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| 氧化三甲胺在H9C2心肌细胞氧糖剥夺/复糖复氧损伤中的作用 |
| Role of trimethylamine N-oxide in oxygen-glucose deprivation/reoxygenation injury of H9C2 cardiomyocytes |
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| DOI：10.12089/jca.2026.08.012 |
| 中文关键词: 心肌细胞 氧化三甲胺 糖尿病 心肌缺血-再灌注损伤 氧糖剥夺/复糖复氧 内质网应激 线粒体相关内质网膜 |
| 英文关键词: Cardiomyocytes Trimethylamine N-oxide Diabetes melitus Myocardial ischemia-reperfusion injury Oxygen-glucose deprivation/reoxygenation Endoplasmic reticulum stress Mitochondria-associated endoplasmic reticulum membranes |
| 基金项目:新疆维吾尔自治区自然科学基金面上项目（2022D01C612） |
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| 摘要点击次数: 19 |
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| 中文摘要: |
目的： 探究氧化三甲胺(TMAO)在H9C2心肌细胞氧糖剥夺／复糖复氧(OGD/R)损伤中的作用及潜在分子机制。
方法： 正常培养的大鼠H9C2心肌细胞，随机分为四组：高糖对照组(HC组）、高糖-OGD/R组(H/R组）、高糖-OGD/R+TMAO组(T组）和高糖-OGD/R+TMAO+1,4,5-三磷酸肌酸受体1型（IP3R1）抑制剂Xestospongin C组(X组）。HC组使用高糖培养基培养H9C2心肌细胞72 h,不进行氧糖剥夺处理。H/R组使用高糖培养基培养H9C2心肌细胞72 h,随后更换为无糖培养基进行4 h缺氧处理，缺氧结束后，再更换为高糖培养基进行4 h复氧处理。T组在氧糖剥夺前72 h加入TMAO 400 μmol/L进行预处理，其余处理同H/R组。X组在氧糖剥夺前72 h加入TMAO 400 μmol/L,并在氧糖剥夺时更换为含IP3R1抑制剂Xestospongin C 1 μmol/L的无糖培养基，其余处理同H/R组。于复氧4 h后，分别采用流式细胞术检测细胞凋亡率、线粒体膜电位(MMP)和活性氧(ROS)水平，采用生化方法检测乳酸脱氢酶(LDH)活性，采用双标记共定位钙成像技术检测内质网钙离子和线粒体钙离子水平，采用免疫荧光共定位法检测IP3R1-GRP75、IP3R1-VDAC1、GRP75-VDAC1共定位水平，采用Western blot法测定GRP78、PERK、IP3R1、GRP75、VDAC1、BAX蛋白相对含量。
结果： 与HC组比较，H/R组、T组和X组细胞凋亡率、LDH活性、ROS水平、线粒体钙离子水平、GRP78、PERK、IP3R1、GRP75、VDAC1、BAX蛋白含量明显升高，IP3R1-GRP75、IP3R1-VDAC1、GRP75-VDAC1共定位水平明显升高，MMP和内质网钙离子水平明显降低(P<0.05)。与H/R组比较，T组细胞凋亡率、LDH活性、ROS水平、线粒体钙离子、GRP78、PERK、IP3R1、GRP75、VDAC1、BAX蛋白含量明显升高，IP3R1-GRP75、IP3R1-VDAC1、GRP75-VDAC1共定位水平明显升高，MMP、内质网钙离子水平明显降低(P<0.05);X组GRP78、PERK、IP3R1蛋白含量明显升高(P<0.05)。与T组比较，X组细胞凋亡率、LDH活性、ROS水平、线粒体钙离子、IP3R1、GRP75、VDAC1、BAX蛋白含量明显降低，IP3R1-GRP75、IP3R1-VDAC1、GRP75-VDAC1共定位水平明显降低，MMP、内质网钙离子水平明显升高(P<0.05)。H/R组和X组细胞凋亡率、LDH活性、MMP、ROS水平、内质网钙离子、线粒体钙离子、GRP75、VDAC1、BAX蛋白含量、IP3R1-GRP75、IP3R1-VDAC1、GRP75-VDAC1共定位水平差异无统计学意义；T组和X组GRP78、PERK蛋白含量差异无统计学意义。
结论： TMAO在H9C2细胞OGD/R损伤中，通过过度激活PERK内质网应激通路，导致内质网-线粒体钙稳态失衡，进而加剧了线粒体功能障碍，其机制可能与IP3R1/GRP75/VDAC1钙通道的激活有关。 |
| 英文摘要: |
Objective: To explore the role of trimethylamine N-oxide (TMAO) in oxygen-glucose deprivation/reoxygenation (OGD/R) injury of H9C2 cardiomyocytes and its potential molecular mechanism.
