姜黄素对高糖诱导的H9C2心肌细胞炎症的保护作用及机制研究

发布时间：2018-06-19 18:02

本文选题：糖尿病心肌病 + 姜黄素　；参考：《南昌大学》2015年硕士论文

【摘要】：目的：1、观察高糖对H9C2心肌细胞生物活性的影响。2、观察姜黄素对高糖诱导的H9C2心肌细胞的生物活性、炎症反应的影响。3、探讨姜黄素抑制高糖诱导的H9C2心肌细胞炎症反应的机制。方法：1、体外正常糖培养H9C2心肌细胞,再分别用高糖、姜黄素、肝X激动剂抑制剂、TLR抑制剂、PP2A抑制剂进行干预。2、实验分组：①Control组：正常对照组,用正常糖培养基(5.5mmol/L)培养H9C2细胞；②HG组：阳性对照组,用高糖培养基(33mmol/L)培养H9C2细胞；③HG+T0901317组：对HG组用肝X激动剂(T0901317终浓度20μmol/L)进行干预；④HG+TAK-242组：对HG组用TLR抑制剂(TAK-242终浓度100nmol/L)进行干预；⑤HG+Curcumin组：对HG组用姜黄素(Curcumin终浓度10μmol/L)进行干预⑥HG+5CPPSS一50组对HG组用肝X抑制剂(5CPPSS-50终浓度15μmol/L)进行干预⑦HG+5CPPSS一50+Curcumin组：在HG+5CPPSS-50组的基础上加用Curcumin进行干预；⑧HG+Okadaic acid组：对HG组用PP2A抑制剂(Okadaic acid终浓度20nmol/L)进行干预;⑨HG+Okadaic acid+Curcumin组：在HG+Okadaic acid组的基础上加用Curcumin进行干预。3、相应组中加入相应药物干预72h后,用CCK-8检测各组H9C2心肌细胞的生长状况；ELISA试剂盒测各组炎症因子(IL6、TNFα、MCP1)分泌量；同时应用qRT-PCR检测各组LXRα/IRAK4/PP2Ac mRNA水平；、Vestern blot检则各组NF-κBp65(总量和核内)、LXRα、IRAK4、p-PP2Ac蛋白的表达并比较。结果：1、CCK-8检测各组H9C2心肌细胞的生长状况结果显示：高糖可使细胞增殖活力下降,LXRs激动剂、TLR抑制剂、姜黄素可以减轻高糖对H9C2心肌细胞增殖活力的抑制,而LXRs抑制剂或PP2A抑制剂干预后细胞增殖活力抑制加剧。在LXRs抑制剂或PP2A抑制剂干预的基础上使用姜黄素不能改善高糖对H9C2心肌细胞增殖活力的抑制。2、ELISA去检测各组细胞上清中炎症因子(IL6、TNFα、MCP1)的分泌量,结果表明：高糖作用下细胞培养上清中炎症因子含量明显增高(P0.05)。T0901317、TAK-242、Curcumin干预后炎症因子分泌量减少(P0.05)。5CPPSS-50或Okadaic acid加剧炎症因子释放(P0.05)。相对HG+Curcumin组,在LXRs抑制剂或PP2A抑制剂干预的基础上使用Curcumin不能降低炎症因子含量(P0.05)。3、qRT-PCR检测各组LXRa, IRAK4, PP2Ac mRNA水平：高糖可使LXRa nRNA水平轻微上升,但无统计学差异(P0.05),而IRAK4和PP2Ac mRNA明显上调(P0.01)。与HG组相比：HG+T0901317组、HG+TAK-242组、IG+Curcumin组LXRa mRNA进一步上调(P0.01),IRAK4 mRNA则明显下降(P0.01)；HG+5CPPSS-50组结果提示LXRαmRNA明显受到抑制(P0.01),RAK4 mRNA进一步上调(P0.01)。在LXRs抑制剂干预的基础上使用Curcumin不能发挥Curcumin上调LXRαmRNA水平的作用。另外与HG组相比还表明：PP2A抑制剂干预将进一步明显上调PP2Ac mRNA水平(P0.01),Curcumin干预测可以明显下调PP2Ac mRNA水平(P0.01),而在PP2A抑制剂干预的基础上使用Curcumin将明显减弱Curcumin下调PP2Ac mRNA水平的作用。4, Western blot检测各组NF-κBp65(总蛋白和核内组分)、LXRa, IRAK4、 p-PP2Ac蛋白的表达结果：各组NF-κBp65总蛋白表达无明显差异(P0.05)。高糖可使LXRa蛋白表达水平轻微上升,但无统计学差异(P0.05),而胞核内NF-κBp65、IRAK4和p-PP2Ac蛋白表达明显上调(P0.01)。与HG组相比：HG+T0901317组、HG+TAK-242组、HG+Curcumin组LXRa蛋白表达则进一步上调(P0.05),IRAK4蛋白和胞核内NF-κBp65蛋白表达则明显下降(P0.05)；HG+5CPPSS-50组结果提示LXRa蛋白表达受到抑制(P0.05),IRAK4蛋白和胞核内NF-κBp65蛋白表达进一步上调(P0.01)。在LXRs抑制剂干预的基础上使用Curcumin不能发挥Curcumin上调LXRa蛋白表达、抑制NF-κBp65入核的作用。另外,与HG组相比较还表明：PP2A抑制剂干预将进一步上调p-PP2Ac、胞核内NF-κBp65蛋白含量(P0.05),Curcumin干预则可以明显下调p-PP2Ac蛋白表达(P0.01),而在PP2A抑制剂干预的基础上使用Curcumin将明显减弱Curcumin下调p-PP2Ac表达、抑制NF-κBp65入核的作用。结论：1、高糖可诱导H9C2心肌细胞发生炎症反应,姜黄素可以抑制高糖诱导的H9C2心肌细胞炎症反应。2、姜黄素能够上调LXRa表达,还可以抑制IRAK4, p-PP2Ac表达,活化PP2A负性调控TLR-NF-κB途径减少NF-κBp65入核进而抑制炎症因子(IL6、TNFα,MCP1)表达,并可能通过这种效应来减轻高糖诱导的H9C2心肌细胞炎症反应。从而提示LXRs、TLR-NF-κB信号通路及相关蛋白激酶和磷酸化酶之间的整合作用可能是姜黄素减轻高糖诱导的H9C2心肌细胞炎症反应的细胞分子机制。
