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Brevilin A, a novel bnip3 inhibitor suppresses osteoclastogenesis and prevents ovariectomy-induced bone loss via impairing mitophagy and mitochondrial metabolism

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Phytomedicine 2025年 143卷 156774页

作者： Zhou, Rui Wang, Yiyuan Liu, Siyi Su, Yuangang Liu, Zhijuan Yang, Baihui Li, Xiangde Zhao, Jinmin Xu, Jiake Liu, Qian Song, Fangming Collaborative Innovation Centre of Regenerative Medicine and Medical BioResource Development and Application Co-constructed by the Province and Ministry Guangxi Medical University Nanning 530021 China Guangxi Key Laboratory of Regenerative Medicine Orthopaedics Trauma and Hand Surgery the First Affiliated Hospital of Guangxi Medical University Nanning 530021 China Life Sciences Institute Guangxi Medical University Nanning 530021 China The First Clinical Medical College Guangxi Medical University Nanning 530021 China School of Biomedical Sciences the University of Western Australia Perth Australia Shenzhen Institute of Advanced Technology Chinese Academy of Sciences Shenzhen 518000 China Shenzhen University of Advanced Technology Shenzhen 518000 China

Background: The mitochondrial dysfunction and overactive osteoclasts is involved in the progress of osteoporosis. Brevilin A (BA), a sesquiterpene lactone, is a compound extracted and purified from Centipeda minima. It exhibits a range of pharmacological activities, such as anti-inflammatory and antioxidant effects. However, its specific impact on osteoporosis remains unclear. The present study is designed to explore BA as a novel osteoclast inhibitor for the treatment of osteoporosis as well as its molecular mechanisms of action via bnip3-mediated mitophagy. Methods: The cytotoxicity of BA in vitro was evaluated using the CCK8 assay, while tartrate-resistant acid phosphatase (TRAcP) staining and bone resorption assays were conducted to examine its effects on osteoclastogenesis and osteoclast function. To elucidate the molecular mechanisms by which BA targets bnip3 in osteoclasts, RNA-seq, molecular docking analysis, Surface plasmon resonance (SPR), qPCR, western blot, mitochondrial oxygen consumption rate (OCR), transmission electron microscopy (TEM), Single cell sequencing and immunofluorescence staining were employed. In addition, a specific bnip3 agonist IOX5, was used to revalidate the inhibitory effect of BA on bnip3. To investigate the effects and protective role of BA in modulating bnip3 on bone loss in osteoporotic mice induced by ovariectomy (OVX), we employed in vivo micro-CT scanning and histological immunostaining techniques. Results: Our study demonstrated that BA inhibited RANKL-induced osteoclastogenesis in a concentration-dependent manner without any cell cytotoxicity. Further, BA abrogated MAPK-related proteins and intracellular and mitochondrial ROS level, subsequently inhibiting NFATc1 activity. RNA-seq analysis revealed that the molecular mechanism by which BA inhibited osteoclasts is closely related to mitophagy and mitochondrial function. Here, we found that BA suppressed oxygen consumption rate and mitochondrial oxidative phosphorylation duri

关键词： bnip3 Brevilin A Mitochondrial function Mitophagy Osteoclast Osteoporosis

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bnip3 and Nix: Atypical regulators of cell fate

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BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 2022年第10期1869卷 119325页

作者： Field, Jared T. Gordon, Joseph W. Univ Manitoba Dept Human Anat & Cell Sci Rady Fac Hlth Sci Winnipeg MB Canada Univ Manitoba Coll Nursing Rady Fac Hlth Sci Winnipeg MB Canada Theme Childrens Hosp Res Inst Manitoba Diabet Res Envisioned & Accomplished Manitoba DRE Winnipeg MB Canada

