黄连素减轻缺氧无血清诱导的骨髓间充质干细胞凋亡以及减轻四氯化碳诱导的急性肝损伤
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摘要
目的
在体外缺氧无血清条件下模拟体内器官的缺血微环境,观察缺氧无血清状态下，大鼠骨髓间充质干细胞的损伤情况。并利用传统中药黄连素预处理观察其对骨髓间充质干细胞(Mesenchymal stem cells，MSCs)损伤的保护作用。
方法
分离培养大鼠MSCs，并建立体外缺氧无血清诱导的MSCs损伤模型，实验分为正常对照组，缺氧无血清组(缺氧无血清处理6小时)，以及黄连素预处理组（缺氧之前1小时给予黄连素）。Hoechst 33342荧光染色和膜联蛋白V/PI双染流式细胞技术观察缺氧无血清是否能够引起MSCs的凋亡。利用细胞活力测定观察不同浓度的黄连素对MSCs的保护作用，同时利用上述凋亡检测技术进一步证实证实黄连素对缺氧无血清诱导的MSCs凋亡的保护作用。
结果
细胞在6小时的缺氧无血清环境下，发生严重损伤。通过细胞形态学分析，Hoechest 33342以及流式细胞检测凋亡发现，细胞发生了大量早期凋亡。利用不同浓度的黄连素预处理一小时发现，黄连素对MSCs的损伤具有保护作用，其保护作用随着黄连素的浓度增加而增强，在10μM的浓度，黄连素具有最强烈的保护作用，几乎能完全逆转MSCs死亡。Hoechest 33342荧光染色和流式细胞凋亡检测进一步证实，黄连素能够通过抑制MSCs的凋亡而发挥保护作用。
结论
我们发现，缺氧无血清处理后，MSCs发生了早期凋亡。不同浓度的黄连素发现其对细胞损伤具有保护作用。这种保护作用是通过抑制缺氧导致的细胞凋亡而发挥作用的。本实验研究为下一步探讨黄连素的具体保护机制打下基础。
目的
建立体外缺氧无血清诱导的大鼠MSCs损伤模型，观察黄连素是否能够通过抑制活性氧簇（Reactive oxygen species, ROS）以及由其引起的c-Jun NH2端激酶c-Jun NH2-terminal kinase，JNK）激活和线粒体依赖的凋亡通路，从而减轻大鼠MSCs的凋亡。同时，研究黄连素可能的其他作用机制。
方法
体外缺氧无血清诱导MSCs凋亡，浓度为10μM的黄连素预处理一小时，细胞缺氧无血清造模，2'，7'-二氯荧光素二乙酸酯探针（2’，7’-d，ichlorodihydrofluorescein diacetate，DCFH-DA）观察黄连素对ROS的释放，JC-1探针观察线粒体通透性变化，蛋白免疫印迹（Western blot）检测JNK的激活，线粒体相关凋亡蛋白细胞色素C（Cytochorome C, Cyt C）的释放，以及凋亡启动蛋白半胱氨酸蛋白酶（Caspase-3）的激活。同时研究一种经典的抗凋亡信号通路磷酸肌醇-3-激酶（Phosphoinositide-3 kinase，PI3K）/蛋白激酶B（Akt）信号通路是否也参与保护作用。利用预先给与PI3K/Akt阻断剂Wortmannin以及LY294002观察其是否能够阻断黄连素的保护作用，从而观察黄连素是否也通过PI3K/Akt信号通路发挥抗凋亡作用，并同时研究PI3K/Akt与黄连素其它抗凋亡作用的关系。
结果
黄连素能够减少缺氧无血清引起的MSCs内ROS的释放，利用抗氧化剂N-乙酰半胱氨酸(acetyl-L-cysteine，NAC)也能够减少ROS的释放并减轻凋亡作用。JC-1探针显示，与缺氧无血清组相比，黄连素还能够减少线粒体膜通透性的下降。Western blot表明黄连素能够抑制JNK的活性，以及线粒体膜通透性下降引起的Cyt C的释放和凋亡蛋白Caspase-3的激活。黄连素还能够增加缺氧无血清早期PI3K/Akt的表达，给与PI3K/Akt阻断剂Wortmannin以及LY294002逆转了黄连素的保护作用。进一步的实验证明，Wortmannin以及LY294002能够抑制黄连素引起的对ROS产生、JNK激活以及随后线粒体凋亡通路的抑制作用，从而阻断黄连素的保护作用。
结论
