缺氧诱导因子1α对失血性休克血管舒张反应性的调控作用及与肌内皮缝隙连接蛋白的关系
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摘要
严重创伤/休克失代偿期常出现血管的低反应性,它表现为全身血管对缩血管物质和舒血管物质的反应降低或不反应,导致血压不能有效提升、组织灌注难以改善,细胞缺氧和损伤进行性加重。研究发现血管低反应性的发生与肾上腺素能受体失敏、一氧化氮(nitric oxide, NO)水平增高、血管平滑肌细胞(vascular smooth muscle cell, VSMC)钾、钙通道功能失常及细胞膜超极化有关。此外,本实验室的前期研究也发现:休克后,VSMC存在钙失敏,VSMC钙失敏在休克后血管低反应性的发生中起重要作用。
HIF-1α是缺氧诱导因子家族优势亚型HIF-1的活性亚基。通过与其调控基因的增加子序列结合,HIF-1广泛参与了哺乳动物细胞对低氧的特异性应答,在缺氧诱导的基因表达调节中起到了关键性的作用,其下游调节基因作用包括红细胞生成、细胞能量代谢、离子代谢、血管舒缩反应、血管生成、细胞周期调控等。
本实验室前期研究证实,在失血性休克后,HIF-1α在血管收缩反应性的调节中有重要的作用,其机制是:HIF-1α通过调控eNOS、iNOS、HO-1、COX-2、ET-1等下游的基因,调节血管活性物质释放而调节血管收缩反应性。但HIF-1α是否参与休克后血管舒张反应性的调节,其调节机制如何,是否与MEGJ有关国内外尚未见报道,需要研究阐明。
为此,本实验利用SD大鼠失血性休克模型和离体缺氧培养血管环,研究了HIF-1α对失血性休克后血管舒张反应性的调控作用以及与MEGJ的关系。具体内容包括:(1) HIF-1α对失血性休克血管舒张反应性的调节作用;(2) HIF-1α对缺血缺氧血管舒张反应性的调节作用与MEGJ的关系;(3) HIF-1α对失血性休克血管钙敏感性的调节作用。主要实验方法:
第一部分HIF-1α对失血性休克血管舒张反应性的调节作用
实验以失血性休克SD大鼠肠系膜上动脉(superior mesenteric artery, SMA)为研究对象,观察HIF-1α特异性抑制剂寡霉素(oligomycin)处理前后,休克大鼠SMA(休克即刻,休克0.5h,休克1h,休克2h,休克3h,休克4h)对非内皮依赖的血管舒张剂硝普钠(sodium nitroprusside ,SNP)和内皮依赖的舒张剂乙酰胆碱(acetylcholine ,Ach)的反应性变化情况。同时测定不同程度休克血管中HIF-1αmRNA的表达变化。
第二部分HIF-1α对缺血缺氧血管舒张反应性的调节作用与MEGJ的关系
实验取正常大鼠SMA血管环,通过缺氧和营养剥夺培养模拟失血性休克体内缺血缺氧环境(缺氧即刻,缺氧0.5h,缺氧1h,缺氧2h,缺氧3h,缺氧4h),观察寡霉素处理前后缺血缺氧处理血管环对Ach的舒张反应性变化规律;观察使用反义寡核苷酸阻断MEGJ后的血管环对Ach的舒张反应性变化规律。分别使用银杏叶提取物EGb761(商品名:金纳多)和寡霉素诱导和阻断HIF-1α的表达,测定Cx40、Cx43 mRNA的表达变化。用免疫共沉淀技术检测HIF-1α与Cx40、Cx43有无直接结合活性。
第三部分HIF-1α对失血性休克血管钙敏感性的调节作用
实验以失血性休克SD大鼠肠系膜上动脉(superior mesenteric artery, SMA)为研究对象,观察HIF-1α特异性抑制剂寡霉素(oligomycin)处理前后,休克大鼠SMA(休克即刻,休克0.5h,休克1h,休克2h,休克3h,休克4h)的钙敏感性变化情况。同时测定不同程度休克血管中MLC20磷酸化水平的变化情况。
主要研究成果: 1. HIF-1α对失血性休克血管舒张反应性的调节作用
各休克组SMA血管环对低浓度SNP(10-9、10-8、10-7mol/L)的舒张反应性降低;寡霉素可进一步降低其最大舒张反应。休克后血管对Ach的舒张反应性呈下降-上升-再下降的趋势,即在休克即刻有一短暂降低,随后呈代偿性增加,休克2小时达峰值,2小时后呈下降趋势;使用寡霉素后,血管对Ach的舒张反应性在休克早期(休克即时-休克1小时)显著下降,而在休克晚期(休克2小时-休克4小时)呈上升趋势。与正常组相比,休克后HIF-1αmRNA的表达增加;与休克早期的内皮依赖的舒张反应呈正相关,而与休克晚期的呈负相关,与内皮非依赖的舒张反应性没有相关性。结果说明:HIF-1α对于非内皮依赖的血管舒张反应性仅在休克早期存在正性调节作用,对内皮依赖的血管舒张反应性,在休克早期呈正性调节作用,而在休克晚期呈负向调节。
2. HIF-1α对缺血缺氧血管舒张反应性的调节作用与MEGJ的关系
(1) HIF-1α对缺血缺氧血管舒张反应性的调控作用:经过体外缺血缺氧处理的大鼠SMA血管环与失血性休克后血管环对Ach的内皮依赖性舒张反应性具有相似的变化趋势,即SMA血管环对Ach的舒张反应性呈先下降,后上升,再下降的趋势。缺氧处理SMA血管环的最大舒张反应在缺氧1h时达到最大,其后逐步下降,以缺氧4h最差。与缺氧对照组比较,寡霉素使SMA血管环最大舒张反应在休克早期下降,而在休克后期上升。寡霉素处理即刻组显著差于缺氧对照组,而3h和4h的寡霉素处理组最大舒张反应均高于缺氧对照组。结果说明:HIF-1α对体外缺血缺氧处理大鼠SMA血管舒张反应性与休克处理在体内具有同样的调节作用,即对内皮依赖性的血管舒张反应性在缺血缺氧早期存在正性调节作用,而在缺血缺氧晚期存在负向调节。
