| BackgroundHepatocellular carcinoma(HCC),a common malignant tumor that originates from hepatocytes,is one of the most common and malignant cancers in the world.Presently,the clinical routinely treatment for HCC are mainly surgical resection,radiotherapy and chemotherapy.Usually,the effect is not ideal and the indications are narrow.Therefore,it is a major problem for us to prevent and treat HCC.Further exploring the molecular mechanism and preventing the occurrence of HCC,timely seeking new targets of drug therapy and delaying or reversing the development of hepatocarcinogenesis are important means to prevent the occurrence and development of HCC.The etiologies of HCC are sophisticated,among which oxidative stress(OS)is one of the most important inducements leading to cell aging and carcinogenesis.Reactive oxygen species(ROS)is the metabolite of oxidative stress response in vivo.It involves in regulating various physiological processes such as cell proliferation and migration.Excessive accumulation of ROS can cause lipid peroxidation,destroy the homeostasis of intracellular environment,promote the change of cell phenotype and then accelerate the occurrence of tumor.Nuclear factor E2 related factor 2(Nrf2)/heme oxygenase-1(HO-1)signal axis is important in anti-inflammatory and antioxidant and maintaining the stability of internal environment.Smad3 is the key protein of TGF-?1/Smad signal transduction pathway.During the development of hepatocarcinogenesis,the cell growth inhibition signal transmitted by pSmad3C is transformed to fibrogenic signal transmitted by pSmad2L/C and the cancer promoting signal by pSmad3L.Astragaloside?(AS-?)is the main active component of Astragalus membranaceus.Clinical and experimental studies have confirmed that AS-? has biological activities such as antiinflammatory,antioxidant and immune regulation.Previous studies have found that ASIV can reduce oxidative stress and inflammatory response by activating Nrf2/HO-1 pathway and protect endothelial cell injury induced by oxidized low density lipoprotein;furthermore,AS-? could delay the pathological process of liver fibrosis in rats by preventing epithelial mesenchymal transformation and inhibiting the invasion of liver cancer cells effectively;Our previous study found that AS-? can regulate TGF?1/Smad2/3 and Nrf2/HO-1 signal axes to inhibit Diethylinitrosamine(DEN)/carbon tetrachloride(CCl4)/C2H5OH-induced liver fibrosis in mice and collagen synthesis of hepatic stellate cell.At the same time,AS-? can regulate pSmad3C/3L,activate Nrf2/HO-1 signal pathway and inhibit TGF-?1-stimulated migration and invasion of hepatoma Huh7 cells.Whereas,TGF-?1/Smad may affect Nrf2/HO-1 pathway to regulate the process of hepatocarcinogenesis,and Smad3 is the key protein of TGF-?1/Smad signal transduction pathway.So,when selectively up regulation of pSmad3C and pSmad3L,how do the changes of Nrf2/HO-1 signal axis affect the proliferation,migration and invasion of HepG2 cells and AS-? intervention?Furthermore,how the Nrf2/HO-1 signal axis change regulate the process of hepatocarcinogenesis after Smad3 C-terminal phosphorylation site mutation and AS-? intervention?Based on the above researches,this project was designed to carry out the following three parts studies:? To observe the mechanism of TGF-?1 induced pSmad3C?pSmad3L on Nrf2/HO-1 signal axis to regulate the proliferation,migration and invasion of HepG2 cells and the intervention of AS-?;?To observe the effect of selectively up regulating the expression of pSmad3C/3L on Nrf2/HO-1 signal axis in inhibiting proliferation,migration and invasion of HepG2 cells and the intervention of AS-?;?To observe the effect of Smad3 C-terminal phosphorylation site mutation on Nrf2/HO-1 signal axis in inhibiting hepatocarcinogenesis on mice and the intervention of AS-?.PART ? Mechanism of TGF-?1 induced pSmad3C?pSmad3L on Nrf2/HO-1 signal axis to regulate proliferation,migration and invasion of HepG2 cells and the intervention of AS-?Objectives:To clarify the anti-hepatoma mechanism of AS-? in inhibiting TGF-?1 induced pSmad3C?pSmad3L to activate Nrf2/HO-1 signal axis and the regulation of proliferation,migration and invasion of HepG2 cells.Methods:? Effect of AS-? on the proliferation of TGF-?1 induced HepG2 cells;The logarithmic stage HepG2 cells were digested with trypsin,made into single cell suspension and inoculated into corresponding well plates.The experiment groups include vehicle control group,40 pM TGF-?1-stimulated group,AS-?