抗氧化剂NAC能够减轻缺氧无血清诱导的骨髓间充质干细胞的凋亡表明ROS在细胞凋亡过程中起到了非常重要的作用。黄连素能够通过抑制ROS的释放，JNK的激活以及线粒体膜电位（mitochondrial membrane potential，ΔΨm）的降低，从而减少凋亡相关蛋白Cyt C的释放以及Caspase-3的激活，最终发挥抗凋亡作用，这种保护作用依赖于PI3K/Akt的激活。本实验为临床联合使用黄连素和骨髓间充质干细胞治疗人类疾病提供相关的实验基础。
目的
通过建立四氯化碳（Carbon tetrachloride，CCl4）引起的急性肝损伤模型，观察抗氧化剂黄连素是否能够减轻急性肝损伤引起的细胞死亡，并研究其是否通过抗氧化以及抗凋亡作用来实现保护作用。
方法
建立CCl4诱导的急性肝损伤模型，药物处理组预先给予50，100，200mg/kg黄连素灌胃7天。造模24小时后，摘取肝脏组织，多聚甲醛固定，石蜡包埋，进行常规病理切片，观察肝脏损伤情况。病理切片同时TUNEL法检测细胞凋亡。全自动生化仪检测血清丙氨酸氨基转移酶（Alanine transaminase，ALT）以及天冬氨酸氨基转移酶（Aspartate Aminotransferase，AST）水平。黄嘌呤氧化酶法检测血清过氧化物岐化酶（Superoxide dismutase，SOD）以及硫代巴比妥法检测血清丙二醛（Malondialdehyde，MDA））观察肝脏氧化状态。
结果
在本实验中，CCl4造模24小时之后，血清ALT和AST急剧升高，黄连素能够减少血清ALT和AST的水平。黄连素给药浓度从50mg /kg到200 mg/kg呈现为浓度依赖的保护作用，并逐渐加强。肝脏氧化状态检测表明，CCl4造模之后，血清SOD急剧降低，而MDA的表达增加，利用黄连素预先处理之后，细胞的SOD增加，而ROS的终产物MDA的活性明显降低，也呈浓度依赖的保护作用，在浓度为200mg/kg时，黄连素的抗氧化作用最强。通过进一步的病理切片和TUNEL凋亡检测进一步证实，与CCl4造模组相比，在用黄连素处理之后，细胞凋亡明显减轻，在200mg/kg浓度达到最大的保护作用。
结论
本文通过建立CCl4诱导的急性肝损伤模型，发现传统的中药黄连素能够减轻急性肝损伤引起的细胞凋亡。我们发现，黄连素能够提高抗氧化剂SOD的水平，同时减少ROS生成（表现为其终产物MDA的水平降低），从而发挥抗凋亡作用。本研究为临床利用黄连素进行干预治疗急性肝损伤提供了实验依据。
Objective
By using an in vitro model of hypoxia consisting of hypoxia and serum deprivation to mimic the in vivo ischemic microenvironment, we sought to investigate whether the Chinese traditional medicine berberine could protect against hypoxia and serum deprivation-induced mesenchymal stem cells injury.
Methods
We isolated mesenchymal stem cells from bone marrow and created in vitro model of hypoxia consisting of hypoxia and serum deprivation for 6 hours to induce mesenchymal stem cell injury. Different concentrations of berberine were given one hour before hypoxia and were still present during the process of hypoxia. Hoechst33342 staining and flow cytometric analysis were determined for cell apoptosis. Cell viability assay was used to test the berberine-induced protective effects against mesenchymal stem cells apoptosis.