Since their discovery nearly 25 years ago, the BCL-2 family members bnip3 and bnip3L (aka Nix) have been labelled 'atypical'. Originally, this was because bnip3 and Nix have divergent BH3 domains compared to other BCL-2 proteins. In addition, this atypical BH3 domain is dispensable for inducing cell death, which is also unusual for a 'death gene'. Instead, bnip3 and Nix utilize a transmembrane domain, which allows for dimerization and insertion into and through organelle membranes to elicit cell death. Much has been learned regarding the biological function of these two atypical death genes, including their role in metabolic stress, where bnip3 is responsive to hypoxia, while Nix responds variably to hypoxia and is also down-stream of PKC signaling and lipotoxic stress. Interestingly, both bnip3 and Nix respond to signals related to cell atrophy. In addition, our current view of regulated cell death has expanded to include forms of necrosis such as necroptosis, pyroptosis, ferroptosis, and permeability transition-mediated cell death where bnip3 and Nix have been shown to play context- and cell-type specific roles. Perhaps the most intriguing discoveries in recent years are the results demonstrating roles for bnip3 and Nix outside of the purview of death genes, such as regulation of proliferation, differentiation/maturation, mitochondrial dynamics, macro- and selective-autophagy. We provide a historical and unbiased overview of these 'death genes', including new information related to alternative splicing and post-translational modification. In addition, we propose to redefine these two atypical members of the BCL-2 family as versatile regulators of cell fate.

关键词： Nix bnip3 Regulated cell death Proliferation Autophagy Mitophagy

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bnip3 enhances pancreatic cancer cell migration and proliferation via modulating autophagy under hypoxia

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HELIYON 2022年第10期8卷 e11190页

作者： Li, Hongmei Zhang, Can Zhang, Qiong Jia, Jiezhi Wang, Xiaojiao Third Mil Med Univ Army Med Univ Dept Oncol Chongqing 400038 Peoples R China Third Mil Med Univ Army Med Univ Southwest Hosp Southwest Canc Ctr Chongqing 400038 Peoples R China Third Mil Med Univ Army Med Univ Southwest Hosp Dept Plast Surg Chongqing 400038 Peoples R China Third Mil Med Univ Army Med Univ Southwest Hosp Inst Burn Res Chongqing 400038 Peoples R China

Chemotherapy and immunotherapy for pancreatic ductal adenocarcinoma (PDAC) have limited success for the intricated surrounding cancer microenvironment. Hypoxic microenvironment in PDAC causes the activation of multiple different molecules and signaling pathways compared with normoxia. We studied the roles of bnip3 for the migration and proliferation of PDAC and Panc1 cells in vitro. In the present study, we found that bnip3 expression was elevated and enhanced the migration and proliferation of CFPAC-1 and Panc1 cells under hypoxia. The upregulation of bnip3 was important for the autophagic activation, while inhibition of autophagy with siRNA targeting Atg5 and Atg7 impaired the hypoxia-induced cell migration and proliferation. Additionally, blocking ERK1/2 mitogen-activated protein kinase (MAPK) signaling with PD98058 significantly down-regulated bnip3 expression, autophagic activation, as well as the migration and proliferation of CFPAC-1 and Panc1 cells under hypoxia. Collectively, our results here uncover a hitherto unknown hypoxia-bnip3-autophagy axis in modulating the migration and proliferation and provide a potential intriguing drug target for the therapy of PDAC.

关键词： bnip3 Autophagy ERK1 2 Pancreatic ductal adenocarcinoma Hypoxia

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bnip3 contributes to cisplatin-induced apoptosis in ovarian cancer cells

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FEBS OPEN BIO 2020年第8期10卷 1463-1473页

