姜黃素激活Nrf2系統(tǒng)緩解線粒體損傷和胰島素抵抗作用機(jī)制研究
發(fā)布時間：2019-07-09 06:22
【摘要】：目的氧化應(yīng)激導(dǎo)致的線粒體功能損害參與胰島素抵抗(Insulin resistance,IR)。姜黃素(Curcumin,Cur)可激活Nrf2系統(tǒng)對抗線粒體氧化應(yīng)激及緩解胰島素抵抗。但其作用機(jī)制尚不明確。本研究圍繞著姜黃素是否以及如何通過調(diào)控Nrf2功能緩解線粒體損傷與IR發(fā)病這一核心問題,進(jìn)一步深度解析Nrf2增敏胰島素的作用途徑、作用靶點以及相關(guān)信號機(jī)理。研究將闡明姜黃素轉(zhuǎn)化應(yīng)用的藥理基礎(chǔ)從而為應(yīng)用于糖尿病的防治奠定理論基礎(chǔ)。方法首先選用C57BL/6J小鼠進(jìn)行高脂飼料干預(yù)誘導(dǎo)IR模型,再給予Cur干預(yù),觀察姜黃素的作用,探討Cur保護(hù)線粒體和逆轉(zhuǎn)IR作用靶點。為深入了解Nrf2抗氧化系統(tǒng)在其中所起作用,利用棕櫚酸誘導(dǎo)HepG2肝細(xì)胞IR模型給予Nrf2抑制劑維甲酸RA處理,觀察對Cur作用影響。由于線粒體形態(tài)調(diào)控與其功能關(guān)系密切。隨后,在此模型上進(jìn)行促線粒體分裂抑制劑Mdivi-1處理,Cur是否通過影響線粒體形態(tài)起到調(diào)控線粒體功能以及與激活Nrf2系統(tǒng)和增強(qiáng)胰島素信號關(guān)系。由于線粒體氧化還原平衡與鈣穩(wěn)態(tài)關(guān)系密切,最后,我們觀察了 Cur以及Nrf2系統(tǒng)對線粒體鈣含量影響,并利用線粒體鈣通道抑制劑,進(jìn)一步探討其對胰島素信號的調(diào)控作用。具體實驗如下:動物IR模型采用60%的高脂飼料飼喂雄性C57BL/6J小鼠并根據(jù)葡萄糖耐量實驗(IPGTT)結(jié)果判定模型是否成功。細(xì)胞IR模型采用人肝癌細(xì)胞HepG2并給予60μM飽和脂肪酸棕櫚酸(Palmitate,PA)處理細(xì)胞誘導(dǎo)IR模型。動物IR模型成功后,連續(xù)2周進(jìn)行Cur灌胃干預(yù),Cur干預(yù)劑量為50mg/kg/d。細(xì)胞用棕櫚酸IR造模時,給予不同的工具藥處理,包括1μM Nrf2抑制劑維甲酸(Retinoic acid,RA)、50μM促線粒體分裂抑制劑 Mdivi-1 處理、30μM釕紅(Ruthenium red,RuR)和10μM Cur干預(yù)。隨后進(jìn)行細(xì)胞線粒體膜電位、ROS、鈣離子含量測定以及WB蛋白含量檢測。蛋白免疫印跡法(Western blot，，WB)檢測線粒體促分裂和融合蛋白(Drp1、Mfr2及OPA1)及相關(guān)調(diào)控因子(NRF1、mtTFA、TFB1M及TFB2M等)在C57BL/6J小鼠組織的變化。磷鉬酸比色法測定小鼠肝臟組織內(nèi)ATP含量及ATPase(Na+ K+-ATPase,Mg2+-ATPase,Ca2+-ATPase,Ca2+ Mg2+-ATPase)的活力,評價 Cur 對 IR 模型小鼠肝臟線粒體能量代謝的影響。細(xì)胞線粒體氧化還原狀態(tài)檢測采用TAB結(jié)合法測定線粒體中脂質(zhì)過氧化指標(biāo)MDA(malondialdehyde)含量;利用JC-1試劑盒測定細(xì)胞線粒體膜電位(Mitochondrial membrane potential,MMP)的改變;MitoSOXTM 試劑盒對 HepG2 肝癌細(xì)胞染色檢測細(xì)胞線粒體內(nèi)ROS的含量進(jìn)行測定;同時,在RA、Mdivi-1和RuR處理細(xì)胞以后,利用WB方法檢測胰島素刺激的PKBSer473磷酸化、GRP78、IκBα及相關(guān)線粒體蛋白及調(diào)控因子的表達(dá)。結(jié)果無論在高脂飼料誘導(dǎo)的動物IR模型中還是在PA誘導(dǎo)的細(xì)胞IR模型中,線粒體融合蛋白均受到抑制(Mfn2、OPA1融合蛋白減少)、促分裂調(diào)控蛋白表達(dá)增多(Drp1促分裂蛋白增加)、轉(zhuǎn)錄調(diào)控蛋白下調(diào)(Nrf1、mtTFA、TFB1M及TFB2M表達(dá)降低),Cur均可以明顯減輕甚至逆轉(zhuǎn)高脂所造成的這些影響。此外,Cur干預(yù)可明顯糾正IR模型小鼠肝臟組織 ATP 含量降低并增強(qiáng) Na+ K+-ATPase,Mg2+-ATPase,Ca2+-ATPase,Ca2+ Mg2+-ATPase的酶活性活性。另外,Cur可部分對抗PA所致的線粒體活性氧ROS含量升高以及線粒體功能損害,即線粒體膜電位降低;反之,對細(xì)胞進(jìn)行Nrf2抑制劑處理后,Cur的上述改善作用受到明顯抑制,說明Cur通過激活Nrf2實現(xiàn)其對線粒體功能以及胰島素信號的調(diào)控作用。進(jìn)一步的研究表明,無論線粒體分裂抑制劑Mdivi-1以及Cur處理處理PAIR細(xì)胞,均能上調(diào)線粒體合成及轉(zhuǎn)錄調(diào)控蛋白,增強(qiáng)線粒體融合蛋白含量,同時抑制線粒體促分裂調(diào)控蛋白表達(dá)。最后,由于姜黃素具有減輕IR細(xì)胞線粒體鈣超載作用。為了進(jìn)一步評價姜黃素這一作用的藥效意義,我們利用線粒體鈣通道抑制劑處理IR細(xì)胞發(fā)現(xiàn):IR細(xì)胞的線粒體鈣超載受到抑制后,可以起到抑制炎癥、內(nèi)質(zhì)網(wǎng)應(yīng)激,同時激活Nrf2系統(tǒng)并增強(qiáng)胰島素信號傳導(dǎo)。結(jié)論Cur無論在高脂飼喂動物以及棕櫚酸誘導(dǎo)的IR細(xì)胞均能上調(diào)促進(jìn)線粒體合成及轉(zhuǎn)錄調(diào)控蛋白,增強(qiáng)線粒體融合蛋白并抑制促分裂調(diào)控蛋白表達(dá),而且這些對線粒體的調(diào)控作用與姜黃素激活Nrf2抗氧化功能以及胰島素信號的調(diào)控作用有關(guān)。因此,姜黃素通過激活Nrf2系統(tǒng)改善線粒體氧化應(yīng)激并對線粒體形態(tài)和功能進(jìn)行調(diào)控,這些作用是其改善胰島素抵抗藥效作用的重要機(jī)理,而姜黃素通過激活Nrf2系統(tǒng)對抗線粒體氧化應(yīng)激并抑制鈣超載誘發(fā)的炎癥激活、內(nèi)質(zhì)網(wǎng)應(yīng)激以及Nrf2系統(tǒng)的功能抑制,并依此改善胰島素敏感性。
