BACKGROUND Finding active lead anti-hepatocellular carcinoma compounds from traditional Chinese medicine has important research value.AIM To assess the detailed mechanism of oxocrebanine,a compound separated from the ...BACKGROUND Finding active lead anti-hepatocellular carcinoma compounds from traditional Chinese medicine has important research value.AIM To assess the detailed mechanism of oxocrebanine,a compound separated from the traditional Chinese medicinal plant Stephania hainanensis H.S.Lo et Y.Tsoong,and to evaluate its inhibition of the proliferation of human hepatocellular carcinoma cells via apoptosis and autophagy.METHODS MTT,BrdU labeling,and colony formation assays were used to assess the inhibitory effect of oxocrebanine on the growth and proliferation of human hepatocellular carcinoma Hep3B2.1-7 cells.Flow cytometry was used to detect the effect of oxocrebanine on the apoptosis of Hep3B2.1-7 cells.Western blotting was used to assess the expression of apoptosis-related proteins in Hep3B2.1-7 cells.The aforementioned methods were also used to evaluate the effects of oxocrebanine on cell proliferation,autophagy markers,and autophagy-related protein expression levels after adding autophagy inhibitor 3-mA.Furthermore,to verify the anti-hepatocellular carcinoma effect of oxocrebanine in vivo,a nude mouse model was used to investigate the inhibitory effect of oxocrebanine treatment and its mechanism.Apoptosis was detected using a TUNEL assay and the expression of microtubule-associated protein 1 LC3 in tumor specimens was assessed using immunohistochemistry.RESULTS Oxocrebanine effectively inhibited the growth of Hep3B2.1-7 cells,whilst upregulating the protein expression of cleaved caspase-3,downregulating poly(ADP-ribose)polymerase 1 protein expression,increasing the levels of Bax and Bcl-2 antagonist/killer 1 protein expression,and decreasing the levels of Bcl-2 and myeloid cell leukemia 1 protein expression,which could promote apoptosis in Hep3B2.1-7 cells.Oxocrebanine promoted the transformation of LC3-I to LC3-II in Hep3B2.1-7 cells,suggesting the occurrence of autophagy,whilst the autophagy inhibitor 3-MA could reverse this process.Oxocrebanine was also shown to reduce the phosphorylation levels of the eukaryotic translation initiation factor 4EBP1 and ribosomal protein S6 kinase B1(P70S6K),two downstream effector molecules in the PI3K/Akt/mTOR pathway,inducing autophagy in Hep3B2.1-7 cells.Moreover,the tumor-bearing nude mouse experiment indicated that oxocrebanine effectively inhibited the growth of Hep3B2.1-7 cells in vivo.The results of the TUNEL assay and immunohistochemistry also revealed that oxocrebanine induced apoptosis in vivo and increased the expression level of LC3,an autophagy marker.CONCLUSION Oxocrebanine can inhibit the proliferation of human hepatocellular carcinoma cells by promoting apoptosis and inducing autophagy in vitro and in vivo.展开更多
目的观察磷脂酰肌醇-3激酶(PI3K)-蛋白质丝氨酸/苏氨酸激酶(AKt)-内皮型一氧化氮合酶(e NOS)信号转导通路在硫化氢(H2S)抑制内皮素-1(endothelin-1,ET-1)诱导心肌肥大过程中的作用。方法体外培养原代心肌细胞,将其随机分为6组,每组4孔,...目的观察磷脂酰肌醇-3激酶(PI3K)-蛋白质丝氨酸/苏氨酸激酶(AKt)-内皮型一氧化氮合酶(e NOS)信号转导通路在硫化氢(H2S)抑制内皮素-1(endothelin-1,ET-1)诱导心肌肥大过程中的作用。方法体外培养原代心肌细胞,将其随机分为6组,每组4孔,1对照组:加入等体积无血清的DMEM培养基;2肥大(ET-1)组:加入终浓度为10-8 mol/L的ET-1;剩余4组为实验组,各组分别加入不同终浓度的H2S供体-Na HS:310-15 M Na HS组:加入10-15 mol/L Na HS+10-8 mol/l ET-1;410-14 M Na HS组:加入10-14 mol/L Na HS+10-8 mol/L ET-1;510-13 M Na HS组:加入10-13 mol/L Na HS+10-8 mol/L ET-1;610-12 M Na HS组:加入10-12 mol/L Na HS+10-8 mol/L ET-1。上述各组药物分别刺激24 h后测定心肌细胞表面积、细胞总蛋白含量、培养液NO含量,RT-PCR检测心肌细胞心房利钠肽(atrial natriuretic peptide,ANP)、脑钠肽(B-type natriuretic peptide,BNP)、磷脂酰肌醇-3激酶(phosphatidylinositol-3-kinase,PI3K)、蛋白激酶B(protein kinase B,PKB/AKt)、e NOS m RNA水平,Western Blot技术检测总AKt和磷酸化AKt蛋白表达含量。结果肥大(ET-1)组的心肌细胞表面积(1933.80±143.06)和细胞总蛋白含量(367.51±25.9)均高于对照组(787.27±107.66,218.55±21.28,P<0.05),ANP及BNP m RNA的表达量也明显增加(P<0.05),但PI3K、AKt、e NOS m RNA表达水平,磷酸化AKt程度和NO的释放量(4.60±0.73)低于对照组(8.63±0.30,P<0.05),各实验组给予不同浓度Na HS刺激后能够浓度依赖性的抑制这种肥大效应(P<0.05),同时上调了PI3K/AKt/e NOS通路各信号分子的表达量(P<0.05)。结论 H2S对ET-1诱导的心肌肥大有一定的抑制作用,这种作用可能与激活PI3K-AKt-e NOS信号通路有关。展开更多
基金Supported by National Natural Science Foundation of China,No.82060778the Hainan Provincial Natural Science Foundation of China,No.820RC776+1 种基金the Hainan Province Health Science and Technology Innovation Joint Project,No.WSJK2024MS162the Heilongjiang Province Scientific Research Project of Traditional Chinese Medicine,No.ZHY2024-098.