作者： Jia, Jinghui Yang, Xiaoxin Zhao, Qing Ying, Feiquan Cai, E. Sun, Si He, Xiaoqi Air Force Gen Hosp Dept Obstet & Gynecol PLA Beijing Peoples R China Huazhong Univ Sci & Technol Union Hosp Tongji Med Coll Dept Obstet & Gynecol Wuhan 430022 Peoples R China WuHan Univ Wuhan Hosp 3 Tongren Hosp Dept Obstet & Gynecol Wuhan Peoples R China Huazhong Univ Sci & Technol Cent Hosp Wuhan Tongji Med Coll Dept Obstet & Gynecol Wuhan Peoples R China

bnip3 is a proapoptotic protein that mediates apoptosis, necrosis and autophagy. However, the involvement of bnip3 in cisplatin-induced apoptosis in ovarian cancer is not clear. In this study, we examined the role of bnip3 in ovarian cancer during cisplatin treatment and its correlation with clinical outcomes. We first measured cisplatin cytotoxicity and bnip3 levels before and after cisplatin exposure for ovarian cancer cell lines A2780, SKOV3, OVCAR4, OV2008, ES2 and HO8910. bnip3 was observed to be differentially expressed in these cell lines, and cisplatin induced a significant increase in bnip3 levels in A2780 and OVCAR4. bnip3 knockdown with siRNA in A2780 and OVCAR4 significantly reduced cisplatin cytotoxicity in these two cell lines and alleviated cisplatin-induced apoptosis. We searched the online databases Gene Expression Omnibus and The Cancer Genome Atlas to analyze the correlation between bnip3 level and overall survival and progression-free survival in patients with ovarian cancer. Pooled analyses showed that higher bnip3 level was correlated with poorer overall survival (95% confidence intervals;hazard ratio = 1.18, 1.04-1.34;P = 0.013) and progression-free survival (95% confidence intervals;hazard ratio = 1.26, 1.10-1.43;P = 0.00049). However, the results of individual datasets and stratification analyses of histology, FIGO (Federation Internationale de Gynecolgie et d'Obstetrique) stage, chemotherapy regimen and P53 mutation status varied. These findings indicate that cisplatin-induced apoptosis is dependent on bnip3 level in ovarian cancer cell lines. Targeting bnip3 may therefore be a potential way of restoring cisplatin sensitivity.

关键词： apoptosis bnip3 cisplatin cisplatin sensitization ovarian cancer

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miR-31-5p对牙髓干细胞HIF-1α/bnip3信号通路及成骨相关因子表达的影响

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上海口腔医学 2022年第3期31卷 237-242页

作者：付洪海孙乐刚丁昌成马向瑞黄玉梅滨州医学院附属医院口腔颌面外科山东滨州256603 滨州医学院附属医院药学部山东滨州256603 滨州医学院附属医院口腔修复科山东滨州256603

目的 :探讨微小RNA-31-5p(miR-31-5p)对牙髓干细胞(DPSCs)低氧诱导因子1α(HIF-1α)/Bcl-2/腺病毒E1B 19-kDa相互作用蛋白3(bnip3)信号通路及成骨相关因子表达的影响。方法 :体外培养人DPSCs,分为对照组(不转染)、mimic NC组(转染negati... 详细信息