[Abstract]:Objective To investigate the role of mitochondrial function induced by oxidative stress in insulin resistance (IR). Curcumin (Curcuin, Cur) can activate the Nrf2 system to counter mitochondrial oxidative stress and to relieve insulin resistance. However, its mechanism of action is not clear. This study focused on whether curcumin and how to alleviate the core problem of mitochondrial injury and IR by regulating the function of Nrf2, and to further analyze the role of Nrf2 to sensitize the insulin, the target of action and the related signal mechanism. The study will clarify the pharmacological basis of curcumin conversion and lay a theoretical foundation for the prevention and treatment of diabetes. Methods C57BL/ 6J mice were first selected for high-fat feed intervention to induce IR, and then Cur intervention was given to observe the effect of curcumin on the protection of mitochondria and reverse the IR target. In order to understand the role of Nrf2 anti-oxidation system, the effect of Nrf2 inhibitor-retinoic acid RA on the effect of Nrf2 on the effect of Nrf2 on the effect of Nrf2 on the effect of Nrf2 on the effect of the anti-oxidation system was observed. The regulation of mitochondrial morphology is closely related to its function. Subsequently, the Mdivi-1 treatment of the mitogen-stimulating inhibitor Mdivi-1 was carried out on this model, and whether the Cur had the function of regulating the mitochondrial function by affecting the mitochondrial morphology and the relationship with the activation of the Nrf2 system and the enhanced insulin signal. In the end, the effects of Cur and Nrf2 on the content of calcium in mitochondria were observed, and the regulation of the insulin signal was further discussed by using the mitochondrial calcium channel inhibitor. The specific experiment is as follows: The animal IR model uses 60% high-fat feed to feed the male C57BL/ 6J mice and determines whether the model is successful according to the results of the glucose tolerance test (IPGTT). The cellular IR model used human liver cancer cell HepG2 and given a 60 & mu; M saturated fatty acid palmitate (Palmitate, PA) to process the cell-induced IR model. After the animal IR model was successful, Cur was given intragastric intervention for 2 weeks, and the intervention dose of Cur was 50 mg/ kg/ day. When the cells were made with palmitic acid IR, different administration treatments were administered, including 1. m u.M Nrf2 inhibitor Retinoic acid (RA),50. m u.M mitogen-mitogen inhibitor Mdivi-1 treatment,30. m u.M of red (RuR) and 10. m u.M Cur intervention. Then the cell mitochondrial membrane potential, the ROS, the calcium ion content determination, and the WB protein content detection were performed. The changes of mitochondrial mitogen and fusion protein (Dr1, Mfr2 and OPPA1) and related regulatory factors (NF1, mtTFA, TFB1M, and TFB2M, etc.) in C57BL/ 6J mice were detected by Western blot and WB. The activity of ATP content and ATPase (Na + K +-ATPase, Mg 2 +-ATPase, Ca 2 +-ATPase, Ca 2 + Mg 2 +-ATPase) in the liver of mice was determined by the P-P-acid colorimetric method, and the effect of Cur on the energy metabolism of liver mitochondria in the mouse liver was evaluated. The content of MDA (malonic dehyde) in the mitochondria was determined by a TAB-binding method, and the changes of the mitochondrial membrane potential (MMP) were determined by the JC-1 kit. The content of ROS in the mitochondria of HepG2 liver cancer cells was determined by the MiteSXTM kit. After the cells were