Results
After 6 hours of hypoxia and serum deprivation, MSCs underwent severe injury. Hoechst 33342 staing showed that there was a lot of early-stage aopotosis in the cellsexposed hypoxia, compared to the control group. Pretreatment of berberine showed that it protected against cell injury in a concentration dependent manner, with the highest effects at a dose of 10μM, which almost completely reversed the cell death. Hoechst 33342 staining and flow cytometry conformed that berberine could protect anainst hypoxia and serum deprivation-induced MSCs apoptosis.
Conclusions
Our data demonstrated that after hypoxia and serum deprivation, MSCs underwent early-stage apoptosis. Different concentrations of berberine could protect against MSCs deaths. Hoechst 33342 staining and flow cytometry confirmed that berberine could attenuate MSCs apoptosis induced by hypoxia and serum deprivation.
Objective
We created a model of hypoxia and serum deprivation-induced MSCs apoptosis,we investigated whether berberine could alleviate reactive oxygen species (ROS)production and inhibit mitochondrial dependent apoptotic pathway, thus protected mesenchymal stem cells apoptosis against hypoxia and serum deprivation. We also explored other mechanisms that could mediate berberine-induced protection.
Methods
Mesenchymal stem cells were exposed to hypoxia and serum deprivation for 6 hours; 10μM berberine was treated 1 hour before hypoxia and was present during the process of hypoxia. Production of cellular ROS was evaluated by analyzing changes in fluorescence intensity resulting from the oxidation of the intracellular fluoroprobe DCFH-DA. Loss of the mitochondrial membrane potential was assessed using the fluorescent probe JC-1. Western blot was used to test the JNK phosphrylation and realease of cytochorome c from the mitochondria to the cytosol. Apoptotic protein caspase-3 was also tested for its activation. At the same time we also used the PI3K/Akt inhibitors wortmannin and LY294002 see if they were also involved the protective effects of berberine.
Results
Berberine could reduce the ROS production of mesenchymal stem cells exposed to hypoxia and serum deprivation, the antioxidant NAC could also inhibit the release of ROS. The JC-1 probe assay showed that berberine could inhibit the loss of mitochondrial membrane potential compared to hypoxia and serum deprivation group. Western blot analysis showed that berberine could inhibit the JNK phosphrylation and realease of cytochorome c from the mitochondria to the cytosol and also berberine reduced the apoptosis related protein cleaved (activated) caspase-3. Our results also showed that berberine could increase the phosphrylation of Akt in the early stage of hypoxia and serum deprivation, and both PI3K/Akt inhibitors wortmannin and LY294002 could block the protective effects of berberine. Further study showed that wortmannin and LY294002 could block berberine induced ROS production JNK activation and subsequent mitochondrial apoptotic pathway, thus blocked the protective effects of berberine.
Conclusions
ROS plays a very important role in hypoxia and serum deprivation-induced MSCs apoptosis. Berberine could inhibit the production of ROS and phosphrylation of JNK, alleviate the release of cytochorome c and activation of caspase-3, and thus confer its
Objective
By creating the model of carbon tetrachloride-induced acute liver injury, we sought to investigate whether an antioxidant berberine could protect against liver cell deaths and the mechanisms within this process.
Methods
Berberine was give orally once everyday for a successive of 7 days, and then mice were inject intraperitoneally with carbon tetrachloride to induce acute liver injury. After 24 hours, mice blood and liver tissue were collected. Serum ALT, AST, SOD and MDA were measured to test the liver function and oxidation. Liver tissue was also fixed with polyformaldehyde and was stained for TUNEL assay.
Results
After 24 hours’treatment, there were huge increase in the serum ALT and AST, as compared to control group. When pretreated with berberine, the ALT and AST level was reduced, with the best effects at the dose of 200mg/kg. The serum SOD was upregulated and MDA was downregulated when pretreated with berberine, compared with the acute liver injury group, with the highest protective effects at the dose of 200mg/kg. The TUNEL assay showed that berberine could significantly reduce the aopotosis.
Conclusions
By creating the carbon tetrachloride-induced acute lung injury model, we found that the antioxidant berberine could alleviate acute liver injury-induced cell apoptosis. We also found that berberine could increase the serum antioxidant SOD level, reduce the ROS production (indicated by the decrease of MDA, which is the end product of ROS) and thus confer its anti-apoptotic effects.