(2) Cx40和Cx43对缺血缺氧血管舒张反应性的调控作用:Cx40AODN和Cx43 AODN转染后,SMA血管组织Cx40和Cx43 mRNA表达明显下降。缺氧处理使SMA血管环对Ach的内皮舒张反应性下降。使用Cx40 AODN阻断Cx40表达后,缺氧处理SMA血管环在缺氧4h对Ach的舒张反应性显著高于空白对照组,而与正常组和缺氧1h组无差异。使用Cx43 AODN后,血管环的舒张反应性在未进行缺氧处理时显著下降,而缺氧处理后与空白对照组无差异。结果说明:MEGJ Cx40和Cx43参与了休克后血管舒张反应性的调节,Cx40降低休克晚期血管舒张反应性,Cx43升高休克早期血管舒张反应性。
(3) HIF-1α调节血管舒张反应性与Cx40和Cx43的关系:使用银杏叶提取物EGb761后,HIF-1αmRNA的表达增高,与空白对照组相比,以正常组和缺氧4h组的增加较为明显。使用EGb761后,各组Cx40 mRNA表达均较空白对照组显著提高;而寡霉素使正常组和缺氧4h组Cx40 mRNA表达显著下降。与空白对照组相比,EGb761使Cx43 mRNA的表达不同程度下降,以正常组和缺氧1h组较为明显;而寡霉素使缺氧4h组Cx43 mRNA表达较空白对照组显著升高。HIF-1α的免疫沉淀可以杂交出各组的Cx43条带,而只能杂交出正常对照组和缺氧1h组微弱的Cx40条带。结果说明:HIF-1α可通过上调Cx40的表达和下调Cx43的表达调节血管舒张反应性的变化;HIF-1α与Cx40、Cx43可能存在直接的结合作用,对Cx43的作用较强,对Cx40的作用相对较弱,其具体结合特性及有无直接作用尚需进一步研究确认。
3. HIF-1α对失血性休克钙敏感性的调节作用
失血性休克后,SMA血管环钙敏感性呈现先升高后下降趋势。血管对钙浓度的量-效曲线在休克0.5 h明显左移,Emax显著增加。在休克2h至4h,血管环对Ca2+敏感性明显下降,Ca2+的量效曲线明显右移,Emax显著降低。使用寡霉素后,SMA血管环对钙浓度的量-效曲线均右移,以休克1h和休克2h组较为显著。寡霉素降低了休克早期的钙敏感性,并使其在休克后期上升;其中,以休克0.5h组和休克1h组下降较为明显。休克后,MLC20磷酸化水平出现先升高后下降的变化趋势。与正常组相比,休克即刻升高明显;随休克时相延长逐步下降,休克3h组和休克4h组明显低于正常组。使用寡霉素抑制HIF-1α后, MLC20磷酸化水平没有出现先升高后下降的趋势,各组间均无显著差异。而与休克组相比,寡霉素处理组在休克即刻的高磷酸化水平受到显著抑制,而休克3h组和休克4h组的磷酸化水平显著升高。结果说明:HIF-1α参与了失血性休克后血管钙敏感性的调节,在休克早期有正性调节作用,而在休克晚期呈负向调节。
结论:
1. HIF-1α参与了失血性休克后血管舒张反应性的调节。对于非内皮依赖的血管舒张反应性仅在休克早期存在正性调节作用;对内皮依赖的血管舒张反应性,在休克早期存在正性调节作用,而在休克晚期存在负向调节作用。
2. MEGJ Cx40可能参与了休克晚期血管舒张反应的下降,Cx43可能参与了休克早期血管舒张反应的升高。HIF-1α可通过上调Cx40和下调Cx43的表达调节血管的舒张反应性。
3. HIF-1α参与了失血性休克后血管钙敏感性的调节,在休克早期有正性调节作用,而在休克晚期呈负向调节。
After sever trauma or shock, vascular reactivity to vasoconstrictors and vasodilators is greatly reduced, which results in hypotension, disorder of perfusion and damage of cell and tissue. Previous studies showed that it may be related to the increased level of nitric oxide (NO), the functional disorder of the K+ and Ca+ channels on the vascular smooth muscle cell (VSMC) membrane and the hyperpolarization of cell membrane. Furthermore, our previous study showed that calcium desensitization (the decrease of force/Ca2+ ratio) existed in the myocardium cell following severe trauma or shock.
