Research On The Mechanism Of Gandoufumu Decoction To Treat Liver Injury Of Wilson Disease By Regulating SLC7A11/GPX4 Pathway To Inhibit Ferroptosis

Objectives:Liver injury is one of the most common manifestations of wilson disease.Without early and effective intervention,it can progress to cirrhosis or fulminant hepatic failure,and even cause death,which brings a heavy burden to the family and society.Early and effective intervention is an important means to treat it.Our previous studies have proved that GandouFuMu Decoction（GDFMD）can effectively improve the symptoms of liver injury in wilson disease.Recent studies have shown that ferroptosis is closely related to the pathogenesis of liver injury.Based on this,this study focused on clarifying whether ferroptosis was involved in the pathogenesis of liver injury in Wilson’s disease.To clarify the mechanism of inhibiting ferroptosis and preventing liver injury in hepatolenticular degeneration by GDFMD in vivo.Based on SLC7A11/GPX4 pathway,the mechanism of inhibition of ferroptosis in HepG2 cells by GDFMD in vitro was investigated.To explore the mechanism of GDFMD in the treatment of liver injury in Wilson disease,and to provide scientific basis for clinical treatment and new drug research and development.Methods:PartⅠ20 TX-j mice were randomly divided into model group,Deferoxamine mesylate（DFO）group,Tetrathiomolybdate（TM）group and TM+DFO group.25 wild-type DL mice with the same genetic background were randomly divided into control group,high copper group,high iron group,high copper+TM group and high iron+DFO group.In TM group,DFO group and TM+DFO group,the purified diet was fed for 28 days,and then injected intraperitoneally with TM,DFO and TM+DFO reagents for 7 days.High copper group and high iron group were fed standard AIN-76A diet for 28 days,respectively,and then given normal saline for intrabitoneal injection for 7 days.High copper+TM group:after feeding high copper diet for 28d according to the method of high copper group,intraperitoneal injection of TM was given according to the method of TM group for 7 days;High iron+DFO group:After feeding high iron feed for 28d according to the method of high iron group,intraperitoneal injection of DFO according to the method of DFO group was given for 7d;Model group and control group were fed ordinary purified feed for 28 days,and then given normal saline intraperitoneally for 7days.ELISA was used to detect four items of serum liver fiber.The contents of serum liver function,copper ion,iron ion,GSH and MDA in liver tissue were determined by colorimetry.ROS in liver tissue was detected by flow cytometry.The histopathological changes of liver were observed by HE and Masson staining.Mitochondrial structure of hepatocytes was observed by transmission electron microscopy.The expression of related proteins in liver tissue was detected by Western Blot.RT-qPCR was used to detect mRNA expression in liver tissues.PartⅡ40 TX-j mice were randomly divided into GDFMD low,medium and high-dose groups,D-penicillamine（PCA）group and model group,and 8 wild-type DL mice with the same genetic background were used as normal control group.According to the principle of equivalent dose conversion between animal and human,the low,medium and high dose groups of GDFMD were given 13.92,6.96 and 3.48g/kg crude drug,respectively.In PCA group,penicillamine tablets were ground into extremely fine powder,which was mixed into suspension with secondary ultra-pure water,and the net dosage of penicillamine at 0.1g/kg body weight was given by gavage.Mice were given0.2m L/10g mouse liquid intragastric administration,and the control group and model group were given equal volume of normal saline intragastric administration,once a day,for consecutive 4 weeks.ELISA was used to measure the serum levels of HA,LN,PC-Ⅲ and C-Ⅳ.The contents of serum AST,ALT,GSH and MDA in liver tissue were determined by colorimetry.ROS in liver tissue was detected by flow cytometry.The histopathological changes of liver were observed by HE and Masson staining.Mitochondrial structure in hepatocytes was observed by transmission electron microscopy.The expression of related proteins in liver tissue was detected by Western Blot.RT-qPCR was used to detect mRNA expression in liver tissues.PartⅢTo construct the ferroptosis cell model of HepG2 cells induced by Erastin.It is divided into three experiments for forward