在体外缺氧无血清条件下模拟体内器官的缺血微环境,观察缺氧无血清状态下，大鼠骨髓间充质干细胞的损伤情况。并利用传统中药黄连素预处理观察其对骨髓间充质干细胞(Mesenchymal stem cells，MSCs)损伤的保护作用。
方法
分离培养大鼠MSCs，并建立体外缺氧无血清诱导的MSCs损伤模型，实验分为正常对照组，缺氧无血清组(缺氧无血清处理6小时)，以及黄连素预处理组（缺氧之前1小时给予黄连素）。Hoechst 33342荧光染色和膜联蛋白V/PI双染流式细胞技术观察缺氧无血清是否能够引起MSCs的凋亡。利用细胞活力测定观察不同浓度的黄连素对MSCs的保护作用，同时利用上述凋亡检测技术进一步证实证实黄连素对缺氧无血清诱导的MSCs凋亡的保护作用。
结果
细胞在6小时的缺氧无血清环境下，发生严重损伤。通过细胞形态学分析，Hoechest 33342以及流式细胞检测凋亡发现，细胞发生了大量早期凋亡。利用不同浓度的黄连素预处理一小时发现，黄连素对MSCs的损伤具有保护作用，其保护作用随着黄连素的浓度增加而增强，在10μM的浓度，黄连素具有最强烈的保护作用，几乎能完全逆转MSCs死亡。Hoechest 33342荧光染色和流式细胞凋亡检测进一步证实，黄连素能够通过抑制MSCs的凋亡而发挥保护作用。
结论
我们发现，缺氧无血清处理后，MSCs发生了早期凋亡。不同浓度的黄连素发现其对细胞损伤具有保护作用。这种保护作用是通过抑制缺氧导致的细胞凋亡而发挥作用的。本实验研究为下一步探讨黄连素的具体保护机制打下基础。
目的
建立体外缺氧无血清诱导的大鼠MSCs损伤模型，观察黄连素是否能够通过抑制活性氧簇（Reactive oxygen species, ROS）以及由其引起的c-Jun NH2端激酶c-Jun NH2-terminal kinase，JNK）激活和线粒体依赖的凋亡通路，从而减轻大鼠MSCs的凋亡。同时，研究黄连素可能的其他作用机制。
方法
体外缺氧无血清诱导MSCs凋亡，浓度为10μM的黄连素预处理一小时，细胞缺氧无血清造模，2'，7'-二氯荧光素二乙酸酯探针（2’，7’-d，ichlorodihydrofluorescein diacetate，DCFH-DA）观察黄连素对ROS的释放，JC-1探针观察线粒体通透性变化，蛋白免疫印迹（Western blot）检测JNK的激活，线粒体相关凋亡蛋白细胞色素C（Cytochorome C, Cyt C）的释放，以及凋亡启动蛋白半胱氨酸蛋白酶（Caspase-3）的激活。同时研究一种经典的抗凋亡信号通路磷酸肌醇-3-激酶（Phosphoinositide-3 kinase，PI3K）/蛋白激酶B（Akt）信号通路是否也参与保护作用。利用预先给与PI3K/Akt阻断剂Wortmannin以及LY294002观察其是否能够阻断黄连素的保护作用，从而观察黄连素是否也通过PI3K/Akt信号通路发挥抗凋亡作用，并同时研究PI3K/Akt与黄连素其它抗凋亡作用的关系。
结果
黄连素能够减少缺氧无血清引起的MSCs内ROS的释放，利用抗氧化剂N-乙酰半胱氨酸(acetyl-L-cysteine，NAC)也能够减少ROS的释放并减轻凋亡作用。JC-1探针显示，与缺氧无血清组相比，黄连素还能够减少线粒体膜通透性的下降。Western blot表明黄连素能够抑制JNK的活性，以及线粒体膜通透性下降引起的Cyt C的释放和凋亡蛋白Caspase-3的激活。黄连素还能够增加缺氧无血清早期PI3K/Akt的表达，给与PI3K/Akt阻断剂Wortmannin以及LY294002逆转了黄连素的保护作用。进一步的实验证明，Wortmannin以及LY294002能够抑制黄连素引起的对ROS产生、JNK激活以及随后线粒体凋亡通路的抑制作用，从而阻断黄连素的保护作用。
结论
抗氧化剂NAC能够减轻缺氧无血清诱导的骨髓间充质干细胞的凋亡表明ROS在细胞凋亡过程中起到了非常重要的作用。黄连素能够通过抑制ROS的释放，JNK的激活以及线粒体膜电位（mitochondrial membrane potential，ΔΨm）的降低，从而减少凋亡相关蛋白Cyt C的释放以及Caspase-3的激活，最终发挥抗凋亡作用，这种保护作用依赖于PI3K/Akt的激活。本实验为临床联合使用黄连素和骨髓间充质干细胞治疗人类疾病提供相关的实验基础。
目的
通过建立四氯化碳（Carbon tetrachloride，CCl4）引起的急性肝损伤模型，观察抗氧化剂黄连素是否能够减轻急性肝损伤引起的细胞死亡，并研究其是否通过抗氧化以及抗凋亡作用来实现保护作用。
方法