treated with RA, Mdivi-1 and RuR, the phosphorylation of PKBSer473, the expression of GRP78, I-B and the related mitochondrial proteins and regulatory factors were detected by WB method. Results In the animal IR model induced by high-fat diet or in the PA-induced cellular IR model, the mitochondrial fusion protein was inhibited (Mfn2, OPA1 fusion protein decreased), the expression of the mitogen-controlled protein was increased (the DDr1 mitogen increased), and the transcription regulatory protein was down-regulated (Nrf1, Both mtTFA, TFB1M, and TFB2M expression decreased, and Cur could significantly reduce or even reverse the effects of high fat. In addition, the activity of the activity of Na + K +-ATPase, Mg2 +-ATPase, Ca2 +-ATPase, Ca2 + Mg2 +-ATPase can be significantly corrected by Cur intervention. in addition, that Cur can partially antagonize the increase in the ROS content of the mitochondria and the damage of the function of the mitochondria, that is, the mitochondrial membrane potential is reduced; on the contrary, after the Nrf2 inhibitor treatment is carried out on the cell, the above-mentioned improvement effect of the Cur is obviously inhibited, The effect of Cur on the function of mitochondria and the regulation of the insulin signal is explained by the activation of Nrf2. The further study shows that the mitochondrial synthesis and transcription regulation protein can be up-regulated, the content of the mitochondrial fusion protein can be enhanced, and the expression of the mitochondrial mitogen-promoting regulatory protein can be inhibited, regardless of the mitochondrial division inhibitor Mdivi-1 and the treatment of PAIR cells by the Cur treatment. And finally, the curcumin has the function of reducing the mitochondrial calcium overload of the IR cells. In order to further evaluate the effect of curcumin, we use the mitochondrial calcium channel inhibitor to treat IR cells, which can inhibit the inflammation, endoplasmic reticulum stress, and activate Nrf2 system and enhance the conduction of insulin signal. Conclusion Cur can upregulate the mitochondrial synthesis and transcriptional regulation protein, enhance the mitochondrial fusion protein and inhibit the expression of mitogen-regulated protein, whether in high-fat feeding animals and palmitic acid-induced IR cells. And the regulatory effects of these on the mitochondria are related to the activation of the anti-oxidation function of the curcumin and the regulation of the insulin signal. Therefore, the curcumin can improve the mitochondrial oxidative stress by activating the Nrf2 system and regulate the shape and function of the mitochondria, which is an important mechanism for improving the effect of insulin resistance, Curcumin inhibits the mitochondrial oxidative stress by activating the Nrf2 system and inhibits the inflammatory activation induced by calcium overload, endoplasmic reticulum stress, and the functional inhibition of the Nrf2 system, and accordingly improves insulin sensitivity.
【學(xué)位授予單位】：福建中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R285.5
本文編號：2511922
