Astragaloside Ⅳ Suppresses Hepatocellular Carcinoma And Regulates PSmad3C/L And Nrf2/HO-1 Pathways

BackgroundsHepatocellular carcinoma（HCC）is one of the malignant tumors with high incidence and mortality.HCC is triggered by a variety of complex hazards,mostly formed on the basis of chronic liver disease or cirrhosis.Currently,there are limited treatments available for HCC and the overall prognosis of most patients is poor.Therefore,more strategies for the prevention and treatment of HCC still need to be further explored.In recent years,studies have shown that some traditional Chinese medicine monomers have significant inhibitory effects on the proliferation,metastasis and angiogenesis of cancer cells.In addition,these traditional Chinese medicine monomers have the advantages of no side effects and adverse reactions.Astragaloside IV（AS-IV）,the monomer of Traditional Chinese medicine Astragalus,has shown attractive anticancer effects in some malignant tumors.At present,only studies have shown that AS-IV has inhibitory effects on hepatoma cells and tumor-bearing mice.However,the effects of AS-IV on the development of DEN/CCl₄/C₂H₅OH-induced HCC in C57BL/6J mice remains unclear.Preliminary studies of our group have shown that Compound Astragalus and Salvia miltiorrhiza extract（CASE）inhibits the progression of HCC by regulating the p Smad3C/L pathway in vitro and in vivo.And whether AS-IV,as one of the active ingredients of CASE,also exerts an inhibitory effect on HCC by regulating the p Smad3C/L pathway remains to be investigated.Meanwhile,one study have found that AS-IV alleviated acetaminophen-induced hepatotoxicity in mice by increasing the transcriptional activation of Nrf2 and enhancing the expression of HO-1.So,does the anti-HCC effect of AS-IV involve the regulation of Nrf2/HO-1 signaling pathway?With these questions,this study used DEN/CCl₄/C₂H₅OH-induced HCC in C57BL/6J mice and TGF-β₁-induced Hu H-7 cells to explore the effects and mechanisms of AS-IV from both in vivo and in vitro perspectives.Objectives1.To observe the effects of AS-IV on DEN/CCl₄/C₂H₅OH-induced HCC in C57BL/6J mice and further investigate whether the mechanism is related to the regulation of p Smad3C/L and Nrf2/HO-1 signaling pathways in vivo.2.To observe the effects of AS-IV on proliferation,migration and invasion of TGF-β₁-induced Hu H-7 cells and further investigate whether the mechanism is related to the regulation of p Smad3C/L and Nrf2/HO-1 signaling pathways in vitro.Methods1.The effects and mechanisms of AS-IV on DEN/CCl₄/C₂H₅OH-induced HCC in C57BL/6J miceGroups:control group,model group,AS-IV（20 mg/kg）group,AS-IV（40 mg/kg）group,AS-IV（80 mg/kg）group,Colchicine（0.1 mg/kg）group.Except for the control group,the other groups were combined with DEN/CCl₄/C₂H₅OH to induce HCC in mice.The AS-IV and Colchicine groups were given the corresponding doses by daily gavage,and the control group and model group were given the corresponding vehicle.The mice were killed to obtain the serum and liver tissue samples at the end of the 20th week.The indexes of TGF-β₁,ALT,AST,SOD and MDA were detected by the corresponding kits.HE staining was used for detection of liver histopathology.The protein levels of GST-P1,AFP,TGF-β₁,p Smad3C,p21,p Smad3L,c-Myc,Nrf2/p Nrf2and HO-1 were detected by Western blot.Immunofluorescence assay was performed to explore the expression of p Smad3C/L.2.The effects and mechanisms of AS-IV on the proliferation,migration and invasion of TGF-β₁-induced Hu H-7 cellsGroups:control group,TGF-β₁ group,TGF-β₁+AS-IV（5μM）group,TGF-β₁+AS-IV（10μM）group,TGF-β₁+AS-IV（20μM）group.Except for the control group,the other groups were induced with TGF-β₁（9 or 40 