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作者：刘思远北京中医药大学

学位级别：硕士

1研究目的与意义阿尔茨海默病(Alzheimer's disease,AD)又称老年性痴呆,是一种老年人常见的中枢神经系统退行性疾病,以学习记忆功能障碍、执行功能障碍、语言功能障碍等为主要症状,严重者可出现人格行为异常等精神症状。AD患病率随... 详细信息

1研究目的与意义阿尔茨海默病(Alzheimer's disease,AD)又称老年性痴呆,是一种老年人常见的中枢神经系统退行性疾病,以学习记忆功能障碍、执行功能障碍、语言功能障碍等为主要症状,严重者可出现人格行为异常等精神症状。AD患病率随着年龄的增长而增加,给国家、社会、家庭都带来沉重的负担。目前在我国,AD已成为了不可忽视的公共卫生、社会经济问题,其发病机制尚不明确。并且,目前暂无可以治愈AD的药物及方法。因此,探讨AD病情进展的机制,寻找新的有效预防和延缓AD的治疗策略具有重要现实意义。本实验通过观察“通督启神”法电针对APP/PS1双转基因AD模型小鼠空间学习、记忆能力,以及额叶HIF-1α、bnip3表达的影响,探讨其治疗AD的作用机制。 2研究方法选用SPF级、健康、雄性、6月龄、APP/PS1双转基因模型小鼠,30只,采用随机数字表法分为3组:模型组(M)、电针组(EA)、电针+抑制剂组(EA+3MA),每组各10只;同背景、同月龄的C57BL6小鼠,10只,为正常对照组(C)。保证相同的饲养环境和饮食:常规、单笼饲养,小鼠自行饮水、进食。先用自制的鼠套,将电针组小鼠束缚固定,腹腔注射PBS缓冲液,30min后,参照“实验动物针灸穴位图谱”,选取“百会”“印堂”“水沟”三穴。选用0.25 mm×13mm的毫针,快速点刺“水沟”穴,不留针;然后交叉平刺“百会”“印堂”穴(两针不可相碰,避免形成短路),进针深度为0.5cm,用胶带固定针柄,并将针柄连接华佗牌电子电疗仪,频率设定为1Hz,电压设定为2V,电流强度设定0.2mA,强度以小鼠的头部微微颤动即可,每天干预1次,每次治疗20min,连续干预15天。电针+抑制剂组小鼠在电针干预前30min,腹腔注射线粒体自噬抑制剂3MA溶液(0.1ml,30mg/kg,PBS缓冲液制备),其余干预手段同电针组,连续干预15天。正常对照组、模型组腹腔注射PBS溶液,30min后,干预治疗时抓取一次,并使用相同的自制鼠套束缚20min,连续干预15天。 3研究结果 3.1 Morris水迷宫测试结果 3.1.1隐蔽平台实验结果各组小鼠的逃避潜伏期随着实验天数的增加呈现出缩短的趋势。正常对照组与模型组相比较,逃避潜伏期的缩短趋势更加明显,差异有统计学意义(P<0.05);第3天开始,电针组与模型组相比较,逃避潜伏时间出现统计学差异(P<0.05),且电针组低于模型组;第5天训练后,电针组与电针+抑制剂组的逃避潜伏时间出现统计学差异(P<0.05),电针组低于电针+抑制剂组。正常对照组的逃避潜伏期时间相对最短,且下降最快;次之为电针组、电针+抑制剂组、模型组。电针+抑制剂组与模型组相比较,逃避潜伏期较短,但差异无统计学意义(P>0.05)。 3.1.2空间探索实验结果正常对照组在目标象限的运动距离和进入次数最多,最少的为模型组。正常对照组与模型组相比较,目标象限进入次数与运动距离均有明显统计学差异(P<0.05)。其余三组两两比较,电针组、电针+抑制剂组的目标象限进入次数与运动距离均高于模型组,且电针组与电针+抑制剂组相比较高,但差异均无统计学意义(P>0.05)。 3.2免疫组化检测结果 HIF-1α、bnip3、Beclin1主要表达于细胞质和细胞膜,其阳性表达呈棕褐色。正常对照组的阳性表达细胞棕褐色着色较深,且表达数量较多;模型组与正常对照组相比较,明显着色变浅,表达数量减少,差异有统计学意义(P<0.05)。电针组与模型组相比较,阳性表达细胞着色较深,表达数量增多,差异有统计学意义(P<0.05)。电针组与电针+抑制剂组相比较,阳性细胞着色深,表达数量多,差异有统计学意义(P<0.05)。电针+抑制剂组阳性表达细胞相比模型组较多,但差异无统计学意义(P>0.05)。 3.3 Western Blot 检测结果与正常对照组相比较,模型组的HIF-1 α、bnip3、Beclin1条带较细,蛋白表达明显较少,PI3K条带增粗,蛋白表达增加,差异均有统计学意义(P<0.05);与模型组相比较,电针组的HIF-1α、bnip3、Beclin1表达均明显增加(P<0.05),电针+抑制剂组的HIF-1α、bnip3、Beclin1表达也有所增加,但差异均无统计学意义(P>0.05);电针+抑制剂组与电针组相比较,三者表达均减少,HIF-1α、bnip3表现出统计学差异(P<0.05),但Beclin1未具备统计学差异(P>0.05);电针组PI3K的表达明显少于模型组和电针+抑制剂组,差异均有统计学意义(P<0.05),电针+抑制剂组表达含量与模型组几乎无差异(P>0.05)。 4结论 (1)“通督启神”法电针,可以明显改善AD的症状,可以明显提高APP/PS1双转基因模型小