[Abstract]:Objective To investigate the role of mitochondrial function induced by oxidative stress in insulin resistance (IR). Curcumin (Curcuin, Cur) can activate the Nrf2 system to counter mitochondrial oxidative stress and to relieve insulin resistance. However, its mechanism of action is not clear. This study focused on whether curcumin and how to alleviate the core problem of mitochondrial injury and IR by regulating the function of Nrf2, and to further analyze the role of Nrf2 to sensitize the insulin, the target of action and the related signal mechanism. The study will clarify the pharmacological basis of curcumin conversion and lay a theoretical foundation for the prevention and treatment of diabetes. Methods C57BL/ 6J mice were first selected for high-fat feed intervention to induce IR, and then Cur intervention was given to observe the effect of curcumin on the protection of mitochondria and reverse the IR target. In order to understand the role of Nrf2 anti-oxidation system, the effect of Nrf2 inhibitor-retinoic acid RA on the effect of Nrf2 on the effect of Nrf2 on the effect of Nrf2 on the effect of Nrf2 on the effect of the anti-oxidation system was observed. The regulation of mitochondrial morphology is closely related to its function. Subsequently, the Mdivi-1 treatment of the mitogen-stimulating inhibitor Mdivi-1 was carried out on this model, and whether the Cur had the function of regulating the mitochondrial function by affecting the mitochondrial morphology and the relationship with the activation of the Nrf2 system and the enhanced insulin signal. In the end, the effects of Cur and Nrf2 on the content of calcium in mitochondria were observed, and the regulation of the insulin signal was further discussed by using the mitochondrial calcium channel inhibitor. The specific experiment is as follows: The animal IR model uses 60% high-fat feed to feed the male C57BL/ 6J mice and determines whether the model is successful according to the results of the glucose tolerance test (IPGTT). The cellular IR model used human liver cancer cell HepG2 and given a 60 & mu; M saturated fatty acid palmitate (Palmitate, PA) to process the cell-induced IR model. After the animal IR model was successful, Cur was given intragastric intervention for 2 weeks, and the intervention dose of Cur was 50 mg/ kg/ day. When the cells were made with palmitic acid IR, different administration treatments were administered, including 1. m u.M Nrf2 inhibitor Retinoic acid (RA),50. m u.M mitogen-mitogen inhibitor Mdivi-1 treatment,30. m u.M of red (RuR) and 10. m u.M Cur intervention. Then the cell mitochondrial membrane potential, the ROS, the calcium ion content determination, and the WB protein content detection were performed. The changes of mitochondrial mitogen and fusion protein (Dr1, Mfr2 and OPPA1) and related regulatory factors (NF1, mtTFA, TFB1M, and TFB2M, etc.) in C57BL/ 6J mice were detected by Western blot and WB. The activity of ATP content and ATPase (Na + K +-ATPase, Mg 2 +-ATPase, Ca 2 +-ATPase, Ca 2 + Mg 2 +-ATPase) in the liver of mice was determined by the P-P-acid colorimetric method, and the effect of Cur on the energy metabolism of liver mitochondria in the mouse liver was evaluated. The content of MDA (malonic dehyde) in the mitochondria was determined by a TAB-binding method, and the