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| 环氧二十碳三烯酸、5-羟色胺与缝隙连接蛋白在慢性缺氧性肺动脉高压形成中的作用 |
| Role of epoxy eicostrioleic acid，5-hydroxytryptamine，gap junction protein in the formation of chronic hypoxic pulmonary hypertension |
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| DOI：10.12089/jca.2022.07.013 |
| 中文关键词: 肺动脉高压 缺氧性肺血管收缩 缝隙连接蛋白 环氧二十碳三烯酸 5-羟色胺 慢性缺氧 |
| 英文关键词: Pulmonary arterial hypertension Hypoxic pulmonary vasoconstriction Gap junction protein Epoxy eicosapentaenoic acid 5-hydroxytryptamine Chronic hypoxia |
| 基金项目:南京市科技局项目（201803068） |
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| 摘要点击次数: 1332 |
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| 中文摘要: |
目的 探讨环氧二十碳三烯酸（EET）、五羟色胺（5-HT）、缝隙连接蛋白40（Cx40）和43（Cx43）在慢性缺氧性肺血管收缩所致肺动脉高压中的作用。
方法 选择SPF级BALB/c小鼠40只，8周龄，体重25～29 g。实验一：采用随机数字表法将小鼠分为两组：对照组（O组，n=8）和慢性缺氧模型组（H组，n=32）。所有小鼠正常饮食，O组在正常环境喂养4周，H组予低氧舱（每天吸入10%氧气10 h）喂养4周后，监测MAP和平均肺动脉压（mPAP）。随后处死小鼠，取心肺组织称量并计算右心室肥厚指数。实验二：O组和H组于显微操作下用胶原酶消化法分离远端肺小动脉血管，采用荧光免疫组化法鉴定肺动脉内皮细胞（PAEC）和肺动脉平滑肌细胞（PASMC）。采用随机数字表法将H组分离得PAEC和PASMC进一步分为三组：缺氧+si-Cx40组（H40组）、缺氧+si-Cx43组（H43组）和缺氧+无义siRNA组（HN组），H40组和H43组PAEC和PASMC分别接受Cx40基因和Cx43基因的特异性siRNA脂质体siPORTNeoFX转染，HN组接受无义siRNA转染。所有细胞在10%氧气条件下进行传代培养。采用qPCR检测Cx40和Cx43 mRNA表达量，MTT实验检测细胞增殖情况，ELISA法检测EET和5-HT浓度，免疫组织化学法检测PASMC中α-平滑肌肌动蛋白（α-SMA）、平滑肌蛋白-22α（SM22α）和2r骨形态发生蛋白（BMP2r）含量。
结果 实验一：H组MAP、mPAP和右心室肥厚指数均明显高于O组（P<0.05）。实验二：与HN组比较，H40组PAEC和PASMC中Cx40 mRNA表达量明显降低（P<0.05）；H43组PAEC和PASMC中Cx43 mRNA表达量明显降低（P<0.05）；H43组和H40组PAEC中EET浓度明显升高（P<0.05），PASMC中EET和5-HT浓度、α-SMA和SM22α含量明显降低（P<0.05），PASMC中BMP2r含量明显升高（P<0.05）。与H40组比较，H43组PAEC中EET浓度明显升高（P<0.05），PASMC中EET和5-HT浓度、α-SMA和SM22α含量明显降低（P<0.05），PASMC中BMP2r含量明显升高（P<0.05）。
结论 PAEC感受缺氧信号，合成分泌增加的EET、5-HT通过Cx40和Cx43转移至PASMC中，促进平滑肌细胞的增殖和收缩蛋白α-SMA、SM22α的表达。通过阻断缝隙连接之间EET、5-HT的转运，有助于减轻缺氧性肺血管收缩和肺动脉高压。 |
| 英文摘要: |
Objective To investigate the role of epoxy eicosapentaenoic acid (EET), 5-hydroxytryptamine (5-HT), connexin 40 (Cx40) and 43 (Cx43) in pulmonary arterial hypertension induced by chronic hypoxic pulmonary vasoconstriction.
Methods Forty SPF BALB/c mice were selected,aged 8 weeks,weighing 25-29 g. In experiment 1, mice were randomly divided into two groups: normoxic control group (group O, n = 8) and chronic hypoxia model group (group H, n = 32). The mice in group O were kept in normoxic environment, and the mice in group H were placed in low oxygen chamber (10% oxygen concentration) for 10 hours every day. Four weeks later, MAP, mean pulmonary artery pressure (mPAP) were monitored. After recording the data, all the mice were sacrificed and its left and right ventricular tissue was weighted separately to calculate the right ventricular hypertrophy index. In experiment 2, the distal pulmonary arterioles of all mice in groups O and H were isolated by collagenase digestion under microscope, pulmonary artery endothelial cells (PAEC) and pulmonary artery smooth muscle cells (PASMC) were identified by fluorescence immunohistochemistry. The identified PAEC and PASMC in group H were randomly divided into three subgroups: hypoxia+si-Cx40 group (group H40), hypoxia+si-Cx43 group (group H43) and hypoxia+nonsense siRNA group (group HN). PAEC and PASMC in groups H40 and H43 were transfected with Cx40 gene and Cx43 gene specific siRNA liposome siPORTNeofx, respectively, and group HN was transfected with nonsense siRNA. Cells were subcultured under 10% oxygen. The expression of Cx40 and Cx43 mRNA were detected by qPCR; the proliferation of PAECs and PASMCs were detected by MTT assay; ELISA was used to detect the concentration of EET and 5-HT; immunohistochemistry was used to detect the expression of α-smooth muscle actin (α-SMA),smooth muscle-22α (SM22α) protein and bone morphogenetic protein 2r (BMP2r) in PASMC.
Results Experiment 1 revealed MAP,mPAP and right ventricular hypertrophy index in group H were significantly higher than that in group O (P < 0.05). Experiment 2 indicated that compared with group HN,the expression of Cx40 mRNA in PAEC and PASMC were significantly down-regulated in group H40 (P < 0.05); the expression of Cx43 mRNA in PAEC and PASMC were significantly down-regulated in group H43 (P < 0.05); the concentrations of EET in PAEC were significantly increased (P < 0.05),the concentrations of EET and 5-HT in PASMC,the expression of α-SMA and SM22α in PASMC were significantly reduced (P < 0.05),the expression of BMP2r in PASMC were significantly increased in groups H40 and H43 (P < 0.05). Compared with group H40,the concentrations of EET in PAEC were significantly increased (P < 0.05),the concentrations of EET and 5-HT in PASMC and the expression of α-SMA and SM22α in PASMC were significantly reduced (P < 0.05),the expression of BMP2r in PASMC were significantly increased in group H43 (P < 0.05).
Conclusion PAEC senses hypoxic signals and transfers EET and 5-HT to PASMC via Cx40 and Cx43, up-regulating the expression of α-SMA and SM22α resulting in sustained pulmonary vasoconstriction and remodeling. Blocking the transport of EET and 5-HT between gap junctions may help to alleviate hypoxic pulmonary vasoconstriction and pulmonary arterial hypertension. |
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