Hypoxia-inducible factor 1α(HIF-1α) is the oxygen-regulated subunit of the transcriptional activator HIF-1, which mediates the changes of gene expression in response to the cellular oxygen concentrations. Our early study revealed that HIF-1αplayed an important role in hemorrhagic shock-induced vasoconstrictive hyporeactivity. And the possible mechanism of HIF-1αregulating vascular reactivity includes the pathway of eNOS/iNOS—NO, HO-1—CO, COX-2—PGI. But it’s not clear whether HIF-1αtakes part in the regulation of vasodilative reactivity following hemorrhagic shock and its mechanism. However, we had found that MEGJ played very important role in the regulation of vasodilative reactivity following hemorrhagic shock before.Thus, using oligomycin as the tool drug and rat hemorrhagic shock model, we obversed the regulatory effect of HIF-1αon vasodilative reactivity and the relationship to MEGJ following hemorrhagic shock in the present study.
Methods:
The experiments were conducted in three parts. In the first part, we observed the role of HIF-1αin the development of vasodilative hyporeactivity following hemorrhagic shock in rats. Superior mesenteric artery (SMA) from rats at different time after shock (shock 0h, 0.5h, 1h, 2h, 3h and 4h) was adopted to assay the vasodilative reactivity via observing the dilatation initiated by sodium nitroprusside (SNP) and acetylcholine (Ach) with isolated organ perfusion system with or without the HIF-1αinhibitor oligomycin. Meanwhile, the changes of HIF-1αmRNA were measured by RT-PCR.
In the second part, we observed the relationship between the role of HIF-1αin the development of vasodilative hyporeactivity and myoendothelial gap junction (MEGJ).We imitated the ischemic SMA of hemorrhagic shock in vitro by using the treatment of oxygen-glucose deprivation. And SMA at different time after oxygen-glucose deprivation (0h, 0.5h, 1h, 2h, 3h and 4h) was adopted to assay the vasodilative reactivity by Ach with isolated organ perfusion system with or without oligomycin.Then we treated the SMA rings with connexin 40 and connexin 43 antisense oligodeoxyuncleotide (Cxs AODN) and measured the vasodilative reactivity by Ach.After using extract of ginkgo biloba leaf EGb761 and oligomycin to induce and inhibit HIF-1αexpression, we semi-quantitated Cx40 and Cx43 mRNA expression of SMA by RT-PCR and observed their relationship to HIF-1αvia co-immunoprecipitaion and immunoblot analysis.