changes of the mitochondrial membrane potential (MMP) were determined by the JC-1 kit. The content of ROS in the mitochondria of HepG2 liver cancer cells was determined by the MiteSXTM kit. After the cells were treated with RA, Mdivi-1 and RuR, the phosphorylation of PKBSer473, the expression of GRP78, I-B and the related mitochondrial proteins and regulatory factors were detected by WB method. Results In the animal IR model induced by high-fat diet or in the PA-induced cellular IR model, the mitochondrial fusion protein was inhibited (Mfn2, OPA1 fusion protein decreased), the expression of the mitogen-controlled protein was increased (the DDr1 mitogen increased), and the transcription regulatory protein was down-regulated (Nrf1, Both mtTFA, TFB1M, and TFB2M expression decreased, and Cur could significantly reduce or even reverse the effects of high fat. In addition, the activity of the activity of Na + K +-ATPase, Mg2 +-ATPase, Ca2 +-ATPase, Ca2 + Mg2 +-ATPase can be significantly corrected by Cur intervention. in addition, that Cur can partially antagonize the increase in the ROS content of the mitochondria and the damage of the function of the mitochondria, that is, the mitochondrial membrane potential is reduced; on the contrary, after the Nrf2 inhibitor treatment is carried out on the cell, the above-mentioned improvement effect of the Cur is obviously inhibited, The effect of Cur on the function of mitochondria and the regulation of the insulin signal is explained by the activation of Nrf2. The further study shows that the mitochondrial synthesis and transcription regulation protein can be up-regulated, the content of the mitochondrial fusion protein can be enhanced, and the expression of the mitochondrial mitogen-promoting regulatory protein can be inhibited, regardless of the mitochondrial division inhibitor Mdivi-1 and the treatment of PAIR cells by the Cur treatment. And finally, the curcumin has the function of reducing the mitochondrial calcium overload of the IR cells. In order to further evaluate the effect of curcumin, we use the mitochondrial calcium channel inhibitor to treat IR cells, which can inhibit the inflammation, endoplasmic reticulum stress, and activate Nrf2 system and enhance the conduction of insulin signal. Conclusion Cur can upregulate the mitochondrial synthesis and transcriptional regulation protein, enhance the mitochondrial fusion protein and inhibit the expression of mitogen-regulated protein, whether in high-fat feeding animals and palmitic acid-induced IR cells. And the regulatory effects of these on the mitochondria are related to the activation of the anti-oxidation function of the curcumin and the regulation of the insulin signal. Therefore, the curcumin can improve the mitochondrial oxidative stress by activating the Nrf2 system and regulate the shape and function of the mitochondria, which is an important mechanism for improving the effect of insulin resistance, Curcumin inhibits the mitochondrial oxidative stress by activating the Nrf2 system and inhibits the inflammatory activation induced by calcium overload, endoplasmic reticulum stress, and the functional inhibition of the Nrf2 system, and accordingly improves insulin sensitivity.