目的 :探讨微小RNA-31-5p(miR-31-5p)对牙髓干细胞(DPSCs)低氧诱导因子1α(HIF-1α)/Bcl-2/腺病毒E1B 19-kDa相互作用蛋白3(bnip3)信号通路及成骨相关因子表达的影响。方法 :体外培养人DPSCs,分为对照组(不转染)、mimic NC组(转染negative control-miR-31-5p)、miR-31-5p mimic组(转染hsa-miR-31-5p mimic)、si RNA NC组(转染nonsense siRNA)和miR-31-5p siRNA组(转染miR-31-5p siRNA)。实时荧光定量PCR(qRT-PCR)检测各组DPSCs细胞中miR-31-5p、HIF-1α、bnip3、碱性磷酸酶(ALP)、Runt相关转录子2(Runx2)mRNA的表达情况,MTT法检测各组DPSCs细胞增殖情况,ALP活性测定试剂盒检测各组DPSCs细胞ALP活性,蛋白印迹(WB)法检测各组DPSCs细胞中HIF-1α、bnip3、Runx2蛋白的表达情况。采用SPSS 24.0软件包对数据进行统计学分析。结果:与对照组、mimic NC组相比,miR-31-5p mimic组DPSCs细胞A值、ALP mRNA表达水平及活性、Runx2 mRNA及蛋白表达水平显著降低(P<0.05),ALP染色明显减弱,miR-31-5p mRNA、HIF-1α、bnip3 mRNA及HIF-1α、bnip3、Beclin1蛋白表达水平显著升高(P<0.05)。与对照组、siRNA NC组相比,miR-31-5p siRNA组DPSCs细胞A值、ALP mRNA表达水平及活性、Runx2 mRNA及蛋白水平显著升高(P<0.05),ALP染色明显增强,miR-31-5p mRNA、HIF-1α、bnip3 mRNA及HIF-1α、bnip3、Beclin1蛋白表达水平显著降低(P<0.05)。结论 :miR-31-5p可以激活HIF-1α/bnip3信号通路,抑制DPSCs细胞的成骨相关因子表达。

关键词：微小RNA-31-5p 牙髓干细胞 HIF-1α bnip3 成骨相关因子表达

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Hyperoside protects against cyclophosphamide induced ovarian damage and reduced fertility by suppressing HIF-1α/bnip3-mediated autophagy

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BIOMEDICINE & PHARMACOTHERAPY 2022年 156卷 113743-113743页

作者： Zhu, Fangfang Gao, Jie Zeng, Fuling Lai, Yuling Ruan, Xiaofeng Deng, Gaopi Guangzhou Univ Chinese Med Clin Med Coll 1 Guangzhou 510080 Peoples R China Guangdong Prov Hosp Chinese Med Dept Gynecol Guangzhou 510120 Peoples R China Guangzhou Univ Chinese Med Affiliated Hosp 1 Dept Gynecol Guangzhou 510405 Peoples R China Guangzhou Univ Chinese Med Affiliated Hosp 1 Dept Gynecol 16 Airport Rd Guangzhou 510405 Peoples R China

Ovarian damage and infertility are the main side effects of chemotherapy for women of childbearing age with cancer. The main objective of this study was to investigate the protective effects and mechanisms of hyperoside against cyclophosphamide (Cy)-induced ovarian damage and reduced fertility. This study consists of two parts: in vivo experiments using Cy intraperitoneal injections to simulate clinical chemotherapy sessions and in vitro experiments using 4-HC, a precursor of an activated form of Cy, to intervene in human granulosa-like cell line (KGN). We found that Cy disrupted the estrous cycle in mice, resulting in decreased serum Anti-Mullerian hormone (AMH) levels, loss of primordial follicles, primary follicle and secondary follicle, increased atretic follicles, and diminished ovarian reserve function. Cy prolonged the time between mating and pregnancy in mice and increased the number of absorbed embryos. Western Blot analysis demonstrate that Cy activated key proteins of HIF-1 alpha/bnip3-associated autophagy both in vivo and in vitro, while in vivo experiments we also found that 4 -HC increased KGN cell apoptosis, damaged mitochondrial membrane potential, and activated autophagic flow. Co-treatment with hyperoside diminished follicular depletion of the primordial follicles, decreased follicular atresia, prevented Cy-induced excessive hypoxia and autophagy activation, increased mitochondrial membrane potential, thereby increasing follicular reserve and rescuing fertility in Cy-treated mice. It suggests that HIF-1 alpha/ bnip3-mediated autophagy is an essential mechanism by which Cy impairs ovarian function and fertility in mice, by blocking this activation, hyperoside shows potential as an ovarian protectant that may be capable of pre-serving fertility in women undergoing chemotherapy.

