| 刘 鹏,曹 露,徐一君,陈志强,温 文.中介素1-53对高磷诱导人主动脉血管平滑肌细胞ERS-线粒体凋亡通路及钙化的影响[J].,2023,(12):2240-2246 |
| 中介素1-53对高磷诱导人主动脉血管平滑肌细胞ERS-线粒体凋亡通路及钙化的影响 |
| Effects of Intermedin 1-53 on ERS Mitochondrial Apoptosis Pathway and Calcification in Human Aortic Vascular Smooth Muscle Cells Induced by High Phosphorus |
| 投稿时间：2023-01-28 修订日期：2023-02-26 |
| DOI：10.13241/j.cnki.pmb.2023.12.007 |
| 中文关键词: 中介素1-53 人主动脉血管平滑肌细胞 内质网应激-线粒体凋亡通路 钙化 |
| 英文关键词: Intermedin 1-53 Human aortic vascular smooth muscle cells Endoplasmic reticulum stress-mitochondrial apoptosis pathway Calcification |
| 基金项目:山西省重点研发项目（201603D321057）；山西省心血管病医院基金资助项目（XYS20170302） |
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| 中文摘要: |
| 摘要 目的：探讨中介素1-53（IMD1-53）对高磷诱导人主动脉血管平滑肌细胞（HA-VSMC）内质网应激（ERS）-线粒体凋亡通路及钙化的影响。方法：体外培养HA-VSMC，MTT法检测IMD1-53对HA-VSMC的毒性作用。HA-VSMC中添加10 mmol/L β-甘油磷酸盐进行高磷诱导，使用0.1 nmol/L或0.5 nmol/L IMD1-53干预，并在0.5 nmol/L IMD1-53干预基础上添加ERS诱导剂衣霉素（TM），流式细胞仪检测HA-VSMC凋亡率，茜素红S染色观察HA-VSMC中钙盐沉积，酞络合酮比色法测定HA-VSMC中钙含量，分光光度法检测HA-VSMC中碱性磷酸酶（ALP）活性，蛋白质印迹法（Western blot）检测HA-VSMC中钙化以及ERS--线粒体凋亡通路相关蛋白表达。结果：IMD1-53浓度≤2.5 nmol/L时，对HA-VSMC细胞活力无明显影响（P＞0.05），故选择对细胞无明显毒副作用的低、高2个剂量（0.1、0.5 nmol/L）进行后续实验。高磷诱导后，HA-VSMC凋亡率、钙含量、ALP活性以及Runt相关转录因子-2（Runx2）、骨桥蛋白（OPN）、Bcl-2相关X蛋白（Bax）、裂解的半胱氨酰天冬氨酸蛋白酶-3（cleaved caspase-3）、葡萄糖调节蛋白78（GRP78）、C/EBP同源蛋白（CHOP）、感受器活化转录因子6（ATF6）、p-需肌醇酶1（IRE1）/IRE1蛋白表达水平升高（P＜0.05），橘红色结节明显增多，骨保护素（OPG）、B细胞淋巴瘤/白血病-2（Bcl-2）蛋白表达水平降低（P＜0.05）。0.1 nmol/L和0.5 nmol/L IMD1-53干预处理均可降低HA-VSMC凋亡率、钙含量、ALP活性以及Runx2、OPN、Bax、cleaved caspase-3、GRP78、CHOP、ATF6、p-IRE1/IRE1蛋白表达水平（P＜0.05），明显减少橘红色结节，升高OPG、Bcl-2蛋白表达水平（P＜0.05）。ERS诱导剂TM可升高HA-VSMC中GRP78、CHOP、ATF6、p-IRE1/IRE1蛋白表达水平，并减弱IMD1-53抑制高磷诱导的HA-VSMC ERS、凋亡和钙化的作用。结论：IMD1-53可减轻高磷诱导的HA-VSMC钙化，其作用机制可能与抑制ERS-线粒体凋亡通路激活，减少细胞凋亡有关。 |
| 英文摘要: |
| ABSTRACT Objective: To investigate the effects of intermedin 1-53 (IMD1-53) on endoplasmic reticulum stress (ERS)-mitochondrial apoptosis pathway and calcification in human aortic vascular smooth muscle cells (HA-VSMC) induced by high phosphorus. Methods: HA-VSMC was cultured in vitro, MTT method was used to detect the toxicity of IMD1-53 on HA-VSMC. 10 mmol/L β-glycerophosphate was added to HA-VSMC for high phosphorus induction, 0.1 nmol/L or 0.5 nmol/L IMD1-53 was used for intervention, and the ERS inducer tunicamycin (TM) was added on the basis of 0.5 nmol/L IMD1-53 intervention, the apoptosis rate of HA-VSMC was detected by flow cytometry, Alizarin red S staining was used to observe calcium salt deposition in HA-VSMC, the calcium content in HA-VSMC was determined by phthalein complexing ketone colorimetry, the activity of alkaline phosphatase (ALP) in HA-VSMC was detected by spectrophotometry, Western blot was used to detect calcification and expression of proteins related to ERS mitochondrial apoptosis pathway in HA-VSMC. Results: When IMD1-53 concentration ≤ 2.5 nmol/L, it had no significant effect on the viability of HA-VSMC cells (P>0.05), therefore, low and high doses (0.1 and 0.5 nmol/L) without obvious toxic and side effects on cells were selected for subsequent experiments. After high phosphorus induction, the apoptosis rate of HA-VSMC, calcium content, ALP activity, the expression levels of Runt related transcription factor-2 (Runx2), osteopontin (OPN), Bcl-2 associated X protein (Bax), cleaved caspase-3, glucose regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), activating transcription factor 6 (ATF6) p-inositol-requiring enzyme 1 (IRE1)/IRE1 proteins increased (P<0.05), orange nodules increased obviously, and the expression levels of osteoprotegerin (OPG) and B cell lymphoma/leukemia 2 (Bcl-2) proteins decreased (P<0.05). 0.1 nmol/L and 0.5 nmol/L IMD1-53 intervention treatment were able to reduce the apoptosis rate of HA-VSMC, calcium content, ALP activity, the expression levels of Runx2, OPN, Bax, cleaved caspase-3, GRP78, CHOP, ATF6, p-IRE1/IRE1 proteins (P<0.05), obviously reduce the orange nodules, and increase the expression levels of OPG and Bcl-2 proteins (P<0.05). The ERS inducer TM was able to increase the expression levels of GRP78, CHOP, ATF6, p-IRE1/IRE1 proteins in HA-VSMC, and weaken the inhibition of IMD1-53 on ERS, apoptosis and calcification induced by hyperphosphate in HA-VSMC. Conclusion: IMD1-53 can reduce the calcification of HA-VSMC induced by high phosphorus, and its mechanism may be related to inhibiting the activation of ERS mitochondrial apoptosis pathway, and reducing cell apoptosis. |
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