关键词：阿尔茨海默病通督启神电针低氧诱导因子1α bnip3

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Angelica polysaccharide moderates hypoxia-evoked apoptosis and autophagy in rat neural stem cells by downregulation of bnip3 (Publication with Expression of Concern)

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ARTIFICIAL CELLS NANOMEDICINE AND BIOTECHNOLOGY 2019年第1期47卷 2492-2499页

作者： Xue, Yongzhen Li, Dongmei Zhang, Yige Gao, Hang Li, Hui East Med Dist Linyi Peoples Hosp Dept Childrens Healthcare Linyi Shandong Peoples R China Shandong Prov Coal Linyi Hot Springs Sanat Hosp Dept Internal Med Linyi Shandong Peoples R China Qingdao Univ Dept Med Classe Six Clin Five Year Syst Qingdao Shandong Peoples R China Jining Med Univ Basic Med Coll Class Eight Clin Med Jining Peoples R China Linyi Womens & Childrens Hosp Dept Childrens Rehabil 1 Qinghe South Rd Linyi 276000 Shandong Peoples R China

Neonatal hypoxia-ischemia is a troublesome disease. Angelica polysaccharide (AP) is proved to have antioxidant effects. Our study was performed to confirm the effects of AP in hypoxia-exposed neural stem cells (NSCs). NSCs were pre-treated with AP and then stimulated with hypoxia. Viability of NSCs was examined by Cell Counting Kit-8 assay. Hypoxia-introduced apoptosis was observed by flow cytometry. Essential regulators of mTOR and Notch signalling pathways were examined by Western blot. mRNA expression was accessed using qRT-PCR. Bcl2/adenovirus EIB 19kD-interacting protein 3 (bnip3) was altered by transfection. We noticed that NSCs were sensitive to hypoxia-induced apoptosis and showed decreased viability. Moreover, Beclin and light chain 3-II was upregulated while p62 was downregulated. However, AP reversed all these results. Similarly, hypoxia decreased the phosphorylation of mTOR and p70S6K and Notch1 expression while AP increased the phosphorylation of mTOR and p70S6K as well as the expression of Notch1. bnip3 was upregulated by hypoxia while downregulated by AP. Further experiments demonstrated that overexpression of bnip3 broken all the effects induced by AP shown in cell viability, apoptosis, autophagy and signalling pathways. Collectively, AP alleviated hypoxia-introduced NSCs damages by maintaining cell viability, blocking apoptosis and autophagy via downregulation of bnip3 with the activation of mTOR and Notch signalling pathways.