关键词： Hyperoside Ovarian damage HIF-1 bnip3 Hypoxia Autophagy

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bnip3 in mitophagy: Novel insights and potential therapeutic target for diseases of secondary mitochondrial dysfunction

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CLINICA CHIMICA ACTA 2020年 506卷 72-83页

作者： Gao, Anbo Jiang, Jinyong Xie, Feng Chen, Linxi Univ South China Hunan Prov Cooperat Innovat Ctr Mol Target New Dr Inst Pharm & Pharmacol Learning Key Lab Pharmacoprote Hengyang 421001 Peoples R China Univ South China Hengyang Med Sch Hengyang 421001 Peoples R China Nanshan Dist Matern & Child Healthcare Hosp Shenz Shenzhen 518052 Peoples R China

The present review is a summary of the recent literature concerning bnip3 expression, function, and regulation, along with its implications in mitochondrial dysfunction, disorders of mitophagy homeostasis, and development of diseases of secondary mitochondrial dysfunction. As a member of the Bcl-2 family of cell death-regulating factors, bnip3 mediates mPTP opening, mitochondrial potential, oxidative stress, calcium overload, mitochondrial respiratory collapse, and ATP shortage of mitochondria from multiple cells. Recent studies have discovered that bnip3 regulates mitochondrial dysfunction, mitochondrial fragmentation, mitophagy, cell apoptosis, and the development of lipid disorder diseases via numerous cellular signaling pathways. In addition, bnip3 promotes the development of cardiac hypertrophy by mediating inflammatory response or the related signaling pathways of cardiomyocytes and is also responsible for raising abnormal mitophagy and apoptosis progression through multiple molecular signaling pathways, inducing the pathogenesis and progress of hepatocellular carcinoma (HCC). Different molecules regulate bnip3 expression at both the transcriptional and post-transcriptional level, leading to mitochondrial dysfunction and unbalance of mitophagy in hepatocytes, which promotes the development of non-alcoholic fatty liver disease (NAFLD). Thus, bnip3 plays an important role in mitochondrial dysfunction and mitophagy homeostasis and has emerged as a promising therapeutic target for diseases of secondary mitochondrial dysfunction.

关键词： bnip3 Mitophagy Cardiac hypertrophy Hepatocellular carcinoma Non-alcoholic fatty liver disease

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bnip3 decreases the LPS-induced inflammation and apoptosis of chondrocytes by promoting the development of autophagy

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JOURNAL OF ORTHOPAEDIC SURGERY AND RESEARCH 2020年第1期15卷 284-284页

作者： Ma, Zetao Wang, Deli Weng, Jian Zhang, Sheng Zhang, Yuanshi PKU Shenzhen Hosp Orthopaed Dept 1120 Rd Lianhua Shenzhen 518036 Guangdong Peoples R China

BackgroundInflammation and apoptosis of chondrocytes are the pathological bases of osteoarthritis. Autophagy could alleviate the symptoms of inflammation and apoptosis. Previous study has shown that BCL2/adenovirus E1B 19kDa protein-interacting protein 3 (bnip3) can induce the occurrence and development of autophagy. However, it is unknown whether autophagy induced by bnip3 can alleviate the inflammation and apoptosis of *** used the lentivirus to construct the overexpression bnip3 chondrocytes. Next, the lipopolysaccharide (LPS) was used to stimulate these cells to simulate the physiological environment of osteoarthritis. After that, the enzyme-linked immunosorbent assays (ELISA) were performed to determine the levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and interleukin-6 (IL-6) and the flow cytometry was performed to detect the apoptosis rates of chondrocytes. At last, the expression of autophagy-related proteins was detected with the western *** expression of bnip3 was suppressed after treatment with LPS. However, overexpression of bnip3 inhibited the secretion of proinflammatory factors (TNF-alpha, IL-1 beta, and IL-6) and decreased the apoptosis of chondrocytes. Furthermore, overexpression of bnip3 led to the upregulation of autophagy-related protein expression including little computer 3 (LC3), autophagy-related protein 7 (ATG7), and Beclin-1. Application of autophagy inhibitor recovered the expression of proinflammatory factors and apoptosis rates of ***3 decreased the LPS-induced inflammation and apoptosis of chondrocytes by activating the autophagy.