(5?M?10?M?20 ?M)+TGF-?1-stimulated group.AS-? or/and TGF-?1 were added to the corresponding groups.MTT assay were applied to detect the effect of different concentrations of AS-? on the proliferation of HepG2 cells;Cell scratch were applied to detect the effect of different concentrations of AS-? on the migration of HepG2 cells;Transwell assay were applied to detect the effect of different concentrations of AS-? on the invasion of HepG2 cells;?Effect of AS-? on the oxidative stress reaction and Nrf2/HO-1 signal pathway related protein expression in TGF-?1-induced HepG2 cells;Cell treatment and experimental grouping are the same as PART ? Methods ?,and the concentration of TGF-?1 is 9 pM.DCFH-DA probe was used to detect the effect of different concentrations of AS-? on the intracellular ROS production;Immunofluorescence assay was applied to detect the effect of different concentrations of AS-? on the expression of Nrf2 and pNrf2 protein;Western blot was adopted to detect the effect of different concentrations of AS-? on the expression of pNrf2,Nrf2,HO-1 and NQO1 proteins in HepG2 cells;?Effect of AS-? on the TGF-?1/Smad2/3 related protein expression in TGF-?1induced HepG2 cells;Cell treatment and experimental grouping are the same as PART? Methods ?.The expression of pSmad3C protein in HepG2 cells was detected by immunofluorescence;Different concentrations of AS-? on pSmad2L/2C,Smad2,pSmad3C/3L,Smad3,?-SMA and PAI-1 proteins expression were detected by Western blot assay;Results:? MTT,Cell scratch,and Transwell assay showed that 5 ?M,10 ?M and 20?M AS-? significantly inhibited the cell proliferation,migration,and invasion of TGF?1-induced HepG2,separately(P<0.05).?DCFH-DA probe assay showed that TGF-?1 could promote ROS production in HepG2 cells,10 ?M and 20 ?M AS-? could significantly inhibit the accumulation of ROS(P<0.05).Immunofluorescence showed that 10 ?M and 20 ?M AS-? could significantly promote the expression of pNrf2 in TGF-?1-induced HepG2 cells;moreover,20 ?M AS-? could significantly promote the expression of Nrf2 in TGF-?1induced HepG2 cells;Western blot showed that 10 ?M and 20 ?M AS-? could significantly up regulate the expression of pNrf2 and NQO1,while 20 ?M AS-? could significantly promote the protein levels of Nrf2 and HO-1(P<0.05).? Immunofluorescence showed that 10 ?M and 20 ?M AS-? could significantly promote the expression of pSmad3C in TGF-pi-induced HepG2 cells;Western blot showed that 10 ?M AS-? could significantly inhibit the protein expression of pSmad2C;20 ?M AS-? could significantly promote the protein expression of pSmad3C;10 ?M and 20 ?M AS-? could significantly inhibit the protein expression of pSmad2L,pSmad3L and PAI-1(P<0.05);There was no significant change in the expression levels of Smad2 and Smad3 proteins;Different concentrations of AS-? can reduce a-SMA protein expression,however,there were no significant differences(P>0.05).Conclusions:TGF-?1 not only promotes pSmad3C ? pSmad3L to enhance the proliferation,migration and invasion of HepG2 cells,but also accompanied by a certain degree of Nr2/HO-1 signal axis inhibition.AS-? can inhibit pSmad3C?pSmad3L and promote the activation of Nrf2/HO-1 signal axis,so as to inhibit the proliferation,migration and invasion of HepG2 cells.PART ? Mechanism of selectively up regulation of pSmad3C/3L on Nrf2/HO-1 signal axis to regulate the proliferation,migration and invasion of hepatoma HepG2 cells and the intervention of AS-?Objectives:To clarify the effect of selectively up regulation of pSmad3C/3L on Nrf2/HO-1 signal axis and the mechanism of AS-? regulating the proliferation,migration and invasion of hepatoma HepG2 cells.Methods:? Western blot was used to verify the protein level of pSmad3C/3L in Smad3 WT,Smad3 EPSM and Smad3 3S-A plasmids stably transfected HepG2 cells;?Effect of AS-? on the proliferation,migration and invasion of TGF-?1-induced three plasmids stably transfected HepG2 cells;The logarithmic stage plasmidtransfected HepG2 cells were digested with trypsin,made into single cell suspension and inoculated into corresponding well plates.The experiment groups include Smad3 WT HepG2 group,Smad3 3S-A-HepG2 group,Smad3 EPSM HepG2 group,Smad3 WT HepG2+TGF-?1(40 pM)-stimulated group,smad3 3S-A-HepG2+TGF-?1stimulated group,Smad3 EPSM HepG2+TGF-?1-stimulated group,Smad3 WT HepG2+AS-?