Grx1 抑制高糖诱导的H9c2细胞中Caspase-8/3和JNK /c-Jun凋亡通路的激活

谷氧还蛋白1（glutaredoxin 1,Grx1）作为一种重要的抗氧化剂,通过响应多种重要蛋白质的活动和功能调节细胞的关键过程．了解Grx1功能,对寻求糖尿病和心肌病等,以凋亡失调和氧化还原稳态改变为发病机制的疾病的新颖治疗策略至关重要．为研究Grx1对高糖诱导的大鼠心肌细胞凋亡的抑制作用及相关信号机制,本研究以高糖诱导大鼠心肌细胞H9c2建立高糖损伤模型,采用免疫印迹实验检测caspase-3、8、9蛋白活性片段的表达和凋亡信号蛋白JNK/c-Jun的磷酸化水平．结果显示,与正常对照组相比,高糖组caspase家族中,剪切的caspase-3、caspase-8、caspase-9相对含量均显著增多,JNK和c-Jun蛋白的磷酸化水平均显著上调. 但给予外源性Grx1保护后,剪切的caspase-3和caspase-8相对含量均显著降低,JNK和c-Jun蛋白的磷酸化水平均显著下调．上述结果表明,高糖通过介导caspase-8/3和caspase-9/3凋亡通路,并激活凋亡相关信号通路JNK/c-Jun诱导H9c2心肌细胞凋亡．给予外源性Grx1保护后,可通过抑制caspase-8/3凋亡通路和JNK /c-Jun信号通路的激活,拮抗高糖诱导的心肌细胞凋亡．     

自噬对高糖诱导的人冠状动脉内皮细胞凋亡的保护作用

目的:研究自噬对高糖诱导的人冠状动脉内皮细胞凋亡的影响。方法:将人冠状动脉内皮细胞,分别用常规培养基(正常对照组)、含30 mmol/L D-葡萄糖的高糖培养基(高糖组)、高糖培养基合并雷帕霉素(Rapamycin,RAPA;100 nmol/L)干预(RAPA组)和高糖培养基合并3-甲基腺嘌呤(3-Methyladenine,3-MA,5 mmol/L)干预(3-MA组)培养。利用CCK-8法检测细胞生长活力,使用流式细胞术检测细胞凋亡水平,western blot检测细胞自噬标记蛋白(Beclin1)的表达水平。结果:(1)高糖溶液刺激内皮细胞24 h后,细胞生长活力为正常组的55.0%(P0.01),自噬标记蛋白Beclin1的表达水平明显增加,凋亡水平为正常组的2.0倍;(2)与高糖组相比,RAPA组细胞生长活力明显增加,Beclin1的表达明显升高(P0.01),凋亡水平为高糖组的70.1%;(3)与高糖组相比,3-MA组细胞生长活力明显减少,Beclin1的表达明显降低(P0.01),凋亡水平为高糖组的1.42倍。结论:细胞自噬可能对高糖诱导的人冠状动脉内皮细胞具有凋亡保护作用。     

神经胶质瘤U251细胞氧化损伤中自噬和凋亡过程的时间顺序

目的：探讨过氧化氢（H2O2）诱导神经胶质瘤U251细胞损伤中自噬和凋亡发生的时间顺序。方法：实验分为4组：正常对照组、1mmol／L H2O2作用（6h、12h、24h）组。应用MTF法检测H202对神经胶质瘤U251细胞生存率的影响;MDC染色检测自噬空泡的变化;流式细胞仪检测细胞凋亡率变化。Western blot检测Beclin1和胞浆cyt c蛋白的表达。结果：与对照组相比,1mmol／L H2O2作用下,U251细胞存活率明显降低,并呈时间依赖性。与对照组相比,1mmol／L H2O2作用后,6h时U251细胞自噬空泡明显增加,自噬相关蛋白Beclin1表达明显增加,12h、24h细胞自噬水平逐渐增强;而6h时未见细胞凋亡率明显变化及cyt c由线粒体向胞浆的释放,12h、24h时细胞凋亡率明显增加,胞浆中cyt c蛋白表达明显增强（P〈0．05）。结论：氧化损伤能够诱导神经胶质瘤U251细胞发生自噬和凋亡,并且自噬发生于凋亡之前。     