建立CCl4诱导的急性肝损伤模型，药物处理组预先给予50，100，200mg/kg黄连素灌胃7天。造模24小时后，摘取肝脏组织，多聚甲醛固定，石蜡包埋，进行常规病理切片，观察肝脏损伤情况。病理切片同时TUNEL法检测细胞凋亡。全自动生化仪检测血清丙氨酸氨基转移酶（Alanine transaminase，ALT）以及天冬氨酸氨基转移酶（Aspartate Aminotransferase，AST）水平。黄嘌呤氧化酶法检测血清过氧化物岐化酶（Superoxide dismutase，SOD）以及硫代巴比妥法检测血清丙二醛（Malondialdehyde，MDA））观察肝脏氧化状态。
结果
在本实验中，CCl4造模24小时之后，血清ALT和AST急剧升高，黄连素能够减少血清ALT和AST的水平。黄连素给药浓度从50mg /kg到200 mg/kg呈现为浓度依赖的保护作用，并逐渐加强。肝脏氧化状态检测表明，CCl4造模之后，血清SOD急剧降低，而MDA的表达增加，利用黄连素预先处理之后，细胞的SOD增加，而ROS的终产物MDA的活性明显降低，也呈浓度依赖的保护作用，在浓度为200mg/kg时，黄连素的抗氧化作用最强。通过进一步的病理切片和TUNEL凋亡检测进一步证实，与CCl4造模组相比，在用黄连素处理之后，细胞凋亡明显减轻，在200mg/kg浓度达到最大的保护作用。
结论
本文通过建立CCl4诱导的急性肝损伤模型，发现传统的中药黄连素能够减轻急性肝损伤引起的细胞凋亡。我们发现，黄连素能够提高抗氧化剂SOD的水平，同时减少ROS生成（表现为其终产物MDA的水平降低），从而发挥抗凋亡作用。本研究为临床利用黄连素进行干预治疗急性肝损伤提供了实验依据。
Objective
By using an in vitro model of hypoxia consisting of hypoxia and serum deprivation to mimic the in vivo ischemic microenvironment, we sought to investigate whether the Chinese traditional medicine berberine could protect against hypoxia and serum deprivation-induced mesenchymal stem cells injury.
Methods
We isolated mesenchymal stem cells from bone marrow and created in vitro model of hypoxia consisting of hypoxia and serum deprivation for 6 hours to induce mesenchymal stem cell injury. Different concentrations of berberine were given one hour before hypoxia and were still present during the process of hypoxia. Hoechst33342 staining and flow cytometric analysis were determined for cell apoptosis. Cell viability assay was used to test the berberine-induced protective effects against mesenchymal stem cells apoptosis.
Results
After 6 hours of hypoxia and serum deprivation, MSCs underwent severe injury. Hoechst 33342 staing showed that there was a lot of early-stage aopotosis in the cellsexposed hypoxia, compared to the control group. Pretreatment of berberine showed that it protected against cell injury in a concentration dependent manner, with the highest effects at a dose of 10μM, which almost completely reversed the cell death. Hoechst 33342 staining and flow cytometry conformed that berberine could protect anainst hypoxia and serum deprivation-induced MSCs apoptosis.