(20 ?M)+TGF-?1-stimulated group,Smad3 EPSM HepG2+AS-IV+TGF-?1-stimulated group,Smad3 3S-A-HepG2+AS-?+TGF-?1-stimulated group.ASIV or/and TGF-?1 were added to the corresponding groups.The effect of AS-? on the proliferation,migration and invasion of HepG2 cells transfected with three plasmids were detected by CCK-8,Cell scratch and Transwell assay,separately;?Effect of AS-? on the oxidative stress response and Nrf2/HO-1 signal pathway related protein expression in TGF-?1-induced three plasmids stably transfected HepG2 cells;Cell treatment and experimental grouping are the same as PART ? Methods?,in which the concentration of TGF-?1 is 9 pM.DCFH-DA probe was used to detect the effect of AS-? on the production of ROS in HepG2 cells transfected with three plasmids;Western blot assay was applied to detect the effects of AS-? on the expression of Nrf2,pNrf2,HO-1 and NQO1 proteins in HepG2 cells transfected with three plasmids;Real time fluorescence quantitative PCR(qPCR)was employed to detect the effect of AS-? on the level of HO-1 mRNA and NQO1 mRNA in HepG2 cells transfected with three plasmids;Results:?Western blot showed that there was no significant change in the expression of Smad3 protein in the three plasmid transfected cells;9 pM TGF-?1 can significantly promote the expression of pSmad3C/3L in Smad3 WT-HepG2 cells;At the same time,the level of pSmad3C in Smad3 EPSM-HepG2 group increased significantly,while the level of pSmad3L in Smad3 3S-A-HepG2 cells increased significantly(P<0.05).? CCK-8,Cell scratch and Transwell assay confirmed that TGF-?1 can significantly accelerate the proliferation,migration and invasion of HepG2 cells transfected with three plasmids,among which Smad3 3S-A-HepG2 group is the most obvious,and its effect is Smad3 3S-A-HepG2>Smad3 WT-HepG2>Smad3 EPSM-HepG2 cells;20?M AS-? can significantly inhibit the proliferation,migration and invasion of TGF-?1stimulated three plasmids transfected HepG2 cells.The inhibition efficiency was Smad3 EPSM-HepG2>Smad3 WT-HepG2>Smad3 3S-A-HepG2(P<0.05).? DCFH-DA probe assay showed that 20 ?M AS-? intervention can significantly inhibit ROS production of TGF-?1-stimulated three plasmids transfected HepG2 cells.The inhibition efficiency of ROS production in HepG2 cells transfected with three plasmids was Smad3 EPSM-HepG2>Smad3 WT-HepG2>Smad3 3S-A-HepG2 cells;qPCR results confirmed that AS-? could boost the expression of HO-1 mRNA and NQO1 mRNA in TGF-?1-stimulated three plasmids transfected HepG2 cells,with the Smad3 EPSM-HepG2 cells being the most obvious,followed by Smad3 WT-HepG2 cells;Western blot showed that AS-? could significantly up regulate the expression of pNrf2,Nrf2,HO-1 and NQO1 proteins in Smad3 WT-HepG2 and Smad3 EPSMHepG2 cells(P<0.05).Conclusions:Selectively upregulation of pSmad3C can activate Nrf2/HO-1 signal axis,enhance the efficiency of AS-? to inhibit the proliferation,migration and invasion of HepG2 cells;While selectively up regulation of pSmad3L had the opposite effect.PART ? Mechanism of pSmad3C+/-on Nrf2/HO-1 signal axis to inhibit hepatocarcinogenesis in mice and the intervention of AS-?Objectives:To clarify the mechanism of pSmad3C+/-on Nrf2/HO-1 signal axis to inhibit hepatocarcinogenesis in mice and the intervention of AS-?.Methods:? Genotyping was confirmed by PCR agarose gel electrophoresis.? Hepatic fibrosis and cancer model were induced by DEN/CCl4/C2H5OH in mice.The mice were randomly grouped into WT-control,WT model,WT-AS-?,HT control,HT model and HT-AS-?,with 20 mice in each group.The control groups were given sterile pellet feed and sterilized water.Mice in other groups were given intraperitoneal injection of 100 mg/kg DEN once a week for two weeks.From the third week to the end of the seventh week,5 ml/kg 20%CCl4 olive oil solution was administered by gavage twice a