[Abstract]:Objective: 1, to observe the effect of high sugar on the biological activity of H9C2 myocardial cells.2, observe the biological activity of curcumin to high glucose induced H9C2 cardiomyocytes, the effect of inflammatory reaction.3, and explore the mechanism of curcumin to inhibit the inflammatory reaction of H9C2 cardiomyocytes induced by high glucose. Methods: 1, the normal glucose is used to cultivate H9C2 cardiac myocytes, and then high glucose is used respectively. Curcumin, liver X agonist inhibitor, TLR inhibitor, and PP2A inhibitor intervention.2, experimental groups: (1) group Control: normal control group, normal glucose medium (5.5mmol/L) culture H9C2 cells; (2) HG group: positive control group, high glucose medium (33mmol/L) culture H9C2 cells; HG+T0901317 group: HG group with liver agonist The final concentration was 20 mol/L) and group HG+TAK-242: group HG+TAK-242: group HG was treated with TLR inhibitor (TAK-242 final concentration 100nmol/L); (5) HG+Curcumin group: intervention on HG group with curcumin (Curcumin final concentration) (Curcumin final concentration of Curcumin) in group 50 Group 0+Curcumin: intervention with Curcumin on the basis of group HG+5CPPSS-50; HG+Okadaic acid group: HG group was intervened with PP2A inhibitor (Okadaic acid terminal concentration 20nmol/L); HG+Okadaic acid+Curcumin group: the corresponding intervention was added to the group based on the intervention, and the corresponding drug intervention was added in the corresponding group 7. After 2h, the growth of H9C2 myocardial cells in each group was detected by CCK-8, and the secretion of inflammatory factors (IL6, TNF alpha, MCP1) in each group was measured by ELISA kit, and the LXR alpha /IRAK4/PP2Ac mRNA level of each group was detected with qRT-PCR. The results of the growth of H9C2 cardiomyocytes in each group showed that high glucose could reduce the proliferation of cell proliferation. LXRs agonist, TLR inhibitor, curcumin could reduce the inhibitory effect of high glucose on the proliferation of H9C2 cardiomyocytes, while LXRs inhibitors or PP2A inhibitors increased the proliferation inhibition of cell proliferation. In LXRs inhibitors or PP2A inhibitors The use of curcumin can not improve the inhibitory effect of high sugar on the proliferation of H9C2 cardiac myocytes on the basis of.2, and ELISA to detect the secretion of inflammatory factors (IL6, TNF, MCP1) in the supernatant of each group. The results show that the content of inflammatory factors in the supernatant of cell culture under the action of high glucose (P0.05).T0901317, TAK-242, Curcumin after intervention, the cause of inflammation P0.05.5CPPSS-50 or Okadaic acid aggravated the release of inflammatory factors (P0.05). Relative HG+Curcumin group, LXRs inhibitor or PP2A inhibitor intervention on the basis of Curcumin can not reduce the