关键词： Neonatal hypoxia-ischemia angelica polysaccharide bnip3 mTOR Notch

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Upregulation of long noncoding RNA RP4 exacerbates hypoxia injury in cardiomyocytes through regulating miR-939/bnip3/Wnt/β-catenin pathway (Publication with Expression of Concern. See vol. 50, pg. 221, 2022)

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ARTIFICIAL CELLS NANOMEDICINE AND BIOTECHNOLOGY 2019年第1期47卷 3013-3020页

作者： Rong, Jingfeng Xu, Jijie Liu, Qian Wu, Yaoyao Guo, Honglin Mu, Ting Zhou, Hua Chi, Hao Zhengzhou Univ Luoyang Cent Hosp Dept Med Cardiovasc Luoyang Henan Peoples R China Shanghai Univ Tradit Chinese Med Shuguang Hosp Inst Cardiovasc Res Shanghai Peoples R China Shanghai Univ Tradit Chinese Med Shuguang Hosp Dept Cardiovasc Shanghai Peoples R China Shanghai Univ Tradit Chinese Med Shuguang Hosp Dept Cardiothorac Surg 528 Zhangheng Rd Shanghai 201499 Peoples R China

This research planned to dig the impacts and potential principles of long noncoding RNA RP4 onH9c2 cell injury induced by hypoxia. The H9c2 cardiac muscle cells were cultured under 3% O-2 concentration to induce hypoxia injury, followed by detection of RP4 expression. RP4 was then over-expressed and silenced to investigate its effects on cell injury induced by hypoxia. The potential correlation between RP4 and miR-939, between miR-939 and bnip3, and between RP4/miR-939/ bnip3 axis and Wnt/beta-catenin pathway activation were explored. Biological processes (suppressed cell viability, migration and invasion, but enhanced cell apoptosis) were changed by hypoxia. Upregulation of RP4 enhanced hypoxia-produced damage in H9c2 cells. Additionally, miR-939 expression was opposite regulated by RP4, and miR-939 mimic abrogated the influences of pc-RP4 on enhanced hypoxia damage in H9c2 cells. Moreover, bnip3 was targeted by miR-939 and their correlation is negative. Furthermore, upregulation of RP4 exacerbated hypoxia-produced injury in H9c2 cells by sensitizing Wnt/beta-catenin signals in H9c2 cells, which was regulated by miR-939/bnip3 axis. Our findings reveal that RP4 is highly expressed in the hypoxia-resulted H9c2 cells. Enhanced expression of RP4 may exacerbate hypoxia injury in cardiomyocytes through regulating miR-939/bnip3 axis-mediated briskness of Wnt/beta-catenin signals. Our study will offer a fresh theoretical basis for the treatment of ischemic myocardial injury.

关键词： Hypoxia-induced myocardial injury long noncoding RNA RP4 miR-939 bnip3 Wnt/beta-catenin pathway

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Testosterone induces renal tubular epithelial cell death through the HIF-1/bnip3 pathway

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JOURNAL OF TRANSLATIONAL MEDICINE 2019年第1期17卷 1-13页

作者： Peng, Yonghan Fang, Ziyu Liu, Min Wang, Zeyu Li, Ling Ming, Shaoxiong Lu, Chaoyue Dong, Hao Zhang, Wenhui Wang, Qi Shen, Rong Xie, Fei Zhang, Weitao Yang, Cheng Gao, Xiaofeng Sun, Yinghao Shanghai Changhai Hosp Dept Urol Shanghai 200433 Peoples R China Fudan Univ Zhongshan Hosp Dept Urol 180 Fenglin Rd Shanghai 200032 Peoples R China Shanghai Key Lab Organ Transplantat 180 Fenglin Rd Shanghai 200032 Peoples R China Fudan Univ Zhangjiang Inst Shanghai 201203 Peoples R China