p M）.AS-IV groups were pretreated with AS-IV at different concentrations（5,10,and 20μM）for 24 h and the other groups were given the equal amount of vehicle.CCK-8,Cell wound scratch and Transwell invasion assays were respectively used to detect cell proliferation,migration and invasion ability.What’s more,the DCFH-DA assay were used to detect the level of ROS in Hu H-7 cells.The protein levels of p Smad3C,p21,p Smad3L,c-Myc,Nrf2/p Nrf2,and HO-1 were detected by Western blot.Immunofluorescence assay were performed to explore the expression and distribution of p Smad3C and Nrf2.Results1.AS-IV alleviated the pathological progression of DEN/CCl₄/C₂H₅OH-induced HCC in miceTumor lesions and granular nodular plaques were scattered in the liver of mice in the model group.While the number of tumor lesions and granular nodular plaques was reduced in the AS-IV administration group.HE staining showed that hepatocytes in the model group were disorganized,with obvious blue staining and heterogeneous nuclei of different sizes,and focal areas of HCC cells were visible in a large area.Compared with the model group,the extent of lesions in mice in the AS-IV administration group was improved.The detection of HCC marker proteins AFP and GST-P1 revealed that the levels of AFP and GST-P1 proteins in liver tissues of DEN/CCl₄/C₂H₅OH-induced mice were significantly increased compared with the control group.While with the increase of AS-IV administration dose,the levels of AFP and GST-P1 proteins in liver tissues of mice gradually decreased,which further verified the inhibitory effect of AS-IV on HCC in mice.In addition,the results of liver function indexes（ALT,AST）and oxidative damage indexes（SOD,MDA）showed that DEN/CCl₄/C₂H₅OH induced significant liver function damage and oxidative stress damage in mice,which were gradually attenuated after AS-IV administration.2.AS-IV regulated p Smad3C/L pathway to alleviate the pathological process of HCC in mice2.1 AS-IV decreased the level of TGF-β₁ in serum and liver tissues of mice with HCC.Both of ELISA kit and Western blot showed that the level of TGF-β₁were significantly increased in the model group.After the administration of different doses of AS-IV（20,40 and 80 mg/kg）,the levels of TGF-β₁ were gradually decreased.The above results suggested that AS-IV can decrease the level of TGF-β₁ in serum and liver tissues of mice with HCC.2.2 Effects of AS-IV on the protein expression of p Smad3C and p21 in liver tissues of mice with HCCWestern blot results showed that the expression of p Smad3C and p21 in AS-IV treatment group was enhanced compared with the model group,especially in the AS-IV（40,80 mg/kg）group.The above results indicated that AS-IV up-regulated the protein level of p Smad3C and p21 in mice liver tissue.Immunofluorescence experiments further showed that AS-IV increased the expression of p Smad3C protein.2.3 Effects of AS-IV on the protein expression of p Smad3L and c-Myc in liver tissues of mice with HCCWestern blot assay showed that the level of p Smad3L and c-Myc in AS-IV treatment group were decreased compared with the model group.And the protein levels of p Smad3L and c-Myc were most significantly decreased in the AS-IV（80mg/kg）group.The above results indicated that AS-IV down-regulated the protein level of p Smad3L and c-Myc in mice liver tissues.The results of Immunofluorescence assay further showed that the expression of p Smad3L was significantly increased in the model group.In contrast,the expression of p Smad3L protein in mouse liver tissues was attenuated after AS-IV intervention.3.Effects of AS-IV on the expression of Nrf2/HO-1 pathway-related proteins in liver tissue of mice with HCCCompared