文摘BACKGROUND Finding active lead anti-hepatocellular carcinoma compounds from traditional Chinese medicine has important research value.AIM To assess the detailed mechanism of oxocrebanine,a compound separated from the traditional Chinese medicinal plant Stephania hainanensis H.S.Lo et Y.Tsoong,and to evaluate its inhibition of the proliferation of human hepatocellular carcinoma cells via apoptosis and autophagy.METHODS MTT,BrdU labeling,and colony formation assays were used to assess the inhibitory effect of oxocrebanine on the growth and proliferation of human hepatocellular carcinoma Hep3B2.1-7 cells.Flow cytometry was used to detect the effect of oxocrebanine on the apoptosis of Hep3B2.1-7 cells.Western blotting was used to assess the expression of apoptosis-related proteins in Hep3B2.1-7 cells.The aforementioned methods were also used to evaluate the effects of oxocrebanine on cell proliferation,autophagy markers,and autophagy-related protein expression levels after adding autophagy inhibitor 3-mA.Furthermore,to verify the anti-hepatocellular carcinoma effect of oxocrebanine in vivo,a nude mouse model was used to investigate the inhibitory effect of oxocrebanine treatment and its mechanism.Apoptosis was detected using a TUNEL assay and the expression of microtubule-associated protein 1 LC3 in tumor specimens was assessed using immunohistochemistry.RESULTS Oxocrebanine effectively inhibited the growth of Hep3B2.1-7 cells,whilst upregulating the protein expression of cleaved caspase-3,downregulating poly(ADP-ribose)polymerase 1 protein expression,increasing the levels of Bax and Bcl-2 antagonist/killer 1 protein expression,and decreasing the levels of Bcl-2 and myeloid cell leukemia 1 protein expression,which could promote apoptosis in Hep3B2.1-7 cells.Oxocrebanine promoted the transformation of LC3-I to LC3-II in Hep3B2.1-7 cells,suggesting the occurrence of autophagy,whilst the autophagy inhibitor 3-MA could reverse this process.Oxocrebanine was also shown to reduce the phosphorylation levels of the eukaryotic translation initiation factor 4EBP1 and ribosomal protein S6 kinase B1(P70S6K),two downstream effector molecules in the PI3K/Akt/mTOR pathway,inducing autophagy in Hep3B2.1-7 cells.Moreover,the tumor-bearing nude mouse experiment indicated that oxocrebanine effectively inhibited the growth of Hep3B2.1-7 cells in vivo.The results of the TUNEL assay and immunohistochemistry also revealed that oxocrebanine induced apoptosis in vivo and increased the expression level of LC3,an autophagy marker.CONCLUSION Oxocrebanine can inhibit the proliferation of human hepatocellular carcinoma cells by promoting apoptosis and inducing autophagy in vitro and in vivo.
文摘目的观察磷脂酰肌醇-3激酶(PI3K)-蛋白质丝氨酸/苏氨酸激酶(AKt)-内皮型一氧化氮合酶(e NOS)信号转导通路在硫化氢(H2S)抑制内皮素-1(endothelin-1,ET-1)诱导心肌肥大过程中的作用。方法体外培养原代心肌细胞,将其随机分为6组,每组4孔,1对照组:加入等体积无血清的DMEM培养基;2肥大(ET-1)组:加入终浓度为10-8 mol/L的ET-1;剩余4组为实验组,各组分别加入不同终浓度的H2S供体-Na HS:310-15 M Na HS组:加入10-15 mol/L Na HS+10-8 mol/l ET-1;410-14 M Na HS组:加入10-14 mol/L Na HS+10-8 mol/L ET-1;510-13 M Na HS组:加入10-13 mol/L Na HS+10-8 mol/L ET-1;610-12 M Na HS组:加入10-12 mol/L Na HS+10-8 mol/L ET-1。上述各组药物分别刺激24 h后测定心肌细胞表面积、细胞总蛋白含量、培养液NO含量,RT-PCR检测心肌细胞心房利钠肽(atrial natriuretic peptide,ANP)、脑钠肽(B-type natriuretic peptide,BNP)、磷脂酰肌醇-3激酶(phosphatidylinositol-3-kinase,PI3K)、蛋白激酶B(protein kinase B,PKB/AKt)、e NOS m RNA水平,Western Blot技术检测总AKt和磷酸化AKt蛋白表达含量。结果肥大(ET-1)组的心肌细胞表面积(1933.80±143.06)和细胞总蛋白含量(367.51±25.9)均高于对照组(787.27±107.66,218.55±21.28,P<0.05),ANP及BNP m RNA的表达量也明显增加(P<0.05),但PI3K、AKt、e NOS m RNA表达水平,磷酸化AKt程度和NO的释放量(4.60±0.73)低于对照组(8.63±0.30,P<0.05),各实验组给予不同浓度Na HS刺激后能够浓度依赖性的抑制这种肥大效应(P<0.05),同时上调了PI3K/AKt/e NOS通路各信号分子的表达量(P<0.05)。结论 H2S对ET-1诱导的心肌肥大有一定的抑制作用,这种作用可能与激活PI3K-AKt-e NOS信号通路有关。