and reverse verification.The forward verification experiment was divided into 5 groups:Control group,model group,GDFMD low,medium and high dose group.Recovery experimentⅠwas divided into 5groups:Control group,model group,GDFMD group,inhibitor group and inhibitor+GDFMD group.Recovery experimentΠwas divided into 6 groups:Control group,model group,GDFMD group,si-SLC7A11 group,si-SLC7A11+GDFMD group and si-NC group.Colorimetric assay was used to detect GSH and MDA.ROS in cells was detected by flow cytometry.Morphological changes of cell mitochondria were observed by transmission electron microscopy.Fluorescence probe was used to examine lipid peroxides and ferrous ions in mitochondria.Immunofluorescence and Western Blot were used to detect the expression of related proteins in liver.RT-qPCR was used to detect mRNA expression in liver tissues.Results:PartⅠ1 Serum AST,ALT):（1）Compared with the control group,AST and ALT in the model group,the high iron group and the high copper group were significantly increased（P<0.01）;（2）Compared with the model group,AST and ALT in the TM,DFO and TM+DFO group decreased significantly（P<0.05 or P<0.01）;（3）Compared with the high iron group,AST in the high iron+DFO group decreased significantly（P<0.01）;（4）Compared with the high copper group,AST and ALT in the high copper+TM group decreased（P<0.05 or P<0.01）.2 Serum HA,LN,PC-Ⅲ,C-Ⅳ:（1）Compared with the control group,the HA,LN,PC-Ⅲ and C-Ⅳ in the model group,the high iron group and the high copper group were increased（P<0.05 or P<0.01）;（2）Compared with the model group,the HA,LN,C-Ⅳ in the TM group decreased（P<0.05）.The HA,LN,PC-Ⅲ,C-Ⅳ in DFO group and TM+DFO group decreased（P<0.05 or P<0.01）.3 Contents of copper ion(Cu²⁺)and iron ion(Fe²⁺)in liver tissue:（1）Compared with the control group,Cu²⁺,Fe²⁺in the model group were increased（P<0.01）,Fe²⁺in the high iron group increased（P<0.01）,Cu²⁺increased in high copper group（P<0.01）;（2）Compared with the model group,the Cu²⁺in the TM group decreased（P<0.01）,Fe²⁺decreased in DFO group（P<0.01）,Cu²⁺and Fe²⁺decreased in TM+DFO group（P<0.01）;（3）Compared with the high iron group,the Fe²⁺of the high iron+DFO group decreased（P<0.01）;（4）Compared with the high copper group,the Cu²⁺of the high copper+TM group decreased（P<0.01）.4 Contents of GSH,MDA and ROS in liver tissue:（1）Compared with the control group,GSH in model group,high iron group and high copper group decreased,MDA and ROS increased（P<0.01）;（2）Compared with the model group,GSH was increased,MDA and ROS were decreased in TM group,DFO group and TM+DFO group（P<0.01）;（3）Compared with the high iron group,GSH increased,MDA and ROS decreased in the high iron+DFO group（P<0.01）;（4）Compared with high copper group,GSH increased,MDA and ROS decreased in high copper+TM group（P<0.01）.5 Liver histopathology（1）HE staining:Microscopically,the nucleus appears blue,while cytoplasm,interstitium,and fibrous tissue appear red.In the control group,the cell structure was normal,the cell size was uniform,the structure of the liver lobule was complete,and no pathological changes were observed.Compared with the control group,degeneration and necrosis of hepatocytes were observed in the model group,high copper group and high iron group,with different sizes,inflammatory cell infiltration,incomplete structure of hepatic lobules,and formation of fibrous septa,among which the lesion degree in the model group was the most serious.The degeneration and necrosis of hepatocytes,inflammatory cell infiltration and the structure of hepatic lobules were improved in the TM group,DFO group,TM+DFO group,high copper+TM group and high iron+DFO group.（2）Masson staining:Under the microscope,the nuclei of the cells were blue-brown,the cytoplasm and muscle fibers were red,and the collagen fibers were blue.In the control group,the cells had normal structure and orderly arrangement,and occasionally a little collagen fiber deposition was observed.Compared with the control group,the degree of collagen fiber deposition in the liver tissue of the model group,the high copper group and the high iron group was increased（P<0.01）;Compared with the model group,TM and DFO groups was reduced（P<0.01）;Compared with the high iron group,the high iron+DFO group was reduced in（P<0.01）;Compared with the high copper group,the high copper+TM group was reduced（P<0.01）.6 Mitochondrial structure of hepatocytes:Under transmission electron microscopy,mitochondrial