In the third part, we observed the role of HIF-1αin the development of calcium desensitization following hemorrhagic shock in rats. Superior mesenteric artery (SMA) from rats at different time after shock (shock 0h, 0.5h, 1h, 2h, 3h and 4h) was adopted to assay the calcium sensitivity via observing the contraction initiated by Ca2+ with isolated organ perfusion system with or without the HIF-1αinhibitor oligomycin. Meanwhile, the phosphorylation level of MLC20 was measured by western blot.
Results:
1. The HIF-1αwas responsible for the regulation of the vasodilative reactivity following hemorrhagic shock: The vascular reactivity of SMA to SNP in each shock group was significantly decreased at lower concentration (10-9, 10-8, 10-7mol/L) of SNP (P<0.05 or P<0.01); oligomycin further depressed the response of SMA to SNP. HIF-1αplayed a positive role to the non-endothelium-dependent vasodilation reactivity in the earlier stage of shock.The response of SMA to Ach in shock groups was transiently decreased after shock, and then began to increase during the compensatory period of shock, which reached the peak level at 2h after shock, and then decreased again. Oligomycin decreased the increase trend of Ach induced relaxing response in the earlier period of hemorrhagic shock, and increased the decrease trend in the late period of shock. HIF-1αmRNA exhibited a time-dependent increase following shock, and peaked at 4h, which was positively correlated with the endothelium-dependent vascular relaxation reactivity in the earlier period of hemorrhagic shock, and negatively correlated with in the late period of shock. HIF-1αplayed a positive role to the endothelium-dependent vasodilation reactivity in the earlier stage of sock, but show negative effect in the late period.
2. The relationship between MEGJ and HIF-1αas regulators of the vasodilative reactivity:
(1) The vascular reactivity of SMA to Ach in oxygen-glucose deprivation has similar changes to that after shock. The response of SMA to Ach reached the peak level at 1h after hypoxia, and then decreased. Oligomycin decreased the increase trend of Ach induced relaxing response in the short time of hypoxia, and increased the decrease trend in long time of hypoxia. The vasodilative reactivity of oligomycin group was lower than those of hypoxia-control group immediately after hypoxia (P<0.01), and higher in 3h and 4h after hypoxia (P<0.05). HIF-1αplayed a positive role to the endothelium-dependent vasodilatation reactivity in the earlier stage of hypoxia, but showed negative effect in the late period.
(2) Cx40 AODN improved the vascular response of SMA to Ach in 4h hypoxia groups (P<0.01), and Cx43 AODN decreased the reactivity in non-hypoxia groups (P<0.05).These results showed that Cx40 and Cx43 took part in the regulation of vascular reactivity after hemorrhagic shock.Cx40 had a suppressing effect on vasodilator reactivity, while Cx43 had an enhancing effect on vasodilator reactivity.
(3) EGb761 incresad the expression of HIF-1αmRNA at non-hypoxia groups and 4h hypoxia groups (P<0.05 or P<0.01). And expression of Cx40 mRNA increased by the effect of EGb761, while expression of Cx43 mRNA decreased. As compared with the control groups, oligomycin made the expression of Cx40 mRNA in non-hypoxia groups and 4h hypoxia groups decrease significantly (P<0.01), and made the expression of Cx43 mRNA in 4h hypoxia groups increase significantly(P<0.01). HIF-1αwas present in the immunoprecipitates of Cx43 in all groups,and HIF-1αwas present weakly in the immunoprecipitates of Cx40 in control groups and 1h hypoxia groups. HIF-1αcan up-regulate the expression of Cx40 mRNA, and down-regulate the expression of Cx43 mRNA. HIF-1αmight directly act on Cx43 and might act on Cx40 weakly following shock.