week,and 10%C2H5OH were given free drink at the same time,which was changed every other day.From the 8th week to the end of the 18th week,6 ml/kg 20%CCl4 olive oil solution was given by gavage twice a week.The modeling was stopped at the 19th week,and regular diet and water were given until the 20th week.From the beginning of modeling to the end of the 18th week,AS-? group was administered by gavage at 40 mg/kg once a day.The gross of liver was observed and the incidence of tumor was evaluated.HE and Masson staining were used to detect the histopathological changes of livers;? Effect of AS-? on oxidative stress response and Nrf2/HO-1 signaling pathway protein in hepatic fibrosis and cancer stage mice with Smad3 C-terminal phosphorylation site mutation;The experimental grouping was the same as PART ?Methods?.The assay kit was used to detect the effect of AS-? on the oxidative stress indexes MDA and SOD in the liver tissues of pSmad3C+/-mutant mice;Immunofluorescence was used to detect the expression and intracellular distribution of pNrf2 protein in the liver tissue of pSmad3C+/-mutant mice;Western blot was used to detect the effect of AS-? on the expression of Nrf2,pNrf2,HO-1 and NQO1 protein in the liver tissue of pSmad3C+/-mutant mice;Results:? At the 12th week after DEN/CCl4/C2H5OH administration,the liver surface was rough and the edge was wrinkled,especially in heterozygous mice,with the formation of punctate particles on the surface.After AS-? treatment,the liver surface became smooth and the edge was sharp.Microscopic observation using HE and Masson staining showed that the liver lobule structure of the model group was damaged,inflammatory cell infiltration and fibrous tissue proliferation.Among them,some hepatocytes showed necrosis,steatosis and structural disorder in the portal area of the heterozygous mice.Hepatic lobules are surrounded by diffusely distributed collagen fibers to form pseudolobular structure.With the treatment of AS-?,the structure of hepatic lobules was improved,and a small amount of inflammatory cell infiltration and some collagen fibers were observed in heterozygotes.At the 20th week,the liver of the model group was dim,yellowish,hard,wrinkled,and granular white nodules were scattered.Among them,hemorrhagic necrotic tumor foci protruding from the liver lobe were formed on the liver surface of heterozygous mice.HE staining showed that the structure of liver lobules in the model group was unclear,hepatocytes were arranged disorderly,a large number of vacuolar necrotic areas were formed,the nuclei were stained differently,and the distribution of liver cancer lesions was more diffuse in the heterozygous mice.The above symptoms were significantly improved after AS-?treatment,and the anti-hepatoma effect of AS-? on wild-type mice was significantly stronger than that of heterozygous mice.Liver biopsy showed that the incidence of liver tumor after administration of DEN/CCl4/C2H5OH was 100%,and the incidence was significantly reduced after AS-? treatment.In particular,the incidence of tumor in wild-type mice was significantly decreased(P<0.05).?Oxidative indicators showed that after DEN/CCl4/C2H5OH administration for 20 weeks,the liver tissues MDA increased significantly,while the SOD value decreased,which was more obvious than that of 12th week.After treatment with 40 mg/kg/d AS-?for 12th and 20th week,the liver tissues MDA decreased,while the SOD value increased significantly.Among them,the change of wild-type group was more obvious than that of heterozygous mice(P<0.05).? Western blot showed that pNrf2,HO-1 and NQO1 protein levels were significantly higher in AS-? group than that of DEN/CCl4/C2H5OH group at 12th week(hepatic fibrosis stage)and 20th week(HCC stage).Immunofluorescence detection of pNrf2 protein expression further verified the above results.The pNrf2 protein level of wildtype mice at 20th week was significantly higher than that of heterozygous mice(P<0.05).Conclusions:Smad3 C-terminal phosphorylation site mutation can inhibit the activation of Nrf2/HO-1 pathway,promote oxidative stress response,and weaken the anti-hepatocarcinogenesis effect of AS-? in mice. |
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