高糖背景下白蛋白抑制肾小管上皮细胞的自噬表达

目的:探讨高糖背景下白蛋白造成肾小管间质损伤的作用及其机制。方法:体外培养大鼠近端肾小管上皮细胞系NRK-52E细胞,观察高糖培养环境下细胞自噬表达的改变;同时观察低浓度牛血清白蛋白(BSA)单独刺激,对肾小管上皮细胞自噬蛋白表达的影响以及细胞凋亡蛋白的表达改变;接着在高糖培养环境下加入低浓度的白蛋白刺激,观察肾小管上皮细胞的损伤效应及自噬表达情况。结果:高糖培养条件下肾小管上皮细胞自噬蛋白Beclin-1表达增加(P0.05),低浓度白蛋白也诱导肾小管上皮细胞自噬蛋白Beclin-1、Atg12表达增加(P0.05),以及细胞凋亡蛋白cleaved caspase3的轻度增加,乳酸脱氢酶活性增加(P0.05);但高糖培养下,少量白蛋白却抑制了肾小管上皮细胞自噬蛋白Beclin-1、Atg12的表达,并且显著增加了肾小管上皮细胞的凋亡蛋白cleaved caspase3的表达(P0.05)。结论:自噬是细胞自身的一种保护机制。在高糖背景下,白蛋白通过影响自噬的自身调节机制,促进了肾小管间质的损害作用。     

mTOR信号通路诱导的自噬在心肌细胞缺氧损伤中的作用研究

目的:探讨自噬在心肌细胞缺氧损伤中的作用及分子机制。方法:体外分离培养乳鼠心肌细胞,体外建立缺氧/去血清(H/SD)模型以模拟在体的缺血环境。分别给予自噬抑制剂3-甲基腺嘌呤(3MA,5 mM)和mTOR抑制剂雷帕霉素(1.0μg/L)调节心肌细胞自噬水平。分别采用TUNEL染色检测心肌细胞凋亡,Western blot方法检测心肌细胞蛋白表达水平。结果:H/SD损伤可以显著诱导心肌细胞自噬水平(P0.05),并且细胞自噬水平可以被3-MA及雷帕霉素调节。同时,H/SD可以显著增加心肌细胞凋亡(P0.05),而给予3-MA抑制自噬水平可以减少细胞凋亡(P0.05)。相反,雷帕霉素增加自噬同样可以加重缺氧导致的心肌细胞凋亡(P0.05)。H/SD损伤过程中,心肌细胞mTOR信号通路被激活,而自噬抑制剂3-MA可以显著提高缺氧条件下心肌细胞中p-mTOR(Ser2448)的表达水平(P0.05),并增加mTOR下游分子p-p70S6k(P0.05)和p-S6(P0.05)的表达。结论:mTOR信号通路诱导的细胞自噬可能参与了缺氧损伤诱导的心肌细胞凋亡。     

Beclin 1对凋亡和自噬的调控作用

Beclin 1是自噬关键调控蛋白之一,参与自噬体膜形成.近期,大量研究结果指出, Beclin 1是caspase家族蛋白酶的全新底物,可被caspase剪切.剪切后的Beclin 1失去自噬调节功能,转而加剧凋亡进程.因而,Beclin 1对细胞凋亡和自噬起着重要的调控作用. 本文主要对细胞凋亡和自噬的相关性,以及Beclin 1在两通路中的调控作用进行了回顾与总结.在此基础上,进一步讨论了Beclin 1与人类疾病如肿瘤、神经系统退行性疾病的关联.最后,简要介绍了实验室常用于Beclin 1研究的工具.     

Beclin 1在凋亡和自噬中的调节作用

Beclin 1是自噬关键调控蛋白之一,参与自噬体膜形成.近期,大量研究结果指出,Beclin 1是caspase家族蛋白酶的全新底物,可被caspase剪切.剪切后的Beclin 1失去自噬调节功能,转而加剧凋亡进程.因而,Beclin 1对细胞凋亡和自噬起着重要的调控作用.本文主要对细胞凋亡和自噬的相关性,以及Beclin 1在两通路中的调控作用进行了回顾与总结.在此基础上,进一步讨论了Beclin 1与人类疾病如肿瘤、神经系统退行性疾病的关联.最后,简要介绍了实验室常用于Beclin 1研究的工具.     

