罗格列酮和肿瘤坏死因子α对脂肪细胞线粒体生物合成及ZAG表达的影响
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摘要
目的:
观察罗格列酮和肿瘤坏死因子α(TNF-α)对脂肪细胞线粒体生物合成和ZAG表达的影响。
方法:
体外培养3T3-L1前脂肪细胞并诱导分化为成熟脂肪细胞,分别以不同浓度的罗格列酮和TNF-α干预48h,提取总RNA,采用RT-PCR方法检测PGC-1α、TFAM、NRF-1/2及ZAG mRNA表达水平。在此实验的基础上,验证罗格列酮和TNF-α对脂肪细胞线粒体合成相关因子及ZAG表达的影响,将分化成熟的脂肪细胞随机分为四组:对照组、罗格列酮组(50μM)、TNF-α组(30ng/mL)、罗格列酮+TNF-α组(RT组),采用RT-PCR和Western Blot技术检测PGC-1α、TFAM、NRF-1/2及ZAG mRNA和蛋白表达;采用线粒体特异性染料Mito Traker Green预染成熟脂肪细胞,激光共聚焦显微镜下观察线粒体荧光强度;采用ELISA法测定细胞培养液上清中FFA浓度。
结果:
1、罗格列酮单独干预分化成熟的3T3-L1脂肪细胞,ZAG及PGC-1α、TFAM、NRF-1/2 mRNA表达水平均高于对照组(P<0.01);
2、TNF-α单独干预分化成熟的3T3-L1脂肪细胞,ZAG及PGC-1α、TFAM、NRF-2 mRNA表达水平均低于对照组(P<0.01),且以TNF-α组(100ng/mL)降低最为显著;TNF-α组(10-100ng/mL)NRF-1mRNA表达水平低于对照组(P<0.01),但TNF-α组(1ng/mL) NRF-1 mRNA表达无显著差异(P>0.05);
3、罗格列酮(50μM)和TNF-α(30ng/mL)干预3T3-L1脂肪细胞48h,罗格列酮组和罗格列酮+TNF-α组线粒体荧光强度高于对照组,而TNF-α组线粒体荧光强度低于对照组;
4、罗格列酮(50μM)和TNF-α(30ng/mL)干预3T3-L1脂肪细胞48h,罗格列酮组ZAG、PGC-1α、TFAM、NRF-1/2 mRNA表达水平均高于对照组(P<0.01);TNF-α组ZAG、PGC-1α、TFAM、NRF-1/2 mRNA表达水平均低于对照组(P<0.01);罗格列酮+TNF-α组ZAG、TFAM、NRF-2 mRNA表达水平高于对照组(P<0.01),而NRF-1mRNA表达水平低于对照组(P<0.05),PGC-1αmRNA表达差异无显著性(P>0.05);
5、罗格列酮(50μM)和TNF-α(30ng/mL)干预3T3-L1脂肪细胞48h,罗格列酮组ZAG、NRF-1和PGC-1α、TFAM蛋白表达水平高于对照组(分别为P<0.05,P<0.01),NRF-2蛋白表达差异无显著性(P>0.05);TNF-α组ZAG、NRF-1和TFAM、NRF-2蛋白表达水平低于对照组(分别为P<0.05,P<0.01),PGC-1α蛋白表达著差异无显性(P>0.05);罗格列酮+TNF-α组ZAG和PGC-1α蛋白表达水平高于对照组(分别为P<0.01,P<0.05),NRF-2蛋白表达水平低于对照组(P<0.01),TFAM、NRF-1蛋白表达差异无显著性(P>0.05);
6、罗格列酮(50μM)和TNF-α(30ng/mL)干预3T3-L1脂肪细胞48h,罗格列酮组FFA浓度低于对照组,TNF-α组FFA浓度高于对照组,差异均有显著性(P<0.05),罗格列酮+TNF-α组浓度表达差异无显著性(P>0.05)。
结论:
1)罗格列酮促进脂肪细胞ZAG和PGC-1α、TFAM、NRF-1/2 mRNA表达;促进ZAG和PGC-1α、TFAM、NRF-1蛋白表达,
2)TNF-α减少ZAG和PGC-1α、TFAM、NRF-1/2 mRNA表达减少,ZAG和TFAM、NRF-1/2蛋白表达,
3)罗格列酮促进线粒体脂肪酸β氧化功能恢复,降低FFA浓度;TNF-α减弱线粒体脂肪酸β氧化功能,增加FFA浓度。
Objective:
To observe effects of rosiglitazone and TNF-αon mitochondrial biogenesis and ZAG expression in adipocytes.