关键词： Autophagy bnip3 Chondrocytes Inflammation Apoptosis

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bnip3通过调控糖酵解促进脑胶质瘤细胞对替莫唑胺治疗抵抗的作用和研究机制

BNIP3通过调控糖酵解促进脑胶质瘤细胞对替莫唑胺治疗抵抗的作用...

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作者：梁琪吉林大学

学位级别：硕士

背景:胶质瘤是中枢神经系统中最常见的恶性原发性肿瘤,其中大部分为高级别胶质瘤,预后差致死率高,是我国目前儿童及成人癌症死亡的主要原因之一。尽管治疗方案通常以手术切除为主,并辅以放疗、化疗和电场治疗等综合疗法,但总体疗效尚不... 详细信息

背景: 胶质瘤是中枢神经系统中最常见的恶性原发性肿瘤,其中大部分为高级别胶质瘤,预后差致死率高,是我国目前儿童及成人癌症死亡的主要原因之一。尽管治疗方案通常以手术切除为主,并辅以放疗、化疗和电场治疗等综合疗法,但总体疗效尚不理想。替莫唑胺(TMZ)作为一种广泛应用且最为有效的化疗药物,在脑胶质瘤术后治疗中占据重要地位,但临床上部分患者对其药物敏感性差,甚至出现耐药性。因此,深入研究替莫唑胺的耐药机制,并探索克服替莫唑胺治疗抵抗的有效策略,对胶质瘤治疗至关重要。有研究发现替莫唑胺耐药的肿瘤出现了线粒体的各方面异常改变,线粒体的异常改变与癌细胞的能量代谢、药物抗性以及细胞死亡机制等重要过程有着紧密的联系。 bnip3是一种位于线粒体外膜的多功能蛋白,可诱导线粒体自噬,选择性地去除受损的线粒体,减少细胞内活性氧(ROS)的积累,并防止线粒体释放促死亡因子,如AIF和细胞色素c,进而使胶质瘤细胞对替莫唑胺的毒性作用产生抵抗,已有大量研究表明bnip3可调节致死性和保护性线粒体自噬。但bnip3对的替莫唑胺治疗抵抗的是否涉及其他机制仍需进一步研究。肿瘤细胞具有Warburg效应,ATP的生产主要依赖于糖酵解,阻断肿瘤细胞能量供给被视为一种潜在的杀死肿瘤细胞的策略。探讨bnip3对胶质瘤糖酵解及ATP产生的影响,以及替莫唑胺治疗作用的影响,不仅拓展bnip3机制研究的思路,同时也有助于为bnip3临床应用提供理论基础。目的: 用人胶质瘤细胞系来研究bnip3对胶质瘤ATP产生的影响以及在替莫唑胺诱导胶质瘤细胞死亡中的作用及机制方法: ***释放实验检测TMZ对胶质瘤细胞死亡率的而影响,再检测使用siRNA沉默bnip3基因后,以及额外加入外源性ATP及丙酮酸,它们分别对胶质瘤细胞的死亡率的影响 2.应用siRNA转染沉默bnip3,并设立对照组从而研究bnip3在胶质瘤细胞中的作用。 3.通过western blotting对4组原发性脑胶质瘤及其周边组织,以及6组不同病理等级的原发性脑胶质瘤组织和2组相应的瘤旁组织样本中的bnip3蛋白表达水平进行了检测。 4.在不同肿瘤细胞中加入TMZ作用时长不同,分别检测细胞ATP和丙酮酸的含量。分为TMZ给药组和未给药组,再细分为bnip3沉默组与未沉默组。分别检测ATP和丙酮酸的含量结果: 1.人胶质瘤肿瘤组织中bnip3的表达水平明显高于瘤周组织,表达水平与胶质瘤病理等级相关。 2.随着替莫唑胺(TMZ)作用的时间增加,bnip3的表达水平显著上升,而与糖酵解关键酶HKII和PKM2的表达水平则显著下降。此外,随着TMZ作用时间的延长,U87和U251细胞内的ATP和丙酮酸含量也显著减少。 ***转染沉默bnip3的表达后,TMZ诱导的肿瘤细胞死亡率显著增加,同时TMZ导致的U87、U251细胞ATP、丙酮酸水平降低进一步加剧。 ***转染沉默bnip3的表达后,可显著加强替莫唑对糖酵解关键酶HKII、PKM2表达水平的下调作用。 5.外源性补充ATP和丙酮酸明显抑制TMZ导致的细胞死亡。结论: ***3在胶质瘤组织中高表达,与胶质瘤病理等级相关 2.替莫唑胺诱导糖酵解障碍导致胶质瘤细胞死亡,并上调bnip3的表达水平 ***3参与调控糖酵解关键酶的表达,并通过促进糖酵解使胶质瘤细胞对TMZ的治疗抵抗。

