Objective To investigate the effects of Niuhuang(Bovis Calculus,BC)and Shexiang(Moschus)(BC-Moschus)on human hepatocellular carcinoma(HCC)cells SMMC-7721 and a nude mouse model of subcutaneous xenografts,and to explor...Objective To investigate the effects of Niuhuang(Bovis Calculus,BC)and Shexiang(Moschus)(BC-Moschus)on human hepatocellular carcinoma(HCC)cells SMMC-7721 and a nude mouse model of subcutaneous xenografts,and to explore its anti-HCC mechanism.Methods The BC-Moschus combination was applied to two liver cancer models in vivo and in vitro.SMMC-7721 was divided into the BC-Moschus group and the control group,and different doses(rude drug dosage 0.625,1.25,2.5,and 5 mg/m L)of BC-Moschus extract were used for the intervention.The proliferation ability of HCC cells was detected using the Cell Counting Kit-8(CCK-8)assay,and the migration ability was detected by a wound healing assay.A subcutaneous xenograft model was prepared using nude mice with human HCC.Specific pathogen-free-grade BALB/c nude mice(5-week-old)were randomly divided into the following groups(n=6 per group):control(0.9%physiological saline 0.2 m L/d),BC-Moschus[BC 45.5 mg/(kg·d)+Moschus 13 mg/(kg·d)],and cisplatin(DDP,intraperitoneal injection5 mg/kg per week)groups.All groups were administered for 14 d.The volume and mass of the subcutaneous xenografts in nude mice were observed.The expression levels of phosphatidylinositol-3 kinase/protein kinase B/mammalian target of rapamycin(PI3K/AKT/mTOR)pathway,apoptosis-associated factor p70 S6 Kinase(S6K),Bax,Bcl-2,caspase-3,and caspase-9 in nude mice subcutaneous xenografts were measured by real-time quantitative PCR(RT-qPCR)and Western blot.Terminal Deoxynucleotidy Transferase-Mediated d UTP NickEnd Labeling(TUNEL)was used for quantitative analysis of apoptotic cells.Results The CCK-8 assay demonstrated that the BC-Moschus combination inhibited HCC cell proliferation in a superior manner to the use of BC and Moschus alone,and the inhibition effect was dose-and time-dependent(P<0.01).The wound healing assay showed that the BC-Moschus combination inhibited HCC cell migration(P<0.01).In the subcutaneous xenograft model of nude mice with human HCC,we found that the tumor volume and weight of the BC-Moschus group were lower than those of the control group(P<0.01).The levels of the PI3K/AKT/m TOR signaling pathway and S6K protein in the BC-Moschus and DDP groups were significantly decreased(P<0.01).The expression level of the anti-apoptotic gene Bcl-2 was downregulated(P<0.05),and the expression of the pro-apoptotic gene Baxand apoptosis-related factors caspase-3 and caspase-9 were significantly upregulated(P<0.01).The TUNEL assays further confirmed that the combination of the BC-Moschuas could promote HCC(P<0.01).Conclusion The BC-Moschus combination inhibited the proliferation and migration ability of HCC cells SMMC-7721 and effectively inhibited the growth of subcutaneous xenografts in nude mice.The mechanism may be closely related to the downregulation of the PI3K/AKT/mTOR pathway,regulation of apoptosis-related protein caspase-3,caspase-9,Bcl-2,and Bax expression,and promotion of apoptosis.展开更多
基金National Natural Science Foundation of China(81473617)Natural Science Foundation of Hunan Province(2020JJ4066)+1 种基金Scientific Research Project of Hunan Education Department(18A266)Hunan Graduate Scientific Research Innovation Project(QL20210173)。
文摘Objective To investigate the effects of Niuhuang(Bovis Calculus,BC)and Shexiang(Moschus)(BC-Moschus)on human hepatocellular carcinoma(HCC)cells SMMC-7721 and a nude mouse model of subcutaneous xenografts,and to explore its anti-HCC mechanism.Methods The BC-Moschus combination was applied to two liver cancer models in vivo and in vitro.SMMC-7721 was divided into the BC-Moschus group and the control group,and different doses(rude drug dosage 0.625,1.25,2.5,and 5 mg/m L)of BC-Moschus extract were used for the intervention.The proliferation ability of HCC cells was detected using the Cell Counting Kit-8(CCK-8)assay,and the migration ability was detected by a wound healing assay.A subcutaneous xenograft model was prepared using nude mice with human HCC.Specific pathogen-free-grade BALB/c nude mice(5-week-old)were randomly divided into the following groups(n=6 per group):control(0.9%physiological saline 0.2 m L/d),BC-Moschus[BC 45.5 mg/(kg·d)+Moschus 13 mg/(kg·d)],and cisplatin(DDP,intraperitoneal injection5 mg/kg per week)groups.All groups were administered for 14 d.The volume and mass of the subcutaneous xenografts in nude mice were observed.The expression levels of phosphatidylinositol-3 kinase/protein kinase B/mammalian target of rapamycin(PI3K/AKT/mTOR)pathway,apoptosis-associated factor p70 S6 Kinase(S6K),Bax,Bcl-2,caspase-3,and caspase-9 in nude mice subcutaneous xenografts were measured by real-time quantitative PCR(RT-qPCR)and Western blot.Terminal Deoxynucleotidy Transferase-Mediated d UTP NickEnd Labeling(TUNEL)was used for quantitative analysis of apoptotic cells.Results The CCK-8 assay demonstrated that the BC-Moschus combination inhibited HCC cell proliferation in a superior manner to the use of BC and Moschus alone,and the inhibition effect was dose-and time-dependent(P<0.01).The wound healing assay showed that the BC-Moschus combination inhibited HCC cell migration(P<0.01).In the subcutaneous xenograft model of nude mice with human HCC,we found that the tumor volume and weight of the BC-Moschus group were lower than those of the control group(P<0.01).The levels of the PI3K/AKT/m TOR signaling pathway and S6K protein in the BC-Moschus and DDP groups were significantly decreased(P<0.01).The expression level of the anti-apoptotic gene Bcl-2 was downregulated(P<0.05),and the expression of the pro-apoptotic gene Baxand apoptosis-related factors caspase-3 and caspase-9 were significantly upregulated(P<0.01).The TUNEL assays further confirmed that the combination of the BC-Moschuas could promote HCC(P<0.01).Conclusion The BC-Moschus combination inhibited the proliferation and migration ability of HCC cells SMMC-7721 and effectively inhibited the growth of subcutaneous xenografts in nude mice.The mechanism may be closely related to the downregulation of the PI3K/AKT/mTOR pathway,regulation of apoptosis-related protein caspase-3,caspase-9,Bcl-2,and Bax expression,and promotion of apoptosis.