自噬在TXNIP过表达诱导的INS-1胰岛细胞凋亡中的作用

硫氧还蛋白相互作用蛋白(thioredoxin interacting protein,TXNIP)是内源性硫氧还蛋白结合抑制蛋白,在糖尿病患者血清和组织中均高表达。本研究观察TXNIP过表达对正常糖脂浓度下培养的INS-1细胞自噬水平的影响,并分析自噬在TXNIP诱导的细胞凋亡中的作用。常规培养的INS-1胰岛细胞分为正常培养组、空病毒(Ad-eGFP)组和TXNIP过表达(Ad-TXNIP-eGFP)组,后两组转染相应腺病毒,48 h后测定TXNIP mRNA和蛋白的表达情况;用Western blot检测各组自噬相关的Beclin-1、LC3和P62的蛋白表达情况,以cleaved caspase 3/caspase 3比值和流式细胞术检测各组细胞凋亡情况;用IF/ICC法检测各组细胞内自噬体数量的变化。结果显示,与Ad-eGFP组相比,Ad-TXNIP-eGFP组TXNIP mRNA和蛋白表达量均明显升高;与Ad-eGFP组相比,Ad-TXNIP-eGFP组LC3-II/LC3-I比值和Beclin-1蛋白表达水平升高,P62蛋白表达降低,自噬体荧光强度增强,细胞凋亡率升高,cleaved caspase 3/caspase 3比值上升。使用自噬抑制剂3-MA干预后,与TXNIP过表达组相比,TXNIP过表达+3-MA组自噬明显受到抑制,同时凋亡明显减轻。以上结果提示,在正常糖脂浓度培养下的INS-1细胞过表达TXNIP可以通过诱导自噬促进细胞凋亡。     

肌肽通过调节JNK和NF-kappaB通路抑制高糖诱导的心肌细胞凋亡

高血糖症是糖尿病并发心血管疾病的重要危险因素. 高血糖诱导产生的活性氧（ROS）能够引起糖尿病心肌病.我们的前期工作已经证实,肌肽对高糖环境下细胞凋亡具有保护作用,但其机制尚未明确.为研究肌肽对高糖诱导的大鼠心肌细胞凋亡的抑制作用及相关信号机制,以高糖诱导心肌细胞H9c2为模型,采用Brdu-ELISA法检测细胞增殖过程中DNA合成情况,流式细胞术检测细胞凋亡率,免疫印迹实验检测JNK/c-Jun、NF-κB的磷酸化水平,RT-PCR检测TNF-α的mRNA表达. 实验结果显示,肌肽能够提高高糖损伤的H9c2细胞增殖能力,降低心肌细胞凋亡,抑制高糖激活的JNK、c-Jun和NF-κB磷酸化水平及TNFα的mRNA表达. 上述结果表明,肌肽拮抗高糖诱导的心肌细胞凋亡机制与抑制p-JNK /p-c-Jun、p-NF-κB水平和TNF-α表达有关.     

线粒体乙醛脱氢酶2(ALDH2)通过AMPK/FOXO3a通路改善高糖导致的心肌细胞凋亡

目的:观察乙醛脱氢酶2(ALDH2)对高糖诱导的H9C2心肌细胞存活及凋亡的影响,并探讨腺苷酸活化蛋白激酶(AMPK)/FOXO3a信号通路在高糖导致的心肌细胞凋亡中的调控作用。方法:以30 mmol/L葡萄糖诱导培养H9C2心肌细胞48 h,经ALDH2激动剂Alda-1及AMPK抑制剂Compound C干预后,用MTT法检测细胞的存活情况,TUNEL试剂盒检测细胞凋亡情况,Western blot检测ALDH2、磷酸化AMPK和FOXO3a蛋白的表达水平。结果:与对照组相比,高浓度葡萄糖培养H9C2心肌细胞后,细胞的存活率显著降低、凋亡指数明显升高,磷酸化AMPK的表达水平明显上调,ALDH2和磷酸化FOXO3a的蛋白表达显著降低(P0.05)。ALDH2的激动剂Alda-1处理可显著提高高糖诱导的H9C2心肌细胞的存活率、降低其凋亡率,减少磷酸化AMPK的蛋白表达,增加ALDH2的表达和FOXO3a蛋白的磷酸化;而进一步采用AMPK的抑制剂Compound C处理,可显著抑制Alda-1对高糖诱导的H9C2心肌细胞的这些影响。结论:ALDH2的激动剂Alda-1对高糖诱导的心肌细胞凋亡具有保护作用,可能与其激活AMPK,进而抑制心肌细胞FOXO3a的活性有关。     

