signal pathway blocker NVP-BEZ235 can partially attenuate the effect of Gandoufumu decoction on the inhibition of autophagy in copper-loaded HepG2 cells3.Gandoufumu decoction may inhibit autophagy through PI3K/AKT/mTOR signal pathway to delay Wilson’s disease liver fibrosis.