damage,mitochondrial ridge breakage and mitochondrial swelling were found in the model group compared with the control group.In TM group,mitochondrial crista was broken and disappeared,and mitochondria became smaller.The mitochondrial crista structure of hepatocytes in DFO group was clear,but the mitochondrial swelling was obvious.In TM+DFO group,the mitochondrial crista structure of hepatocytes was still clear,and the mitochondrial morphology was not swollen and the number was relatively reduced.In addition,the crista structure of mitochondria of liver cells in the high copper group was clear,and the mitochondria were slightly swollen;the crista structure of mitochondria of liver cells in the high iron group was destroyed and disappeared,and the mitochondrial volume was reduced.The crista structure of fine mitochondria of liver cells in the high copper+TM group was not swollen,while the crista structure of mitochondria of liver cells in the high iron+DFO group was reasonable.7 Protein expression of SLC7A11/GPX4 pathway in liver tissue:（1）Compared with the control group,the expression of P53 protein in the model group and the high iron group increased,while the expression of SLC7A11 and GPX4 protein decreased（P<0.01）;（2）Compared with the model group,the expression of P53 protein in DFO group and TM+DFO group decreased,while the expression of SLC7A11 and GPX4protein increased（P<0.05 or P<0.01）;（3）Compared with the high iron group,the expression of P53 protein in the high iron+DFO group decreased,and the expression of SLC7A11 protein increased（P<0.05 or P<0.01）.8 mRNA expression of SLC7A11/GPX4 pathway in liver tissue:（1）Compared with the control group,the expression of P53 mRNA in the model group and the high iron group increased,while the expression of SLC7A11 and GPX4 protein decreased（P<0.01）;（2）Compared with the model group,the expression of P53 mRNA in DFO group and TM+DFO group decreased（P<0.05）,SLC7A11 and GPX4 mRNA expression increased（P<0.01）;（3）Compared with the high iron group,the expression of P53 mRNA in the high iron+DFO group decreased,while the expression of SLC7A11and GPX4 mRNA increased（P<0.01）.PartⅡ（1）AST,ALT:（1）Compared with the control group,AST and ALT in the model group were significantly increased（P<0.01）;（2）Compared with model group,AST and ALT in GDFMD-L,GDFMD-M,GDFMD-H and PCA groups were significantly decreased（P<0.01）.（2）HA,LN,PC-Ⅲ,C-Ⅳ:（1）Compared with the control group,HA,LN,PC-Ⅲ,C-Ⅳin the model group were increased（P<0.01）;（2）Compared with the model group,HA,LN,PC-Ⅲ,C-Ⅳin GDFMD-M,GDFMD-H and PCA groups decreased（P<0.05or P<0.01）.2 Effects of GDFMD on levels of GSH,MDA and ROS in liver tissue of mice in each group:（1）Compared with the control group,GSH in the model group decreased,MDA and ROS increased（P<0.01）;（2）Compared with the model group,MDA and ROS in the GDFMD-L group decreased（P<0.05 or P<0.01）;GSH increased and MDA and ROS decreased in GDFMD-M,GDFMD-H and PCA groups（P<0.01）.3 Effect of GDFMD on the liver histopathology of mice in each group（1）HE staining:The nucleus appears blue,while cytoplasm,interstitium,and fibrous tissue appear red.Compared with control group,the model group showed degeneration and necrosis of hepatocytes with different sizes,inflammatory cell infiltration,incomplete structure of hepatic lobules and formation of fibrous septa.GDFMD-L,GDFMD-M,GDFMD-H and PCA groups showed hepatocyte degeneration and necrosis,inflammatory cell infiltration,and the structure of hepatic lobules were improved to varying degrees.（2）Masson staining:The nuclei of the cells were blue-brown,the cytoplasm and muscle fibers were red,and the collagen fibers were blue.Compared with the control group,the degree of collagen fiber deposition in the liver tissue of the model group was increased（P<0.01）;Compared with the model group,the GDFTD-L,GDFTD-M,GDFTD-H and PCA groups was reduced（P<0.01）.4 Effects of GDFMD on mitochondrial structure of hepatocytes of mice in each group:Mitochondria were destroyed,mitochondrial crists were broken and disappeared,and mitochondria were swollen in model group compared with control group.The mitochondrial ridge breakage and disappearance of hepatoma cells in GDFMD-L,GDFMD-M,GDFMD-H and PCA groups were improved to varying degrees.5 Effects of GDFMD on the expression of SLC7A11/GPX4 pathway in liver tissue of mice in each group:（1）Compared with the control group,the