with the model group,the level of p Nrf2 in the AS-IV（40 and 80mg/kg）group was significantly increased.At the same time,AS-IV administration also enhanced the expression of Nrf2 downstream target protein HO-1,and the AS-IV（80mg/kg）group had a significant statistical difference（P<0.01）.The above results suggested that AS-IV up-regulated the expression of Nrf2/HO-1 pathway-related proteins in mouse liver tissue.4.AS-IV inhibited proliferation,migration and invasion of TGF-β₁-induced Hu H-7 cellsThe stimulation of TGF-β₁（40 p M）facilitated the proliferation,migration and invasion of Hu H-7 cells.Co-treatment with AS-IV suppressed the proliferation,migration,and invasion of TGF-β₁-induced Hu H-7 cells in a dose-dependent fashion.It is suggested that AS-IV can exert anti-HCC effects in vitro by inhibiting the proliferation,migration and invasion of TGF-β₁-induced Hu H-7 cells.5.AS-IV reduced ROS production in TGF-β₁-induced Hu H-7 cellsThe results of DCFH-DA assay showed that the ROS production of the TGF-β₁group was markedly elevated compared to the control group.AS-IV treatment attenuated this elevation with increasing AS-IV concentration,which indicated that AS-IV could inhibit the production of ROS in Hu H-7 cells stimulated by TGF-β₁ and alleviate oxidative stress injury.6.AS-IV regulated the p Smad3C/L pathway in TGF-β₁-induced Hu H-7 cells6.1 Effects of AS-IV on the protein expression of p Smad3C and p21 in TGF-β₁-induced Hu H-7 cellsWestern blot results revealed that TGF-β₁ stimulation significantly increased the protein level of p Smad3C compared to the control group.AS-IV treatment further enhanced the expression of p Smad3C.Additionally,the levels of p Smad3C downstream target proteins p21 were also increased with the treatment of AS-IV.The above results indicated that AS-IV contributed to the enhancement of the p Smad3C and p21 protein in TGF-β₁-induced Hu H-7 cells.The Immunofluorescence assay further displayed that AS-IV increased the nuclear expression of p Smad3C protein.6.2 Effects of AS-IV on the protein expression of p Smad3L and c-Myc in TGF-β₁-induced Hu H-7 cellsWestern blot results showed that TGF-β₁ stimulation significantly increased the protein level of p Smad3L compared to the control group.However,after AS-IV treatment,the levels of p Smad3L and its downstream target protein c-Myc were decreased,among which TGF-β₁+AS-IV（10μM）group and TGF-β₁+AS-IV（20μM）group had significant statistical difference.The results implied that AS-IV suppressed the expression of p Smad3L and c-Myc proteins in TGF-β₁-induced Hu H-7 cells.7.Effects of AS-IV on the expression of Nrf2/HO-1 pathway-related proteins in TGF-β₁-induced Hu H-7 cellsWestern blot assay showed that the levels of Nrf2 and p Nrf2 were low in the TGF-β₁ group.However,with the co-treatment of AS-IV,the protein level of Nrf2 and p Nrf2 were gradually increased in a concentration dependent manner.Meanwhile,the expression of HO-1,the downstream target protein of Nrf2 was also significantly elevated by AS-IV pretreatment in TGF-β₁-induced Hu H-7 cells.This suggested that AS-IV activated the Nrf2/HO-1 pathway in TGF-β₁-induced Hu H-7 cells.Immunofluorescence assay further proved that AS-IV increased the nuclear expression of Nrf2 protein in a dose-dependent manner.Conclusions1.AS-IV inhibited the development of hepatocellular carcinoma in C57BL/6J mice induced by DEN/CCl₄/C₂H₅OH,and AS-IV may exert anti-HCC effects by regulating p Smad3C/L and Nrf2/HO-1 signaling pathways.2.AS-IV inhibited the proliferation,migration,invasion,and ROS production of TGF-β₁-induced Hu H-7 cells,and AS-IV may exert anti-HCC effects in vitro by p Smad3C/L and Nrf2/HO-1 signaling pathways.