BackgroundThe morbidity of nephrolithiasis is 2-3 times higher in males than in females, suggesting that androgen plays a key role in nephrolithiasis. The death of renal tubular epithelial cells (TECs) is an important pathophysiological process contributing to the development of nephrolithiasis. Therefore, the aim of this study is to investigate whether androgen directly induces TECs apoptosis and necrosis and its underlying mechanisms in kidney stone *** and methodsWe compared serum testosterone level between male and female healthy volunteers and kidney stone patients. The in vivo nephrolithiasis model was established using glyoxylic acid, and calcium deposits were detected by van Kossa staining. In the in vitro study using mouse TECs (TCMK-1 cells) and human TECs (HK-2 cells), apoptosis, necrosis, and the expression of BH3-only protein Bcl-2-like 19kDa-interacting protein 3 (bnip3) were examined incubated with different doses of testosterone using flow cytometry. Levels of apoptosis-related proteins transfected with the bnip3 siRNA were examined by western blotting. The mitochondrial potential (m) was detected by JC-1 staining and flow cytometry. We monitored bnip3 expression in the testosterone-induced TECs injury model after treatment with hypoxia inducible factor 1 (HIF-1) and/or hypoxia inducible factor 2 (HIF-2) inhibitors to determine the upstream protein regulating bnip3 expression. Additionally, ChIP and luciferase assays were performed to confirm the interaction between HIF-1 and *** male and female patients have significantly higher testosterones compared with healthy volunteers. More calcium deposits in the medulla were detected in male mice compared to female and castrated male mice. Testosterone induced TECs apoptosis and necrosis and increased bnip3 expression in a dose-dependent manner. Testosterone also increased Bax expression, decreased Bcl-2 expression and induced a loss of m. This effect was reversed by bnip3 knoc

关键词： Androgen Testosterone Kidney Tubular epithelial cell bnip3 Cell death

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A crosstalk between phosphorylation and ubiquitination of bnip3 regulates mitophagy under hypoxia

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Autophagy reports 2023年第1期2卷 2197637页

作者： Yun-Ling He Jian Li Yan Cao Hai-Tao Wu Li-Ying Wu Department of Neurobiology Beijing Institute of Basic Medical Sciences Beijing China. Beijing Institute of Microbiology and Epidemiology Beijing China. State Key Laboratory of Proteomics Beijing Proteome Research Center Beijing Institute of Radiation Medicine Beijing China. Chinese Institute for Brain Research Beijing China.

bnip3 (BCL2/adenovirus e1B 19 kDa protein interacting protein 3) is a mitochondrial outer membrane protein that is sensitive to hypoxia and mediates mitophagy, a process important for mitochondrial quality control and to maintain energetic and redox homeostasis under hypoxia. It has been reported that up-regulation of bnip3, which acts as mitophagy receptor, promotes mitophagy. In our recent study, we found that the post-translational modification of bnip3 is crucial to induce mitophagy, and that a crosstalk between phosphorylation/dephosphorylation and ubiquitination acts as a switch to control bnip3-mediated mitophagy under hypoxia. We demonstrated that the phosphorylation of bnip3 at S60 and T66 by MAPK8/9 (mitogen-activated protein kinase 8/9) under hypoxia blocks the degradation of bnip3 via the ubiquitin-proteasome pathway and enhances its interaction with MAP1LC3 (microtubule associated protein 1 light chain 3), thereby promoting mitophagy. In contrast, dephosphorylation of bnip3 by members of the PP1/2A (protein phosphatase PP1 and PP2A) phosphatase subfamily under hypoxia accelerates degradation of bnip3 via the ubiquitin-proteasome pathway, thereby suppressing mitophagy. Altogether, these findings provide knowledge necessary to devise intervention strategies for hypoxia-related diseases and/or hypoxia-related developmental processes. BCL2: BCL2 apoptosis regulator; BCL2L1: BCL2 like 1; BECN1: beclin 1, autophagy related; BH3: BCL2 homology 3; bnip3: BCL2/adenovirus e1B 19 kDa protein interacting protein 3; LIR: MAP1LC3-interacting region; MAP1LC3: microtubule associated protein 1 light chain 3; MAPK8: mitogen-activated protein kinase 8; MAPK9: mitogen-activated protein kinase 9; PEST: rich in amino acids P, E, S, T, and D; PP1: protein phosphatase 1; PP2A: protein phosphatase 2A; PEST: rich in amino acids P, E, S, T, and D.