3. The HIF-1αwas responsible for the regulation of the calcium sensitivity following hemorrhagic shock.As compared with the control group, the cumulative dose-response curves of SMA to Ca2+at 0.5h after shock shifted to the left, the maximal contractions (Emax) of Ca2+ were increased significantly (P<0.05 or P<0.01). But the cumulative dose-response curves of SMA to Ca2+ at late shock(2h to 4h after shock) shifted to the right, Emax of Ca2+ were significantly decreased (P<0.05 or P<0.01). Oligomycin made the cumulative dose-response curves of SMA to Ca2+ at 1h and 2h after shock shift to the right, and the calcium sensitivity was decreased in the period of early shock (0.5h and 1h after shock) (P<0.05),and increased in the period of late shock. The phosphorylation of MLC20 was increased during the compensatory period of shock, and then decreased. As compared with the shock group, oligomycin inhibited the high phosphorylation level of MLC20 at early shock (immediate after shock) significantly (P<0.05), and the phosphorylation of MLC20 was increased at late shock (3h and 4h after shock) significantly (P<0.01) by the treatment of oligomycin. HIF-1αup-regulates the calcium sensitivity in the earlier period of hemorrhagic shock, and down-regulates it in the late stage.
Conclusions:
1. The endothelium-dependent vascular relaxation reactivity in the earlier period of hemorrhagic shock is increased, and decreased in the late stage. Endothelium-independent vascular relaxation reactivity is markedly decreased following hemorrhagic shock. HIF-1αplays a positive role to the non-endothelium-dependent vasodilatation reactivity in the earlier stage of sock, and plays a positive role to the endothelium-dependent vasodilatation reactivity in the earlier stage of sock, but show negative effect in the late period.
2. Cx40 can depress endothelium-dependent vasodilator in late hemorrhagic shock, Cx43 can enhance it in earlier stage. HIF-1αhas an obivious effect on Cx40 and Cx43, which can up-regulate the expression of Cx40 mRNA, and down-regulate the expression of Cx43 mRNA. HIF-1αmight directly act on Cx43 and might act on Cx40 weakly following shock.
3. HIF-1αup-regulates the calcium sensitivity in the earlier period of hemorrhagic shock, and down-regulates it in the late stage through regulating the phosphorylation level of MLC20.