Methods:
3T3-L1 preadipocytes were cultured in vitro and induced to differentiate into matured adipocytes. Adipocytes were treated with different concentrations of rosiglitazone and TNF-αfor 48h respectively. MRNA levels of ZAG, PGC-1α, TFAM, NRF-1/2 genes were detected by RT-PCR. Based on above results, 3T3-L1 adipocytes were randomly divided into four groups: control group, rosiglitazone group (50μM), TNF-αgroup (30ng/mL) and mixed group (rosiglitazone with TNF-αgroup). Expression of mitochondrial biogenesis genes such as PGC-1α, TFAM, NRF-1/2 and ZAG were detected by RT-PCR and Western Blot. Mito Traker Green were used to evaluate the flourescence intensity of mitochondria by laser scanning confocal microscop(LSCM). FFA concentrations in the cell culture supernatants were measured by ELISA.
Results:
1. Compared with control group, the levels of ZAG, PGC-1α, TFAM, NRF-1/2 genes mRNA expression were increased when mature adipocytes were treated with Rosiglitazone (0.1-50μM) (P<0.01).
2. Compared with control group, the levels of ZAG, PGC-1α, TFAM, NRF-2 genes mRNA expression were decreased when mature adipocytes were treated with TNF-α(1-100ng/mL ) (P<0.01). The most significant decline group was the TNF-αgroup (100ng/mL).The levels of NRF-1 mRNA expression were decreased in the TNF-αgroups (10-100ng/mL) (P< 0.01). There was no significant differences between 1ng/mL group and control group (P>0.05).
3. Rosiglitazone (50μM) increased and TNF-αgroup (30ng/mL) decreased the fluorescence of mitochondria in adipocytes.
4. 3T3-L1 adipocytes were treated with rosiglitazone (50μM) and TNF-α(30ng/mL) for 48h. Compared with the control group, the levels of ZAG, PGC-1α, TFAM, NRF-1/2 genes mRNA expression were increased in the Rosiglitazone group (50μM) (P<0.01). But mRNA expression of ZAG, PGC-1α, TFAM, NRF-1/2 genes were decreased in the TNF-αgroup (30ng/mL) (P<0.01). Compared with the control group, the level of ZAG、TFAM、NRF-2 genes mRNA expression was increased in the RT group (P<0.01), as the level of NRF-1 mRNA expression was decreased in the RT group (P<0.05). But the level of PGC-1αmRNA expression were no significant differences in the RT group (P>0.05).
5. 3T3-L1 adipocytes were treated with rosiglitazone (50μM) and TNF-α(30ng/mL) for 48h. Compared with the control group, the levels of ZAG, NRF-1 and PGC-1α, TFAM proteins expression were increased in the Rosiglitazone group (50μM) (P<0.05 and P<0.01, respectively). And the level of NRF-2 protein expression was no significant difference in this group (P>0.05). The proteins expression levels of ZAG, NRF-1 and TFAM, NRF-2 were decreased in the TNF-αgroup (30ng/mL) (P<0.05 and P<0.01, respectively). And the level of PGC-1αprotein expression was no significant difference in this group (P>0.05). Compared with the control group, the levels of ZAG and PGC-1αproteins expression were increased in the RT group(P<0.01 and P<0.05, respectively) and the level of NRF-2 protein expression was decreased in the RT group (P<0.01).
6. Compared with the control group, the FFA concentrations were increased in the TNF-αgroup (30ng/mL) while decreasing in the Rosiglitazone group (50μM) and RT group (P<0.05).
Conclusion:
1. Rosiglitazone can promote mRNA expression of ZAG, PGC-1α, TFAM, NRF-1/2 genes and proteins expression of ZAG, PGC-1α, TFAM, NRF-1;
2. TNF-αcan decrease the mRNA expression of ZAG, PGC-1α, TFAM, NRF-1/2 genes and proteins expression of ZAG, TFAM, NRF-1/2 ;
3. Rosiglitazone can recover the mitochondrial function in fatty acid oxidation which lead to the low FFA concentrations. TNF-αcould reduced mitochondrial function in fatty acid oxidation which lead to the high FFA concentrations.