expression of P53 protein in the model group increased,while the expression of SLC7A11 and GPX4protein decreased（P<0.01）;（2）Compared with the model group,the expression of P53protein in the GDFMD-L group decreased（P<0.05）,GPX4 protein expression significantly increased（P<0.01）,P53 protein expression decreased,SLC7A11 and GPX4 protein expression increased significantly in GDFMD-M,GDFMD-H and PCA groups（P<0.01）.6 Effects of GDFMD on SLC7A11/GPX4 pathway mRNA expression in liver tissues of mice in each group:（1）Compared with the control group,the expression of P53 mRNA in the model group was significantly increased,while the expression of SLC7A11 and GPX4 mRNA was significantly decreased（P<0.01）;（2）Compared with the model group,the expression of SLC7A11 and GPX4 mRNA was increased in the GDFMD-M group（P<0.01）;GDFMD-H and PCA groups showed decreased P53mRNA expression and increased SLC7A11 and GPX4 mRNA expression（P<0.01）.PartⅢ1 Effect of GDFMD on Erastin-induced ferroptosis of HepG2 cells by regulating SLC7A11/GPX4 pathway（1）Effects of GDFMD on the content of GSH,MDA and ROS in HepG2 cells induced by Erastin:（1）Compared with the control group,GSH in the model group decreased,and the MDA and ROS increased（P<0.01）;（2）Compared with the model group,the GDFMD-L,GDFMD-M and GDFMD-H groups had higher GSH and lower MDA and ROS（P<0.01）.（2）Effects of GDFMD on the expression of P53,SLC7A11 and GPX4 protein in HepG2 cells induced by Erastin:（1）Compared with the control group,the expression of P53 protein in the model group was increased,while the expression of SLC7A11 and GPX4 protein was decreased（P<0.01）;（2）Compared with the model group,the expression of SLC7A11 and GPX4 protein in the GDFMD-L group increased（P<0.01）.GDFMD-M and GDFMD-H groups showed significantly decreased expression of P53 protein and increased expression of SLC7A11 and GPX4 protein（P<0.01）.（3）Effects of GDFMD on the mRNA expressions of P53,SLC7A11 and GPX4in Erastin-induced HepG2 cells:（1）Compared with the control group,the expression of P53 mRNA in the model group was increased,while the expression of SLC7A11 and GPX4 mRNA was decreased（P<0.01）;（2）Compared with the model group,the expression of P53 mRNA in GDFMD-L,GDFMD-M and GDFMD-H groups decreased,while the expression of SLC7A11 and GPX4 mRNA increased（P<0.01）.2 Effect of GDFMD on Erastin-induced ferroptosis in HepG2 cells by counteracting or silencing SLC7A11 expression（1）Effects of GDFMD on GSH,MDA and ROS contents of HepG2 model cells with SLC7A11 expression inhibited or silencedRecovery experimentⅠ:Compared with the control group,the GSH in the model group decreased,and the MDA and ROS increased（P<0.01）;（2）Compared with the model group,the GSH in the GDFMD group increased,and the MDA and ROS decreased（P<0.01）,the GSH decreased and MDA and ROS levels increased in the inhibitor group（P<0.01）;（3）Compared with the inhibitor group,the GSH of the inhibitor+GDFMD group increased,and the MDA and ROS decreased（P<0.01）.Recovery experimentⅡ:（1）Compared with the control group,the GSH in the model group decreased,and the MDA and ROS increased（P<0.01）;（2）Compared with the model group,the GSH in the GDFMD group increased,and the MDA and ROS decreased（P<0.01）.The GSH in si-SLC7A11 group decreased,while MDA and ROS levels increased（P<0.01）;（3）Compared with the si-SLC7A11 group,the GSH of the si-SLC7A11+GDFMD group increased,and the MDA and ROS decreased（P<0.01）.（2）Effect of GDFMD on mitochondrial lipid peroxides of HepG2 model cells with SLC7A11 expression inhibited or silencedRecovery experimentⅠ:（1）Compared with the control group,the lipid peroxide（P<0.01）;（2）Compared with the model group,GDFMD decreased in the GDFMD inhibitor group and increased in the GDFMD inhibitor group（P<0.05 or P<0.01）;（3）Compared with the inhibitor group,the inhibitor+GDFMD group decreased（P<0.01）.Recovery experimentⅡ:（1）Compared with the control group,the lipid peroxide（P<0.01）;（2）Compared with the model group,the GDFMD group decreased,and the si-SLC7A11 group increased（P<0.05 or P<0.01）;（3）Compared with the si-SLC7A11group,the si-SLC7A11+GDFMD group decreased（P<0.01）.（3）Effect of GDFMD on mitochondrial morphological changes of HepG2model cells with SLC7A11 expression inhibited or silencedRecovery experimentⅠ:（1）Compared with the control group,mitochondrial crista was broken and disappeared in the model group.