关键词：脑胶质瘤替莫唑胺 bnip3 糖酵解 ATP

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缺氧微环境下HIF-1α/bnip3信号通路介导的细胞自噬在牙髓炎发病机制中的研究

缺氧微环境下HIF-1α/BNIP3信号通路介导的细胞自噬在牙髓炎发病...

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作者：王小禾新疆医科大学

学位级别：博士

目的:组织缺氧和细胞自噬与牙髓炎密切相关,但其具体作用及调控机制尚未完全阐明。本研究旨在临床组织标本及体内外实验的基础上,对牙髓炎发生发展过程中,缺氧引起的细胞损伤、自噬及作用进行评估,并初步探讨HIF-1α/bnip3信号通路参与... 详细信息

目的:组织缺氧和细胞自噬与牙髓炎密切相关,但其具体作用及调控机制尚未完全阐明。本研究旨在临床组织标本及体内外实验的基础上,对牙髓炎发生发展过程中,缺氧引起的细胞损伤、自噬及作用进行评估,并初步探讨HIF-1α/bnip3信号通路参与自噬调控的相关机制。方法:1.基于临床组织标本探讨牙髓炎中缺氧状态及自噬水平:(1)收集40例20-35岁患者第三磨牙牙髓组织标本,其中牙髓炎和正常对照组各20例。从两组中随机选取5例用于转录组学测序,并通过GO、KEGG富集及构建PPI网络对两组间的差异表达基因、相关信号通路及核心基因蛋白进行生物信息学分析;(2)运用H&E染色检测牙髓组织的炎症特征,并通过电子透射显微镜对其中超微结构的变化进行观察。同时采用免疫组化染色技术对转录组测序富集到的关键通路、核心基因蛋白的表达进行验证。2.利用动物模型在体内水平验证牙髓炎的缺氧状态和自噬水平:(1)构建大鼠牙髓炎模型,30只6～8周龄的SD雄性大鼠,随机分为空白对照组、髓腔开放1、3、5、7天组,每组6只。其中空白对照组大鼠不做任何处理,模型组大鼠对其左侧上颌第一磨牙进行开髓处理,根据相应造模时间,收集并制作标本;(2)采用H&E染色判断大鼠牙髓组织的炎症进展;(3)ELISA及MOD、SDA试剂盒法用于检测大鼠血清中炎症和氧化应激水平的变化;(4)通过免疫组化染色确定缺氧及自噬相关蛋白HIF-1α、bnip3、LC3B及P62在大鼠牙髓组织中的表达。3.在体外细胞模型中探讨缺氧对人牙髓细胞的损伤效应及自噬的作用:(1)从人牙髓组织中分离、提取原代细胞并进行鉴定;(2)构建人牙髓细胞缺氧模型,评价细胞状态变化:通过光学显微镜观察细胞形态,CCK-8法检测细胞增殖活性,流式细胞术、JC-1染色法检测细胞凋亡水平,采用DCFH-DA标记细胞,借助流式细胞术和荧光染色检测细胞内活性氧的水平,ELISA法被用于检测细胞内炎症因子IL-6、IL-1β、TNF-α的变化,同时,使用Western blot检测凋亡和炎症相关蛋白Bax、Bcl-2及TNF-α、NF-κB的水平;(3)判断缺氧引起的细胞自噬及自噬流的变化:利用MDC染色和电子透射显微镜观察细胞中的自噬结构,并通过免疫荧光染色和Western blot对细胞内自噬标志性蛋白LC3B、P62的表达水平进行检测。