【學(xué)位授予單位】：福建中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R285.5
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 劉穎;傅晗;師騰瑞;劉江正;王欣;海春旭;;高脂飼料喂飼C57小鼠誘發(fā)胰島素抵抗過程中肝臟氧化應(yīng)激水平及Nrf2、NRF-1和mtTFA蛋白表達(dá)的變化[J];癌變·畸變·突變;2015年03期
2 陳娜;鄧倩;譚秋榮;李莉明;喻南慧;蔣濤;;線粒體功能障礙與2型糖尿病[J];中南藥學(xué);2014年09期
3 許榮波;門秀麗;宋旭東;;線粒體動力學(xué)與2型糖尿病胰島素抵抗的研究進(jìn)展[J];生理科學(xué)進(jìn)展;2014年02期
4 劉莉;譚波濤;李昱;余剛;;姜黃素對大鼠大腦皮質(zhì)缺血再灌注損傷的保護(hù)作用及其機(jī)制研究[J];解放軍醫(yī)學(xué)雜志;2013年03期
5 時麗麗;張莉;譚初兵;徐為人;杜冠華;;線粒體功能損傷與胰島素抵抗[J];中國藥理學(xué)通報;2012年11期
6 門秀麗;張麗萍;吳靜;劉虹麟;張文健;婁晉寧;;線粒體形態(tài)與細(xì)胞凋亡的關(guān)系[J];生理科學(xué)進(jìn)展;2012年05期
7 何惠君;王國玉;高淵;于志文;;姜黃素活化肝細(xì)胞Nrf2抗氧化系統(tǒng)效應(yīng)及緩解胰島素抵抗作用機(jī)制研究[J];熱帶醫(yī)學(xué)雜志;2012年07期
8 ;Curcumin attenuates Nrf2 signaling defect, oxidative stress in muscle and glucose intolerance in high fat diet-fed mice[J];World Journal of Diabetes;2012年05期
9 孫魯寧;趙明沂;宋曉宇;鄭寧寧;李罡;張海鵬;;代謝綜合征大鼠肝細(xì)胞線粒體損傷和mtDNA編碼蛋白的表達(dá)變化[J];解剖科學(xué)進(jìn)展;2011年03期
10 趙曙光;李強(qiáng);王景杰;王旭霞;劉震雄;聞勤生;;姜黃素誘導(dǎo)Nrf2核轉(zhuǎn)位對氧化應(yīng)激誘導(dǎo)人肝細(xì)胞胰島素抵抗的影響[J];現(xiàn)代生物醫(yī)學(xué)進(jìn)展;2011年01期
相關(guān)碩士學(xué)位論文 前2條
1 劉宏;線粒體DNA編碼蛋白質(zhì)在炎癥反應(yīng)中作用的初步探討[D];中國人民解放軍醫(yī)學(xué)院;2013年
2 孫靜;姜黃素改善HepG2細(xì)胞胰島素抵抗的機(jī)制研究[D];蘭州大學(xué);2009年
本文編號：2511922
本文鏈接：http://sikaile.net/shoufeilunwen/mpalunwen/2511922.html
最近更新
教材專著