关键词： bnip3 hypoxia mitophagy phosphorylation ubiquitination

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bnip3介导骨骼肌线粒体自噬：不同强度运动的影响

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中国组织工程研究 2020年第5期24卷 682-688页

作者：于亮赵泽铭赵斌婷李琳刘子铭王祯王瑞元北京体育大学运动人体科学学院

背景:不同强度运动对机体可产生不同的影响,运动后骨骼肌的变化也不明确,而运动中机体的生理变化机制更是目前研究的热点。目的:探讨不同强度运动对大鼠骨骼肌质量的影响以及bnip3介导的骨骼肌线粒体自噬在维持骨骼肌质量中的作用。方法... 详细信息

背景:不同强度运动对机体可产生不同的影响,运动后骨骼肌的变化也不明确,而运动中机体的生理变化机制更是目前研究的热点。目的:探讨不同强度运动对大鼠骨骼肌质量的影响以及bnip3介导的骨骼肌线粒体自噬在维持骨骼肌质量中的作用。方法:实验方案经北京体育大学动物实验伦理委员会批准。8周龄雄性SD大鼠24只,随机分为对照组、中等强度运动组(5°,15m/min,1 h,60%VO2max)和大强度运动组(5°,35m/min,20min,85%VO2max),每组8只,每周运动6次。4周运动后取大鼠比目鱼肌和腓肠肌,称量湿质量,免疫荧光检测肌纤维横截面积,Western Blot检测比目鱼肌和腓肠肌bnip3、p62和LC3蛋白的表达。结果与结论:(1)腓肠肌湿质量大强度运动组和中等强度运动组显著低于对照组(P<0.01);(2)比目鱼肌肌纤维横截面积大强度运动组显著小于对照组(P <0.01);腓肠肌肌纤维横截面积中等强度运动组和大强度运动组均显著大于对照组(P<0.01);(3)中等强度运动诱导线粒体自噬增加,表现为bnip3、LC3-Ⅱ/LC3-Ⅰ值相较对照组表达增加(P <0.05),而p62较对照组表达下降(P <0.05);大强度运动组LC3-Ⅱ/LC3-Ⅰ、p62的表达量相较4周中等强度运动分别为上升和下降(P<0.05),但其bnip3的表达量却下降(P<0.05);(4)结果表明,4周中等强度运动可促进骨骼肌通过bnip3途径的线粒体自噬清除损伤线粒体,维持骨骼肌功能。4周大强度运动自噬水平更高,但可能对骨骼肌产生不利影响。

关键词：线粒体自噬 bnip3 骨骼肌运动强度

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Roles of HIF1α- and HIF2α-regulated bnip3 in hypoxia-induced injury of neurons

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PATHOLOGY RESEARCH AND PRACTICE 2019年第4期215卷 822-827页

作者： Zhu, Lei Qi, Boxiang Hou, Daorong Xuzhou Med Univ Xuzhou Childrens Hosp Dept Intens Care Unit Xuzhou 227006 Jiangsu Peoples R China Nanjing Med Univ Key Lab Model Anim Res Anim Core Facil 101 Longmian Ave Nanjing 211166 Jiangsu Peoples R China