L-Ascorbate Protects Against Methamphetamine-Induced Neurotoxicity of Cortical Cells via Inhibiting Oxidative Stress,Autophagy, and Apoptosis

Methamphetamine (METH)-induced cell death contributes to the pathogenesis of neurotoxicity; however, the relative roles of oxidative stress, apoptosis, and autophagy remain unclear. L-Ascorbate, also called vitamin (Vit.) C, confers partial protection against METH neurotoxicity via induction of heme oxygenase-1. We further investigated the role of Vit. C in METH-induced oxidative stress, apoptosis, and autophagy in cortical cells. Exposure to lower concentrations (0.1, 0.5, 1 mM) of METH had insignificant effects on ROS production, whereas cells exposed to 5 mM METH exhibited ROS production in a time-dependent manner. We confirmed METH-induced apoptosis (by nuclear morphology revealed by Hoechst 33258 staining and Western blot showing the protein levels of pro-caspase 3 and cleaved caspase 3) and autophagy (by Western blot showing the protein levels of Belin-1 and conversion of microtubule-associated light chain (LC)3-I to LC3-II and autophagosome staining by monodansylcadaverine). The apoptosis as revealed by cleaved caspase-3 expression marked an increase at 18 h after METH exposure while both autophagic markers, Beclin 1 and LC3-II, marked an increase in cells exposed to METH for 6 and 24 h, respectively. Treating cells with Vit. C 30 min before METH exposure time-dependently attenuated the production of ROS. Vitamin C also attenuated METH-induced Beclin 1 and LC3-II expression and METH toxicity. Treatment of cells with Vit. C before METH exposure attenuated the expression of cleaved caspase-3 and reduced the number of METH-induced apoptotic cells. We suggest that the protective effect of Vit. C against METH toxicity might be through attenuation of ROS production, autophagy, and apoptosis.     

Cardiac-specific overexpression of catalase attenuates lipopolysaccharide-induced myocardial contractile dysfunction: Role of autophagy

Lipopolysaccharide (LPS) from gram-negative bacteria is a major initiator of sepsis, leading to cardiovascular collapse. Accumulating evidence has indicated a role of reactive oxygen species (ROS) in cardiovascular complications in sepsis. This study was designed to examine the effect of cardiac-specific overexpression of catalase in LPS-induced cardiac contractile dysfunction and the underlying mechanism(s) with a focus on autophagy. Catalase transgenic and wild-type FVB mice were challenged with LPS (6mg/kg) and cardiac function was evaluated. Levels of oxidative stress, autophagy, apoptosis, and protein damage were examined using fluorescence microscopy, Western blot, TUNEL assay, caspase-3 activity, and carbonyl formation. A Kaplan-Meier curve was constructed for survival after LPS treatment. Our results revealed a lower mortality in catalase mice compared with FVB mice after LPS challenge. LPS injection led to depressed cardiac contractile capacity as evidenced by echocardiography and cardiomyocyte contractile function, the effect of which was ablated by catalase overexpression. LPS treatment induced elevated TNF-α level, autophagy, apoptosis (TUNEL, caspase-3 activation, cleaved caspase-3), production of ROS and O(2)(-), and protein carbonyl formation, the effects of which were significantly attenuated by catalase overexpression. Electron microscopy revealed focal myocardial damage characterized by mitochondrial injury after LPS treatment, which was less severe in catalase mice. Interestingly, LPS-induced cardiomyocyte contractile dysfunction was prevented by the antioxidant N-acetylcysteine and the autophagy inhibitor 3-methyladenine. Taken together, our data revealed that catalase protects against LPS-induced cardiac dysfunction and mortality, which may be associated with inhibition of oxidative stress and autophagy.     