Methods: Normally cultured rat H9C2 cardiomyocytes were randomly divided into four groups: high glucose control group (group HC), high glucose-OGD/R group (group H/R), high glucose-OGD/R + TMAO group (group T), and high glucose-OGD/R + TMAO + IP3R1 inhibitor Xestospongin C group (group X). Group HC was cultured with high glucose medium for 72 hours without OGD/R treatment. Group H/R was cultured with high glucose medium for 72 hours, followed by 4 hours of hypoxia using glucose-free medium, then reoxygenated with high glucose medium for another 4 hours. Group T received a pre-treatment with TMAO 400 μmol/L 72 hours before OGD/R, with all other procedures identical to group H/R. Group X received TMAO 400 μmol/L 72 hours prior to OGD/R and was switched to glucose-free medium containing IP3R1 inhibitor Xestospongin C 1 μmol/L during the OGD phase, all other treatments were the same as in group H/R. At 4 hours after reoxygenation, cell apoptosis rate, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) levels were measured by flow cytometry, lactate dehydrogenase (LDH) activity was assessed biochemically, endoplasmic reticulum calcium and mitochondrial calcium levels were analyzed using dual-label co-localization calcium imaging, co-localization levels of IP3R1-GRP75, IP3R1-VDAC1, and GRP75-VDAC1 were detected by immunofluorescence co-localization, and relative protein expression levels of GRP78, PERK, IP3R1, GRP75, VDAC1, and BAX were determined by Western blot.
Results: Compared with group HC, groups H/R, T, and X showed significantly increased cell apoptosis rate, LDH activity, ROS levels, mitochondrial calcium levels, and protein expression of GRP78, PERK, IP3R1, GRP75, VDAC1, and BAX, along with significantly elevated co-localization levels of IP3R1-GRP75, IP3R1-VDAC1, and GRP75-VDAC1, and significantly decreased MMP and endoplasmic reticulum calcium levels (P < 0.05). Compared with group H/R, group T exhibited significantly higher cell apoptosis rate, LDH activity, ROS levels, mitochondrial calcium levels, and protein expression of GRP78, PERK, IP3R1, GRP75, VDAC1, and BAX, as well as elevated co-localization levels of IP3R1-GRP75, IP3R1-VDAC1, and GRP75-VDAC1, and lower MMP and endoplasmic reticulum calcium levels (P < 0.05), while group X showed increased protein expression of GRP78, PERK, and IP3R1 (P < 0.05). Compared with group T, group X demonstrated significantly reduced cell apoptosis rate, LDH activity, ROS levels, mitochondrial calcium levels, and protein expression of IP3R1, GRP75, VDAC1, and BAX, along with decreased co-localization levels of IP3R1-GRP75, IP3R1-VDAC1, and GRP75-VDAC1, and significantly increased MMP and endoplasmic reticulum calcium levels (P < 0.05). There were no statistically significant differences in apoptosis rate, LDH activity, MMP, ROS levels, endoplasmic reticulum calcium ions, mitochondrial calcium ions, GRP75, VDAC1, BAX protein content, or co-localization levels of IP3R1-GRP75, IP3R1-VDAC1, and GRP75-VDAC1 between group H/R and group X. Similarly, there were no statistical differences in GRP78 and PERK protein content between group T and group X.
Conclusion: In H9C2 cells subjected to OGD/R injury, TMAO exacerbates mitochondrial dysfunction by excessively activating the PERK endoplasmic reticulum stress pathway, leading to disruption of endoplasmic reticulum-mitochondrial calcium homeostasis. This mechanism may be associated with the activation of the IP3R1/GRP75/VDAC1 calcium channel complex. |
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