Background: To explore the roles of HIF1 alpha- and HIF2 alpha-regulated bnip3 in hypoxia-induced injury of neurons. Methods: The sera of neonates with hypoxic-ischemic encephalopathy (HIE) within 24 h after birth and full-term healthy newborns (n = 40) were collected. The bnip3 levels were detected by ELISA. *** cells were cultured in 1% O-2 at 37 degrees C. The apoptosis of cells treated with 1, 5 and 10 ng/ml bnip3 for 48 h was detected by flow cytometry. The proliferation of cells transfected with sibnip3 was detected by CCK-8 assay. The mRNA level of bnip3 in cells under hypoxic conditions was measured by RT-PCR. The protein level of bnip3 in cells cultured under hypoxic conditions after pretreatment with HIF1 alpha or HIF2 alpha inhibitor was measured by Western blot. Results: The serum bnip3 concentration of HIE neonates ((4.5 +/- 2.1) ng/ml) was significantly higher than that of healthy neonates ((1.2 +/- 0.5) ng/ml) (P < 0.001). Compared with untreated group, the number of apoptotic *** cells treated with bnip3 significantly increased (P < 0.05). Under hypoxic conditions (1%), the mRNA and protein levels of bnip3 increased significantly with prolonged time. After pretreatment with HIF1 alpha or HIF2 alpha inhibitor and hypoxic culture, bnip3 expression was significantly lower than that of cells hypoxically cultured only. Inhibiting the expression of HIF1 alpha or HIF2 alpha or transfecting with sibnip3 before hypoxic treatment significantly reduced the number of apoptotic cells. Under hypoxic conditions, HIF1 alpha or HIF2 alpha bound bnip3 promoter, which did not occur under normal culture conditions. HIF1 alpha or HIF2 alpha was significantly enriched near the hypoxia response element (HRE) site of bnip3 promoter. Conclusions: bnip3 was involved in the apoptosis of cells undergoing HIE. The HRE site of bnip3 promoter bound HIF to promote its transcription.

关键词： Neonate Hypoxic-ischemic encephalopathy bnip3 Apoptosis HIF

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miR-96-5p regulates wound healing by targeting bnip3/FAK pathway

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JOURNAL OF CELLULAR BIOCHEMISTRY 2019年第8期120卷 12904-12911页

作者： Wu, Peng Cao, Yongqian Zhao, Ran Wang, Yibing Shandong Univ Shandong Prov Hosp Dept Burns & Plast Surg 324 Jingwu Rd Jinan 250021 Shandong Peoples R China Linyi Peoples Hosp Dept Burns & Plast Surg Linyi Shandong Peoples R China

Cutaneous wound healing is a highly orchestrated basic biological process and one of the key processes in restoring skin integrity. The role of microRNAs (miRNAs) during this process has raised numerous attention and is poorly explored. The aim of this study is to investigate the potential function of BCL2 interacting protein (bnip3) and its target miRNA, miR-96-5p, in cutaneous wound healing. The results demonstrated that bnip3 was significantly increased and miR-96-5p was obviously decreased during wound healing. Overexpression of bnip3 significantly increased, while inhibition of bnip3 decreased cell proliferation and migration of human primary keratinocytes. miR-96-5p was predicted to be a target miRNA for bnip3 and luciferase reporter assay confirmed that miR-96-5p directly targeted the 3 '-untranslated region of bnip3. Moreover, miR-96-5p overexpression significantly decreased, while miR-96-5p inhibition dramatically increased bnip3 protein expression and focal adhesion kinase (FAK) pathway activation. Furthermore, miR-96-5p inhibited cell proliferation and migration of human primary keratinocytes. Overall, our findings suggest that miR-96-5p might be critical in the regulation of wound healing by mediating bnip3 and FAK pathway.

关键词： bnip3 FAK pathway human primary keratinocyte miR-96-5p wound healing

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MicroRNA-210/bnip3通过影响线粒体自噬-凋亡调控缺氧条件下心肌与内皮细胞损伤的机制研究

MicroRNA-210/BNIP3通过影响线粒体自噬-凋亡调控缺氧条件下心肌...