去乙酰化酶抑制剂TSA介导Ku70乙酰化对结肠癌HT29细胞凋亡和自噬的影响

目的:探讨不同浓度组蛋白去乙酰化酶抑制剂TSA对结肠癌HT29细胞的增殖、凋亡和自噬影响及其机制研究。方法:取对数生长期人结肠癌HT29细胞,采用MTT法检测不同浓度TSA处理对其细胞活力影响,并根据IC50值确定适宜给药浓度;采用流式细胞术检测不同浓度TSA处理后结肠癌HT29细胞的凋亡情况;Western blot验证空白对照组与TSA给药处理组中凋亡标志蛋白Ku70、acetrl-Ku70、Caspase3、Bax、Bcl-2和自噬标志蛋白LC3和Beclin1的表达。结果:MTT法实验结果表明TSA对结肠癌HT29细胞具有时间和浓度依赖性抑制作用,根据IC50=1.12μM,本研究中TSA的给药浓度为0.5μM和1μM;流式细胞凋亡检测结果表明TSA能够显著促进结肠癌HT29细胞凋亡,且其促凋亡作用存在浓度依赖性;此外,Western blot检测结果证实,与空白对照组相比,TSA给药处理可显著上调上述细胞中acetrl-Ku70以及促凋亡蛋白Caspase3、Bax和自噬标志蛋白LC3和Beclin1的表达,下调抗凋亡蛋白Bcl-2的表达(P<0.05)。结论:组蛋白去乙酰化酶抑制剂(TSA)的体外抗结肠癌细胞的增殖、促进细胞凋亡和自噬作用与其上调Ku70蛋白乙酰化密切相关,有望成为临床潜在抗癌靶点。     

The role of Beclin 1 in IR-induced crosstalk between autophagy and G2/M cell cycle arrest

ObjectivesBeclin 1 is a well-established core mammalian autophagy protein. Autophagy has been demonstrated to play roles in cellular responses to DNA damage, such as cell cycle regulation and apoptosis. In the present study, we investigated the exact mechanism by which Beclin 1 acts as a bridge between autophagy and cell cycle, when cells are exposed to ionizing radiation (IR).Materials and methodsWestern blotting and coimmunoprecipitation were performed to investigate protein expression levels and interactions. Immunofluorescence was used to monitor the localization and distribution of the indicated proteins. The levels of apoptosis and cell cycle changes were evaluated by flow cytometry. Double thymidine deoxyribonucleoside (TdR) blocking was conducted to differentiate G2 from mitotic delay. In vitro kinase assays using ATM kinase were performed to elucidate the specific phosphorylation site in Beclin 1.ResultsIn this study, we show that Beclin 1 knockdown reduces IR-induced autophagy. IR enhanced Beclin 1/PIK3CIII complex activity as demonstrated by the results of coimmunoprecipitation and immunofluorescence assays. An investigation to assess the possible relationship between autophagy and G2/M arrest showed that, similar to the autophagy inhibitor 3MA, Beclin 1 knockdown delayed IR-induced G2/M arrest. Furthermore, the interactions between Beclin 1 and several G2/M checkpoint-related proteins, namely, PLK1 and CDC25C, were observed to increase. In addition, we observed that both 3MA and Beclin 1 inhibition decreased IR-induced apoptosis. Regarding the potential mechanism associated with this phenomenon, we showed that IR induced the interaction between Beclin 1 and Tip60 as well as their redistribution. Furthermore, we demonstrated that Beclin 1 T57 may be a targeted phosphorylation site for ATM.ConclusionsIn the present study, we demonstrate the crucial and intricate roles of Beclin 1 in IR-induced autophagy, G2/M cell cycle arrest, and apoptosis. Additionally, Tip60 and ATM were identified as important molecular regulators of Beclin 1. Our findings show the precise mechanism of crosstalk between IR-induced autophagy and G2/M cell cycle arrest.     

Epigallocatechin gallate reduces hypoxia-induced apoptosis in human hepatoma cells

Cell detachment from extracellular matrix is closely related to induction of apoptosis. Epigallocatechin gallate (EGCG) has been shown to have antioxidant effect and to protect hypoxia-induced damage. We investigated whether EGCG reduced hypoxia-induced apoptosis and cell detachment in HepG2 cells. EGCG prevented cell death by hypoxia (0.5% O2) in a dose-dependent manner (hypoxic cell viability, 54.67%). RT-PCR and caspase3 activity assay showed that the hypoxia-induced cell death was caused by apoptosis increasing mRNA level of BAX, CASP3, and caspase3 activity. EGCG reduced increase of these mRNA and caspase3 activity. Western blot analysis and immunocytochemistry showed that EGCG increased cell adhesion proteins including E-cadherin (CDH1), tumor-associated calcium signal transducer 1 (TACSTD1), and protein tyrosine kinase 2 (PTK2) decreased by hypoxia. Hypoxia-induced apoptosis in HepG2 cells, and EGCG contributed to the HepG2 cell survival by attenuating the apoptosis.     