利用抑制剂3-MA、CQ干预细胞后,通过自噬结构和相关蛋白水平的表达变化,判断自噬流的活化;(4)研究自噬在缺氧微环境下的具体作用:分别在常氧和缺氧的条件下给予抑制剂3-MA、CQ干预细胞,通过一系列分子生物学检测手段,进一步对细胞增殖、凋亡水平,细胞内活性氧、线粒体膜电位和炎症因子的水平,以及相关蛋白的表达水平进行检测。4.在牙髓炎大鼠模型的基础上给予缺氧干预,并结合细胞缺氧模型,研究自噬的分子调控机制:(1)24只6～8周龄SD雄性大鼠,随机分为4组,每组6只。其中,空白对照组在常规动物房中饲养;髓腔开放组,左侧上颌第一磨牙行开髓术后,放入常规动物房中饲养;缺氧组大鼠放入西北地区特殊环境低氧舱(DYC-3013M)中饲养,不施加任何其他干预方式;缺氧+髓腔开放组,左侧上颌第一磨牙行开髓术后,再置于低氧舱内饲养。各组干预时间均为7天;(2)运用H&E染色观察大鼠牙髓组织的炎症特征,免疫组化染色检测缺氧和自噬相关蛋白HIF-1α、bnip3和LC3B在牙髓组织中的定位及表达水平;(3)在细胞缺氧模型的基础上,使用HIF-1α化学抑制剂YC-1进行干预,免疫荧光染色和Western blot用来检测缺氧和自噬相关蛋白的表达水平。结果:1.人炎症牙髓组织中缺氧和自噬水平上调:(1)转录组学测序共鉴定出1351个差异表达基因,这些基因分别与炎症、免疫、自噬及线粒体自噬等过程密切相关,其中一类与缺氧和自噬同时相关的蛋白bnip3表达在牙髓炎组中差异性上调;(2)与正常牙髓组织相比,炎症组中出现牙髓组织结构破坏、病变区域血管扩张充血及大量炎性细胞浸润的改变,同时典型的自噬体超微结构在炎症组织中显著表达;(3)缺氧相关蛋白HIF-1α、bnip3及自噬相关蛋白LC3B、P62、bnip3在牙髓炎组的表达水平较正常组显著增加(P<0.05)。2.牙髓炎大鼠体内存在缺氧状态和显著的自噬现象:(1)空白对照组,牙髓腔内可见正常组织结构,无炎性渗出及血管扩张现象。模型组中,随时间推移,炎症逐渐从牙髓冠部向根中部蔓延,并呈现加重趋势;(2)血清学检测结果显示,与空白对照组相比,模型组中IL-6、IL-1β以及TNF-α含量呈明显上升趋势(P<0.05),同时,SOD活性显著下降,MDA水平升高,二者比值减小(P<0.05);(3)模型组大鼠牙髓组织的成牙本质细胞、多细胞层及成纤维细胞中可见大量HIF-1α、LC3B及B

关键词：缺氧自噬牙髓炎 HIF-1α bnip3

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