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作者：杨一博吉林大学

学位级别：博士

研究背景心血管疾病是全世界范围内导致死亡的首要原因,而缺血性心脏病是最为常见的心血管系统疾病。在缺血性心脏病中,各种原因造成的血管狭窄、血流减少或中断会导致心脏局部组织缺血缺氧,进而造成电生理、功能、代谢和结构等方面的... 详细信息

研究背景心血管疾病是全世界范围内导致死亡的首要原因,而缺血性心脏病是最为常见的心血管系统疾病。在缺血性心脏病中,各种原因造成的血管狭窄、血流减少或中断会导致心脏局部组织缺血缺氧,进而造成电生理、功能、代谢和结构等方面的紊乱。线粒体是细胞能量代谢,维持生命活动的中心,是缺氧损伤的主要靶点之一。当缺氧发生时,细胞的代谢方式从氧化磷酸化向无氧糖酵解转变,能量供应不足,作为电子受体的氧分子缺乏,导致电子传递链复合物产生大量的活性氧。这些活性氧会破坏蛋白质、脂膜分子和核酸,造成线粒体损伤和功能失调。受损线粒体的内容物释放入胞浆,通过激活下游的Caspase级联反应,触发线粒体相关的细胞凋亡,进而引起细胞损伤。缺氧情况下,稳定的线粒体功能与结构是细胞存活的关键因素。线粒体损伤除了诱导细胞凋亡外,有时还会引起线粒体自噬的发生。线粒体自噬是选择性自噬的一种形式,可以通过线粒体-溶酶体途径分解功能障碍、结构损伤的线粒体,维持细胞中线粒体的功能,促进线粒体重组,参与线粒体的质量控制,在多种生物学过程中发挥重要作用。在某些情况下,线粒体自噬能清除功能失调的线粒体,减少活性氧的持续产生,减轻细胞损伤,发挥一定的细胞保护作用;但持续或严重的损伤过程可能诱发过度的线粒体自噬,引起线粒体的过度破坏,反而加重损伤,加快细胞死亡。鉴于线粒体自噬在缺氧损伤中发挥的作用尚不清楚,其机制也未完全探明,因此,进一步明确其功能和机制并寻找可能的调控因子很有必要。在线粒体自噬及细胞凋亡的相关机制研究中,bnip3是重要的分子之一。bnip3在缺氧条件下被激活并定位于线粒体,通过调节多种生物学反应参与多种疾病的发生发展过程。bnip3在发现之初被认为是BH3-only亚家族的一员,通过BH3-only区域与跨膜结构域发挥促细胞凋亡的作用。但随着研究的深入,bnip3与线粒体自噬也密切相关,它运用自身的LC3相互作用区与LC3识别,促进线粒体自噬的发生,发挥不同的效应。因此,bnip3在缺血性心脏病中发挥的具体作用仍需进一步探索。实验室的前期工作发现,micro RNA-210(miR-210)能够抑制氧化应激损伤诱导的凋亡,发挥细胞保护作用,但其具体机制尚需进一步研究。bnip3是miR-210的下游靶分子,研究miR-210是否通过bnip3影响线粒体自噬和凋亡,有利于揭示miR-210保护缺氧细胞的机制,为寻找心血管疾病的治疗手段或靶点提供相应的理论基础。研究目的本实验以心肌细胞H9c2与冠状动脉内皮细胞HCAECs为研究对象,以bnip3为切入点,探讨线粒体自噬-凋亡途径在缺氧诱导心血管细胞损伤过程中的作用和机制,明确miR-210/bnip3在缺血性心脏病中发挥保护作用的机制,为缺血性心脏病的治疗提供理论依据。研究方法1.缺氧袋培养细胞(H9c2细胞与HCAECs),CCK-8检测细胞存活率,比色法观察ATP的水平,DCFH-DA探针标记细胞中ROS水平,电镜下观察线粒体微观形态,评估细胞与线粒体的损伤。Western blot检测凋亡相关蛋白(Caspase-3与Caspase-9裂解片段)的水平,评估细胞凋亡。同时应用western blot检测线粒体自噬相关蛋白(LC3、TOM20、P62)水平,并应用Mito Tracker与Lyso Tracker分别标记线粒体与溶酶体,共聚焦显微镜观察它们在胞质中的共定位情况,评价线粒体自噬的水平。2.应用线粒体自噬抑制剂环孢素A(CsA)预处理缺氧细胞,western blot检测蛋白(LC3、TOM20、P62),线粒体与溶酶体共定位染色评估线粒体自噬水平。检测应用CsA后的细胞存活率,ATP水平与凋亡相关蛋白(Caspase-3与Caspase-9裂解片段)水平,评估抑制线粒体自噬后细胞损伤与凋亡的变化。3.应用western blot与实时定量PCR检测bnip3在缺氧细胞中的蛋白与RNA水平。利用sibnip3质粒构建bnip3沉默模型。CCK-8检测细胞存活率,比色法检测ATP水平,透射电镜观察线粒体微观结构,JC-1染色描述线粒体膜电位,评估bnip3对线粒体形态与功能的影响。应用western blot检测凋亡相关蛋白(Caspase-3与Caspase-9裂解片段)的水平,TUNEL染色标记凋亡细胞共同评估凋亡的发生,探究bnip3对细胞凋亡的影响。同时检测线粒体自噬相关蛋白(LC3、TOM20、P62)水平,线粒体与溶酶体的共定位,并应用免疫荧光染色标记P62观察其点状聚集情况并将P62和TOM20共染,研究bnip3对线粒体自噬的调控。应用western blot观察CsA抑制线粒体自噬后bnip3的表达变化,研究线粒体自噬水平对bnip3表达的影响,探究bnip3在缺氧H9c2细胞与HCAECs线