Allopurinol reduces oxidative stress and activates Nrf2/p62 to attenuate diabetic cardiomyopathy in rats

Allopurinol (ALP) attenuates oxidative stress and diabetic cardiomyopathy (DCM), but the mechanism is unclear. Activation of nuclear factor erythroid 2‐related factor 2 (Nrf2) following the disassociation with its repressor Keap1 under oxidative stress can maintain inner redox homeostasis and attenuate DCM with concomitant attenuation of autophagy. We postulated that ALP treatment may activate Nrf2 to mitigate autophagy over‐activation and consequently attenuate DCM. Streptozotocin‐induced type 1 diabetic rats were untreated or treated with ALP (100 mg/kg/d) for 4 weeks and terminated after heart function measurements by echocardiography and pressure‐volume conductance system. Cardiomyocyte H9C2 cells infected with Nrf2 siRNA or not were incubated with high glucose (HG, 25 mmol/L) concomitantly with ALP treatment. Cell viability, lactate dehydrogenase, 15‐F2t‐Isoprostane and superoxide dismutase (SOD) were measured with colorimetric enzyme‐linked immunosorbent assays. ROS, apoptosis, was assessed by dihydroethidium staining and TUNEL, respectively. The Western blot and qRT‐PCR were used to assess protein and mRNA variations. Diabetic rats showed significant reductions in heart rate (HR), left ventricular eject fraction (LVEF), stroke work (SW) and cardiac output (CO), left ventricular end‐systolic volume (LVVs) as compared to non‐diabetic control and ALP improved or normalized HR, LVEF, SW, CO and LVVs in diabetic rats (all P < .05). Hearts of diabetic rats displayed excessive oxidative stress manifested as increased levels of 15‐F2t‐Isoprostane and superoxide anion production, increased apoptotic cell death and cardiomyocytes autophagy that were concomitant with reduced expressions of Nrf2, heme oxygenase‐1 (HO‐1) and Keap1. ALP reverted all the above‐mentioned diabetes‐induced biochemical changes except that it did not affect the levels of Keap1. In vitro, ALP increased Nrf2 and reduced the hyperglycaemia‐induced increases of H9C2 cardiomyocyte hypertrophy, oxidative stress, apoptosis and autophagy, and enhanced cellular viability. Nrf2 gene silence cancelled these protective effects of ALP in H9C2 cells. Activation of Nrf2 subsequent to the suppression of Keap1 and the mitigation of autophagy over‐activation may represent major mechanisms whereby ALP attenuates DCM.     

Lactate accelerates calcification in VSMCs through suppression of BNIP3-mediated mitophagy

Arterial media calcification is one of the major complications of diabetes mellitus, which is related to oxidative stress and apoptosis. Mitophagy is a special regulation of mitochondrial homeostasis and takes control of intracellular ROS generation and apoptotic pathways. High circulating levels of lactate usually accompanies diabetes. The potential link between lactate, mitophagy and vascular calcification is investigated in this study. Lactate treatment accelerated VSMC calcification, evaluated by measuring the calcium content, ALP activity, RUNX2, BMP-2 protein levels, and Alizarin red S staining. Lactate exposure caused excessive intracellular ROS generation and VSMC apoptosis. Lactate also impaired mitochondrial function, determined by mPTP opening rate, mitochondrial membrane potential and mitochondrial biogenesis markers. Western blot analysis of LC3-II and p62 and mRFP-GFP-LC3 adenovirus detection for autophagy flux revealed that lactate blocked autophagy flux. LC3-II co-staining with LAMP-1 and autophagosome quantification revealed lactate inhibited autophagy. Furthermore, lactate inhibited mitophagy, which was confirmed by TOMM20 and BNIP3 protein levels, LC3-II colocalization with BNIP3 and TEM assays. In addition, BNIP3-mediated mitophagy played a protective role against VSMC calcification in the presence of lactate. This study suggests that lactate accelerates osteoblastic phenotype transition of VSMC and calcium deposition partly through the BNIP3-mediated mitophagy deficiency induced oxidative stress and apoptosis.