观察罗格列酮和肿瘤坏死因子α(TNF-α)对脂肪细胞线粒体生物合成和ZAG表达的影响。
方法:
体外培养3T3-L1前脂肪细胞并诱导分化为成熟脂肪细胞,分别以不同浓度的罗格列酮和TNF-α干预48h,提取总RNA,采用RT-PCR方法检测PGC-1α、TFAM、NRF-1/2及ZAG mRNA表达水平。在此实验的基础上,验证罗格列酮和TNF-α对脂肪细胞线粒体合成相关因子及ZAG表达的影响,将分化成熟的脂肪细胞随机分为四组:对照组、罗格列酮组(50μM)、TNF-α组(30ng/mL)、罗格列酮+TNF-α组(RT组),采用RT-PCR和Western Blot技术检测PGC-1α、TFAM、NRF-1/2及ZAG mRNA和蛋白表达;采用线粒体特异性染料Mito Traker Green预染成熟脂肪细胞,激光共聚焦显微镜下观察线粒体荧光强度;采用ELISA法测定细胞培养液上清中FFA浓度。
结果:
1、罗格列酮单独干预分化成熟的3T3-L1脂肪细胞,ZAG及PGC-1α、TFAM、NRF-1/2 mRNA表达水平均高于对照组(P<0.01);
2、TNF-α单独干预分化成熟的3T3-L1脂肪细胞,ZAG及PGC-1α、TFAM、NRF-2 mRNA表达水平均低于对照组(P<0.01),且以TNF-α组(100ng/mL)降低最为显著;TNF-α组(10-100ng/mL)NRF-1mRNA表达水平低于对照组(P<0.01),但TNF-α组(1ng/mL) NRF-1 mRNA表达无显著差异(P>0.05);
3、罗格列酮(50μM)和TNF-α(30ng/mL)干预3T3-L1脂肪细胞48h,罗格列酮组和罗格列酮+TNF-α组线粒体荧光强度高于对照组,而TNF-α组线粒体荧光强度低于对照组;
4、罗格列酮(50μM)和TNF-α(30ng/mL)干预3T3-L1脂肪细胞48h,罗格列酮组ZAG、PGC-1α、TFAM、NRF-1/2 mRNA表达水平均高于对照组(P<0.01);TNF-α组ZAG、PGC-1α、TFAM、NRF-1/2 mRNA表达水平均低于对照组(P<0.01);罗格列酮+TNF-α组ZAG、TFAM、NRF-2 mRNA表达水平高于对照组(P<0.01),而NRF-1mRNA表达水平低于对照组(P<0.05),PGC-1αmRNA表达差异无显著性(P>0.05);
5、罗格列酮(50μM)和TNF-α(30ng/mL)干预3T3-L1脂肪细胞48h,罗格列酮组ZAG、NRF-1和PGC-1α、TFAM蛋白表达水平高于对照组(分别为P<0.05,P<0.01),NRF-2蛋白表达差异无显著性(P>0.05);TNF-α组ZAG、NRF-1和TFAM、NRF-2蛋白表达水平低于对照组(分别为P<0.05,P<0.01),PGC-1α蛋白表达著差异无显性(P>0.05);罗格列酮+TNF-α组ZAG和PGC-1α蛋白表达水平高于对照组(分别为P<0.01,P<0.05),NRF-2蛋白表达水平低于对照组(P<0.01),TFAM、NRF-1蛋白表达差异无显著性(P>0.05);
6、罗格列酮(50μM)和TNF-α(30ng/mL)干预3T3-L1脂肪细胞48h,罗格列酮组FFA浓度低于对照组,TNF-α组FFA浓度高于对照组,差异均有显著性(P<0.05),罗格列酮+TNF-α组浓度表达差异无显著性(P>0.05)。
结论:
1)罗格列酮促进脂肪细胞ZAG和PGC-1α、TFAM、NRF-1/2 mRNA表达;促进ZAG和PGC-1α、TFAM、NRF-1蛋白表达,
2)TNF-α减少ZAG和PGC-1α、TFAM、NRF-1/2 mRNA表达减少,ZAG和TFAM、NRF-1/2蛋白表达,
3)罗格列酮促进线粒体脂肪酸β氧化功能恢复,降低FFA浓度;TNF-α减弱线粒体脂肪酸β氧化功能,增加FFA浓度。
Objective:
To observe effects of rosiglitazone and TNF-αon mitochondrial biogenesis and ZAG expression in adipocytes.