关键词：缺血性心脏病缺氧线粒体自噬细胞凋亡 bnip3 微小RNA-210

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bnip3 mediates doxorubicin-induced cardiomyocyte pyroptosis via caspase-3/GSDME

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LIFE SCIENCES 2020年第0期242卷 117186-000页

作者： Zheng, Xinbin Zhong, Ting Ma, Yeshuo Wan, Xiaoya Qin, Anna Yao, Bifeng Zou, Huajiao Song, Yan Yin, Deling Cent S Univ Xiangya Sch Pharmaceut Sci Changsha 41008 Hunan Peoples R China East Tennessee State Univ Dept Internal Med Coll Med Johnson City TN 37614 USA Zhengzhou Univ Affiliated Hosp 1 Dept Vasc Surg Zhengzhou 450052 Henan Peoples R China

Aims: This study was aimed to investigate the role of GSDME-mediated pyroptosis in cardiac injury induced by Doxorubicin (DOX), and to evaluate the role of BH3-only protein Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 (bnip3) in regulation of DOX-induced pyroptosis. Main methods: HL-1 cardiomyocytes and C57BL/6J mice were treated by DOX to establish DOX-induced cardiotoxicity in vitro and in vivo models, respectively. Cell transfection was applied to regulate the expression of caspase-3, GSDME and bnip3. Western blot was used for measuring expression of protein level. LDH-cytotoxicity assay was used to detect the LDH release. The Flow cytometry analysis was used to detect the cell death. Echocardiography was used to determine the cardiac function. HE staining was used for observing pathological feature of heart tissues. Key findings: Our results showed that GSDME-mediated pyroptosis was involved in DOX-induced cardiotoxicity in vivo. We showed that HL-1 cardiomyocytes exposed to DOX exhibited morphological features of pyroptosis in vitro. We also showed that DOX induced activation of caspase-3 and eventually triggered GSDME-dependent pyroptosis, which was reduced by the silence or inhibitor of caspase-3. We further showed that knockdown of GSDME inhibited DOX-induced cardiomyocyte pyroptosis in vitro. Finally, DOX increased the expression of bnip3, whereas silencing of bnip3 blunted cardiomyocyte pyroptosis induced by DOX, which was regulated through caspase-3 activation and GSDME cleavage. Significance: Our findings revealed a novel pathway that cardiomyocyte pyroptosis is regulated through bnip3-caspase-3-GSDME pathway following DOX treatment, suggesting that bnip3-dependent pyroptosis may offer a novel therapeutic strategy to reduce cardiotoxicity induced by DOX.

关键词： Pyroptosis bnip3 GSDME Caspase-3 Doxorubicin

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