inflammatory factor content (P0.05).3. But there was no statistical difference (P0.05), while IRAK4 and PP2Ac mRNA were obviously up-regulated (P0.01). Compared with the HG group, HG+T0901317 group, HG+TAK-242 group and IG+Curcumin group LXRa mRNA were further up-regulated (P0.01). On the basis of inhibitor intervention, the use of Curcumin can not play the role of Curcumin up regulation of LXR alpha mRNA. In addition, compared with the HG group, the intervention of PP2A inhibitors will further increase the level of PP2Ac mRNA (P0.01), and Curcumin dry prediction can obviously reduce PP2Ac mRNA level. The expression of NF- kappa Bp65 (total protein and intra nuclear component), LXRa, IRAK4, and p-PP2Ac protein expression of each group was significantly weakened by in, and Western blot was used to detect the expression of NF- kappa Bp65 (total protein and intra nuclear components), LXRa, IRAK4, and p-PP2Ac protein. The expression of NF- kappa Bp65, IRAK4 and p-PP2Ac protein in the nucleus increased significantly (P0.01). Compared with the HG group, the expression of LXRa protein in the HG+T0901317 group, HG+TAK-242 group and HG+Curcumin group was further up-regulated (P0.05), and the expression of IRAK4 protein and nucleus kappa protein expression decreased significantly. The expression of AK4 protein and NF- kappa Bp65 protein in the nucleus was further up-regulated (P0.01). On the basis of LXRs inhibitor intervention, Curcumin could not play the role of Curcumin up regulation of LXRa protein expression and inhibit the nucleation of NF- kappa Bp65. P0.05), Curcumin intervention can obviously reduce the expression of p-PP2Ac protein (P0.01), while Curcumin will obviously weaken the Curcumin down regulation of p-PP2Ac expression on the basis of PP2A inhibitor intervention and inhibit the effect of NF- kappa Bp65 entry. Conclusion: 1, high glucose can induce the inflammatory reaction of H9C2 myocardial cells, curcumin can inhibit the high glucose induced H9C2 heart. .2, curcumin can up regulate the expression of LXRa, inhibit the expression of IRAK4, p-PP2Ac, and activate PP2A negative regulation of TLR-NF- kappa B pathway to reduce NF- kappa Bp65 into the nucleus and inhibit the expression of inflammatory factors (IL6, TNF alpha, MCP1), and may reduce the inflammatory reaction induced by high glucose induced cardiomyocytes by this effect. The integration of NF- kappa B signaling pathway and related protein kinase and phosphorylase may be the molecular mechanism of curcumin to reduce the inflammatory response of H9C2 cardiomyocytes induced by high glucose.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.2

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