Conclusions
Our data demonstrated that after hypoxia and serum deprivation, MSCs underwent early-stage apoptosis. Different concentrations of berberine could protect against MSCs deaths. Hoechst 33342 staining and flow cytometry confirmed that berberine could attenuate MSCs apoptosis induced by hypoxia and serum deprivation.
Objective
We created a model of hypoxia and serum deprivation-induced MSCs apoptosis,we investigated whether berberine could alleviate reactive oxygen species (ROS)production and inhibit mitochondrial dependent apoptotic pathway, thus protected mesenchymal stem cells apoptosis against hypoxia and serum deprivation. We also explored other mechanisms that could mediate berberine-induced protection.
Methods
Mesenchymal stem cells were exposed to hypoxia and serum deprivation for 6 hours; 10μM berberine was treated 1 hour before hypoxia and was present during the process of hypoxia. Production of cellular ROS was evaluated by analyzing changes in fluorescence intensity resulting from the oxidation of the intracellular fluoroprobe DCFH-DA. Loss of the mitochondrial membrane potential was assessed using the fluorescent probe JC-1. Western blot was used to test the JNK phosphrylation and realease of cytochorome c from the mitochondria to the cytosol. Apoptotic protein caspase-3 was also tested for its activation. At the same time we also used the PI3K/Akt inhibitors wortmannin and LY294002 see if they were also involved the protective effects of berberine.
Results
Berberine could reduce the ROS production of mesenchymal stem cells exposed to hypoxia and serum deprivation, the antioxidant NAC could also inhibit the release of ROS. The JC-1 probe assay showed that berberine could inhibit the loss of mitochondrial membrane potential compared to hypoxia and serum deprivation group. Western blot analysis showed that berberine could inhibit the JNK phosphrylation and realease of cytochorome c from the mitochondria to the cytosol and also berberine reduced the apoptosis related protein cleaved (activated) caspase-3. Our results also showed that berberine could increase the phosphrylation of Akt in the early stage of hypoxia and serum deprivation, and both PI3K/Akt inhibitors wortmannin and LY294002 could block the protective effects of berberine. Further study showed that wortmannin and LY294002 could block berberine induced ROS production JNK activation and subsequent mitochondrial apoptotic pathway, thus blocked the protective effects of berberine.
Conclusions
ROS plays a very important role in hypoxia and serum deprivation-induced MSCs apoptosis. Berberine could inhibit the production of ROS and phosphrylation of JNK, alleviate the release of cytochorome c and activation of caspase-3, and thus confer its
Objective
By creating the model of carbon tetrachloride-induced acute liver injury, we sought to investigate whether an antioxidant berberine could protect against liver cell deaths and the mechanisms within this process.
Methods
Berberine was give orally once everyday for a successive of 7 days, and then mice were inject intraperitoneally with carbon tetrachloride to induce acute liver injury. After 24 hours, mice blood and liver tissue were collected. Serum ALT, AST, SOD and MDA were measured to test the liver function and oxidation. Liver tissue was also fixed with polyformaldehyde and was stained for TUNEL assay.
Results
After 24 hours’treatment, there were huge increase in the serum ALT and AST, as compared to control group. When pretreated with berberine, the ALT and AST level was reduced, with the best effects at the dose of 200mg/kg. The serum SOD was upregulated and MDA was downregulated when pretreated with berberine, compared with the acute liver injury group, with the highest protective effects at the dose of 200mg/kg. The TUNEL assay showed that berberine could significantly reduce the aopotosis.
Conclusions
By creating the carbon tetrachloride-induced acute lung injury model, we found that the antioxidant berberine could alleviate acute liver injury-induced cell apoptosis. We also found that berberine could increase the serum antioxidant SOD level, reduce the ROS production (indicated by the decrease of MDA, which is the end product of ROS) and thus confer its anti-apoptotic effects.
引文
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