本文選題：糖尿病心肌病 + 姜黃素��； 參考：《南昌大學(xué)》2015年碩士論文

【摘要】：目的：1、觀察高糖對H9C2心肌細(xì)胞生物活性的影響。2、觀察姜黃素對高糖誘導(dǎo)的H9C2心肌細(xì)胞的生物活性、炎癥反應(yīng)的影響。3、探討姜黃素抑制高糖誘導(dǎo)的H9C2心肌細(xì)胞炎癥反應(yīng)的機(jī)制。方法：1、體外正常糖培養(yǎng)H9C2心肌細(xì)胞,再分別用高糖、姜黃素、肝X激動劑抑制劑、TLR抑制劑、PP2A抑制劑進(jìn)行干預(yù)。2、實(shí)驗(yàn)分組：①Control組：正常對照組,用正常糖培養(yǎng)基(5.5mmol/L)培養(yǎng)H9C2細(xì)胞；②HG組：陽性對照組,用高糖培養(yǎng)基(33mmol/L)培養(yǎng)H9C2細(xì)胞；③HG+T0901317組：對HG組用肝X激動劑(T0901317終濃度20μmol/L)進(jìn)行干預(yù)；④HG+TAK-242組：對HG組用TLR抑制劑(TAK-242終濃度100nmol/L)進(jìn)行干預(yù)；⑤HG+Curcumin組：對HG組用姜黃素(Curcumin終濃度10μmol/L)進(jìn)行干預(yù)⑥HG+5CPPSS一50組對HG組用肝X抑制劑(5CPPSS-50終濃度15μmol/L)進(jìn)行干預(yù)⑦HG+5CPPSS一50+Curcumin組：在HG+5CPPSS-50組的基礎(chǔ)上加用Curcumin進(jìn)行干預(yù)；⑧HG+Okadaic acid組：對HG組用PP2A抑制劑(Okadaic acid終濃度20nmol/L)進(jìn)行干預(yù);⑨HG+Okadaic acid+Curcumin組：在HG+Okadaic acid組的基礎(chǔ)上加用Curcumin進(jìn)行干預(yù)。3、相應(yīng)組中加入相應(yīng)藥物干預(yù)72h后,用CCK-8檢測各組H9C2心肌細(xì)胞的生長狀況；ELISA試劑盒測各組炎癥因子(IL6、TNFα、MCP1)分泌量；同時(shí)應(yīng)用qRT-PCR檢測各組LXRα/IRAK4/PP2Ac mRNA水平；、Vestern blot檢則各組NF-κBp65(總量和核內(nèi))、LXRα、IRAK4、p-PP2Ac蛋白的表達(dá)并比較。結(jié)果：1、CCK-8檢測各組H9C2心肌細(xì)胞的生長狀況結(jié)果顯示：高糖可使細(xì)胞增殖活力下降,LXRs激動劑、TLR抑制劑、姜黃素可以減輕高糖對H9C2心肌細(xì)胞增殖活力的抑制,而LXRs抑制劑或PP2A抑制劑干預(yù)后細(xì)胞增殖活力抑制加劇。在LXRs抑制劑或PP2A抑制劑干預(yù)的基礎(chǔ)上使用姜黃素不能改善高糖對H9C2心肌細(xì)胞增殖活力的抑制。2、ELISA去檢測各組細(xì)胞上清中炎癥因子(IL6、TNFα、MCP1)的分泌量,結(jié)果表明：高糖作用下細(xì)胞培養(yǎng)上清中炎癥因子含量明顯增高(P0.05)。T0901317、TAK-242、Curcumin干預(yù)后炎癥因子分泌量減少(P0.05)。5CPPSS-50或Okadaic acid加劇炎癥因子釋放(P0.05)。相對HG+Curcumin組,在LXRs抑制劑或PP2A抑制劑干預(yù)的基礎(chǔ)上使用Curcumin不能降低炎癥因子含量(P0.05)。3、qRT-PCR檢測各組LXRa, IRAK4, PP2Ac mRNA水平：高糖可使LXRa nRNA水平輕微上升,但無統(tǒng)計(jì)學(xué)差異(P0.05),而IRAK4和PP2Ac mRNA明顯上調(diào)(P0.01)。與HG組相比：HG+T0901317組、HG+TAK-242組、IG+Curcumin組LXRa mRNA進(jìn)一步上調(diào)(P0.01),IRAK4 mRNA則明顯下降(P0.01)；HG+5CPPSS-50組結(jié)果提示LXRαmRNA明顯受到抑制(P0.01),RAK4 mRNA進(jìn)一步上調(diào)(P0.01)。