（2）Compared with the model group,the mitochondrial crista in the GDFMD group was improved,while the mitochondrial damage in the inhibitor group was more serious and the crista disappeared.（3）Compared with inhibitor group,mitochondrial cristae in inhibitor+GDFMD group was improved.Recovery experimentⅡ:（1）Compared with the control group,mitochondrial crista was broken and disappeared in the model group.（2）Compared with the model group,the mitochondrial cristae of the GDFMD group was improved,the mitochondrial damage of the si-SLC7A11 group was more serious.（3）Compared with si-SLC7A11group,the mitochondrial cristae in si-SLC7A11+GDFMD group was improved.（4）Effect of GDFMD on the content of ferrous ions in mitochondria of HepG2model cells with SLC7A11 expression inhibited or silencedRecovery experimentⅠ:（1）Compared with the control group,the content of mitochondrial Fe²⁺in the model group was increased（P<0.01）;（2）Compared with the model group,GDFMD decreased in the GDFMD inhibitor group and increased in the GDFMD inhibitor group（P<0.01）;（3）Compared with the inhibitor group,the inhibitor+GDFMD group decreased（P<0.01）.Recovery experimentⅡ:（1）Compared with the control group,the content of mitochondrial Fe²⁺in the model group was increased（P<0.01）;（2）Compared with the model group,the GDFMD group decreased,and the si-SLC7A11 group increased（P<0.01）;（3）Compared with the si-SLC7A11 group,the si-SLC7A11+GDFMD group decreased（P<0.01）.（5）Effect of GDFMD on the expression of P53,SLC7A11 and GPX4 proteins of HepG2 model cells with SLC7A11 expression inhibited or silencedRecovery experimentⅠ:（1）Compared with the control group,the protein expression of P53 was significantly increased,and the protein expressions of SLC7A11 and GPX4were decreased in model group（P<0.01）;（2）Compared with model group,the expression of P53 protein decreased,SLC7A11 and GPX4 protein increased in GDFMD group（P<0.01）,SLC7A11 and GPX4 protein expression decreased（P<0.01）;（3）Compared with the inhibitor group,the expression of P53 protein decreased,SLC7A11 and GPX4protein increased in the inhibitor+GDFMD group（P<0.01）.Recovery experimentⅡ:（1）Compared with the control group,the protein expression of P53 was significantly increased,and the protein expressions of SLC7A11and GPX4 were decreased in model group（P<0.01）;（2）Compared with model group,the expression of P53 protein decreased,SLC7A11 and GPX4 protein increased in GDFMD group（P<0.01）,SLC7A11 and GPX4 protein expression decreased in si-SLC7A11 group（P<0.01）,the protein expressions of P53,SLC7A11 and GPX4 were not significant in si-NC group（P<0.05）;（3）Compared with the si-SLC7A11 group,the expression of P53 protein was decreased,and the expression of SLC7A11 and GPX4protein was increased in the si-SLC7A11+GDFMD group（P<0.01）.（6）Effect of GDFMD on the expression of P53,SLC7A11 and GPX4 mRNA of HepG2 model cells with SLC7A11 expression inhibited or silencedRecovery experimentⅠ:（1）Compared with the control group,the mRNA expressions of P53 were increased,and the mRNA expressions of SLC7A11 and GPX4were decreased in the model group（P<0.01）;（2）Compared with model group,the mRNA expression of P53 decreased,SLC7A11 and GPX4 increased in GDFMD group（P<0.01）,SLC7A11 and GPX4 mRNA expression decreased in inhibitor group（P<0.01）;（3）Compared with inhibitor group,P53 mRNA expression decreased,SLC7A11 and GPX4 mRNA expression increased in inhibitor+GDFMD group（P<0.05 or P<0.01）.Recovery experimentⅡ:（1）Compared with the control group,the mRNA expressions of P53 were increased,and the mRNA expressions of SLC7A11 and GPX4were decreased in the model group（P<0.01）;（2）Compared with model group,the mRNA expression of P53 decreased,SLC7A11 and GPX4 increased in GDFMD group（P<0.01）,SLC7A11 and GPX4 mRNA expression decreased in si-SLC7A11 group（P<0.01）;（3）Compared with the si-SLC7A11 group,the mRNA expression of P53 was decreased,and the mRNA expressions of SLC7A11 and GPX4 were increased in the si-SLC7A11+GDFMD group（P<0.01）.Conclusions:There is abnormal iron metabolism in WD animal model TX-j mice,and ferroptosis is involved in the pathogenesis of WD liver injury.GDFMD can effectively improve the liver injury of WD,improve the antioxidant capacity of liver tissue,and restore mitochondrial morphology.The mechanism is related to up-regulating the expression of SLC7A11/GPX4 pathway and inhibiting ferroptosis of hepatocytes.Therefore,SLC7A11/GPX4 pathway may be a new target for GDFMD treatment of WD liver injury.