Methods:
3T3-L1 preadipocytes were cultured in vitro and induced to differentiate into matured adipocytes. Adipocytes were treated with different concentrations of rosiglitazone and TNF-αfor 48h respectively. MRNA levels of ZAG, PGC-1α, TFAM, NRF-1/2 genes were detected by RT-PCR. Based on above results, 3T3-L1 adipocytes were randomly divided into four groups: control group, rosiglitazone group (50μM), TNF-αgroup (30ng/mL) and mixed group (rosiglitazone with TNF-αgroup). Expression of mitochondrial biogenesis genes such as PGC-1α, TFAM, NRF-1/2 and ZAG were detected by RT-PCR and Western Blot. Mito Traker Green were used to evaluate the flourescence intensity of mitochondria by laser scanning confocal microscop(LSCM). FFA concentrations in the cell culture supernatants were measured by ELISA.
Results:
1. Compared with control group, the levels of ZAG, PGC-1α, TFAM, NRF-1/2 genes mRNA expression were increased when mature adipocytes were treated with Rosiglitazone (0.1-50μM) (P<0.01).
2. Compared with control group, the levels of ZAG, PGC-1α, TFAM, NRF-2 genes mRNA expression were decreased when mature adipocytes were treated with TNF-α(1-100ng/mL ) (P<0.01). The most significant decline group was the TNF-αgroup (100ng/mL).The levels of NRF-1 mRNA expression were decreased in the TNF-αgroups (10-100ng/mL) (P< 0.01). There was no significant differences between 1ng/mL group and control group (P>0.05).
3. Rosiglitazone (50μM) increased and TNF-αgroup (30ng/mL) decreased the fluorescence of mitochondria in adipocytes.
4. 3T3-L1 adipocytes were treated with rosiglitazone (50μM) and TNF-α(30ng/mL) for 48h. Compared with the control group, the levels of ZAG, PGC-1α, TFAM, NRF-1/2 genes mRNA expression were increased in the Rosiglitazone group (50μM) (P<0.01). But mRNA expression of ZAG, PGC-1α, TFAM, NRF-1/2 genes were decreased in the TNF-αgroup (30ng/mL) (P<0.01). Compared with the control group, the level of ZAG、TFAM、NRF-2 genes mRNA expression was increased in the RT group (P<0.01), as the level of NRF-1 mRNA expression was decreased in the RT group (P<0.05). But the level of PGC-1αmRNA expression were no significant differences in the RT group (P>0.05).
5. 3T3-L1 adipocytes were treated with rosiglitazone (50μM) and TNF-α(30ng/mL) for 48h. Compared with the control group, the levels of ZAG, NRF-1 and PGC-1α, TFAM proteins expression were increased in the Rosiglitazone group (50μM) (P<0.05 and P<0.01, respectively). And the level of NRF-2 protein expression was no significant difference in this group (P>0.05). The proteins expression levels of ZAG, NRF-1 and TFAM, NRF-2 were decreased in the TNF-αgroup (30ng/mL) (P<0.05 and P<0.01, respectively). And the level of PGC-1αprotein expression was no significant difference in this group (P>0.05). Compared with the control group, the levels of ZAG and PGC-1αproteins expression were increased in the RT group(P<0.01 and P<0.05, respectively) and the level of NRF-2 protein expression was decreased in the RT group (P<0.01).
6. Compared with the control group, the FFA concentrations were increased in the TNF-αgroup (30ng/mL) while decreasing in the Rosiglitazone group (50μM) and RT group (P<0.05).
Conclusion:
1. Rosiglitazone can promote mRNA expression of ZAG, PGC-1α, TFAM, NRF-1/2 genes and proteins expression of ZAG, PGC-1α, TFAM, NRF-1;
2. TNF-αcan decrease the mRNA expression of ZAG, PGC-1α, TFAM, NRF-1/2 genes and proteins expression of ZAG, TFAM, NRF-1/2 ;
3. Rosiglitazone can recover the mitochondrial function in fatty acid oxidation which lead to the low FFA concentrations. TNF-αcould reduced mitochondrial function in fatty acid oxidation which lead to the high FFA concentrations.
引文
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