在LXRs抑制劑干預(yù)的基礎(chǔ)上使用Curcumin不能發(fā)揮Curcumin上調(diào)LXRαmRNA水平的作用。另外與HG組相比還表明：PP2A抑制劑干預(yù)將進(jìn)一步明顯上調(diào)PP2Ac mRNA水平(P0.01),Curcumin干預(yù)測可以明顯下調(diào)PP2Ac mRNA水平(P0.01),而在PP2A抑制劑干預(yù)的基礎(chǔ)上使用Curcumin將明顯減弱Curcumin下調(diào)PP2Ac mRNA水平的作用。4, Western blot檢測各組NF-κBp65(總蛋白和核內(nèi)組分)、LXRa, IRAK4、 p-PP2Ac蛋白的表達(dá)結(jié)果：各組NF-κBp65總蛋白表達(dá)無明顯差異(P0.05)。高糖可使LXRa蛋白表達(dá)水平輕微上升,但無統(tǒng)計(jì)學(xué)差異(P0.05),而胞核內(nèi)NF-κBp65、IRAK4和p-PP2Ac蛋白表達(dá)明顯上調(diào)(P0.01)。與HG組相比：HG+T0901317組、HG+TAK-242組、HG+Curcumin組LXRa蛋白表達(dá)則進(jìn)一步上調(diào)(P0.05),IRAK4蛋白和胞核內(nèi)NF-κBp65蛋白表達(dá)則明顯下降(P0.05)；HG+5CPPSS-50組結(jié)果提示LXRa蛋白表達(dá)受到抑制(P0.05),IRAK4蛋白和胞核內(nèi)NF-κBp65蛋白表達(dá)進(jìn)一步上調(diào)(P0.01)。在LXRs抑制劑干預(yù)的基礎(chǔ)上使用Curcumin不能發(fā)揮Curcumin上調(diào)LXRa蛋白表達(dá)、抑制NF-κBp65入核的作用。另外,與HG組相比較還表明：PP2A抑制劑干預(yù)將進(jìn)一步上調(diào)p-PP2Ac、胞核內(nèi)NF-κBp65蛋白含量(P0.05),Curcumin干預(yù)則可以明顯下調(diào)p-PP2Ac蛋白表達(dá)(P0.01),而在PP2A抑制劑干預(yù)的基礎(chǔ)上使用Curcumin將明顯減弱Curcumin下調(diào)p-PP2Ac表達(dá)、抑制NF-κBp65入核的作用。結(jié)論：1、高糖可誘導(dǎo)H9C2心肌細(xì)胞發(fā)生炎癥反應(yīng),姜黃素可以抑制高糖誘導(dǎo)的H9C2心肌細(xì)胞炎癥反應(yīng)。2、姜黃素能夠上調(diào)LXRa表達(dá),還可以抑制IRAK4, p-PP2Ac表達(dá),活化PP2A負(fù)性調(diào)控TLR-NF-κB途徑減少NF-κBp65入核進(jìn)而抑制炎癥因子(IL6、TNFα,MCP1)表達(dá),并可能通過這種效應(yīng)來減輕高糖誘導(dǎo)的H9C2心肌細(xì)胞炎癥反應(yīng)。從而提示LXRs、TLR-NF-κB信號通路及相關(guān)蛋白激酶和磷酸化酶之間的整合作用可能是姜黃素減輕高糖誘導(dǎo)的H9C2心肌細(xì)胞炎癥反應(yīng)的細(xì)胞分子機(jī)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.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R587.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 宋利娜;柳茵;李琳;劉維軍;于志文;;姜黃素改善糖尿病小鼠心臟抗氧化及葡萄糖攝取能力[J];醫(yī)學(xué)研究雜志;2014年08期

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本文編號：2040785