HIF-1α是缺氧诱导因子家族优势亚型HIF-1的活性亚基。通过与其调控基因的增加子序列结合,HIF-1广泛参与了哺乳动物细胞对低氧的特异性应答,在缺氧诱导的基因表达调节中起到了关键性的作用,其下游调节基因作用包括红细胞生成、细胞能量代谢、离子代谢、血管舒缩反应、血管生成、细胞周期调控等。
本实验室前期研究证实,在失血性休克后,HIF-1α在血管收缩反应性的调节中有重要的作用,其机制是:HIF-1α通过调控eNOS、iNOS、HO-1、COX-2、ET-1等下游的基因,调节血管活性物质释放而调节血管收缩反应性。但HIF-1α是否参与休克后血管舒张反应性的调节,其调节机制如何,是否与MEGJ有关国内外尚未见报道,需要研究阐明。
为此,本实验利用SD大鼠失血性休克模型和离体缺氧培养血管环,研究了HIF-1α对失血性休克后血管舒张反应性的调控作用以及与MEGJ的关系。具体内容包括:(1) HIF-1α对失血性休克血管舒张反应性的调节作用;(2) HIF-1α对缺血缺氧血管舒张反应性的调节作用与MEGJ的关系;(3) HIF-1α对失血性休克血管钙敏感性的调节作用。主要实验方法:
第一部分HIF-1α对失血性休克血管舒张反应性的调节作用
实验以失血性休克SD大鼠肠系膜上动脉(superior mesenteric artery, SMA)为研究对象,观察HIF-1α特异性抑制剂寡霉素(oligomycin)处理前后,休克大鼠SMA(休克即刻,休克0.5h,休克1h,休克2h,休克3h,休克4h)对非内皮依赖的血管舒张剂硝普钠(sodium nitroprusside ,SNP)和内皮依赖的舒张剂乙酰胆碱(acetylcholine ,Ach)的反应性变化情况。同时测定不同程度休克血管中HIF-1αmRNA的表达变化。
第二部分HIF-1α对缺血缺氧血管舒张反应性的调节作用与MEGJ的关系
实验取正常大鼠SMA血管环,通过缺氧和营养剥夺培养模拟失血性休克体内缺血缺氧环境(缺氧即刻,缺氧0.5h,缺氧1h,缺氧2h,缺氧3h,缺氧4h),观察寡霉素处理前后缺血缺氧处理血管环对Ach的舒张反应性变化规律;观察使用反义寡核苷酸阻断MEGJ后的血管环对Ach的舒张反应性变化规律。分别使用银杏叶提取物EGb761(商品名:金纳多)和寡霉素诱导和阻断HIF-1α的表达,测定Cx40、Cx43 mRNA的表达变化。用免疫共沉淀技术检测HIF-1α与Cx40、Cx43有无直接结合活性。
第三部分HIF-1α对失血性休克血管钙敏感性的调节作用
实验以失血性休克SD大鼠肠系膜上动脉(superior mesenteric artery, SMA)为研究对象,观察HIF-1α特异性抑制剂寡霉素(oligomycin)处理前后,休克大鼠SMA(休克即刻,休克0.5h,休克1h,休克2h,休克3h,休克4h)的钙敏感性变化情况。同时测定不同程度休克血管中MLC20磷酸化水平的变化情况。
主要研究成果: 1. HIF-1α对失血性休克血管舒张反应性的调节作用
各休克组SMA血管环对低浓度SNP(10-9、10-8、10-7mol/L)的舒张反应性降低;寡霉素可进一步降低其最大舒张反应。休克后血管对Ach的舒张反应性呈下降-上升-再下降的趋势,即在休克即刻有一短暂降低,随后呈代偿性增加,休克2小时达峰值,2小时后呈下降趋势;使用寡霉素后,血管对Ach的舒张反应性在休克早期(休克即时-休克1小时)显著下降,而在休克晚期(休克2小时-休克4小时)呈上升趋势。与正常组相比,休克后HIF-1αmRNA的表达增加;与休克早期的内皮依赖的舒张反应呈正相关,而与休克晚期的呈负相关,与内皮非依赖的舒张反应性没有相关性。结果说明:HIF-1α对于非内皮依赖的血管舒张反应性仅在休克早期存在正性调节作用,对内皮依赖的血管舒张反应性,在休克早期呈正性调节作用,而在休克晚期呈负向调节。
2. HIF-1α对缺血缺氧血管舒张反应性的调节作用与MEGJ的关系
(1) HIF-1α对缺血缺氧血管舒张反应性的调控作用:经过体外缺血缺氧处理的大鼠SMA血管环与失血性休克后血管环对Ach的内皮依赖性舒张反应性具有相似的变化趋势,即SMA血管环对Ach的舒张反应性呈先下降,后上升,再下降的趋势。缺氧处理SMA血管环的最大舒张反应在缺氧1h时达到最大,其后逐步下降,以缺氧4h最差。与缺氧对照组比较,寡霉素使SMA血管环最大舒张反应在休克早期下降,而在休克后期上升。寡霉素处理即刻组显著差于缺氧对照组,而3h和4h的寡霉素处理组最大舒张反应均高于缺氧对照组。结果说明:HIF-1α对体外缺血缺氧处理大鼠SMA血管舒张反应性与休克处理在体内具有同样的调节作用,即对内皮依赖性的血管舒张反应性在缺血缺氧早期存在正性调节作用,而在缺血缺氧晚期存在负向调节。
(2) Cx40和Cx43对缺血缺氧血管舒张反应性的调控作用:Cx40AODN和Cx43 AODN转染后,SMA血管组织Cx40和Cx43 mRNA表达明显下降。缺氧处理使SMA血管环对Ach的内皮舒张反应性下降。使用Cx40 AODN阻断Cx40表达后,缺氧处理SMA血管环在缺氧4h对Ach的舒张反应性显著高于空白对照组,而与正常组和缺氧1h组无差异。使用Cx43 AODN后,血管环的舒张反应性在未进行缺氧处理时显著下降,而缺氧处理后与空白对照组无差异。结果说明:MEGJ Cx40和Cx43参与了休克后血管舒张反应性的调节,Cx40降低休克晚期血管舒张反应性,Cx43升高休克早期血管舒张反应性。
(3) HIF-1α调节血管舒张反应性与Cx40和Cx43的关系:使用银杏叶提取物EGb761后,HIF-1αmRNA的表达增高,与空白对照组相比,以正常组和缺氧4h组的增加较为明显。使用EGb761后,各组Cx40 mRNA表达均较空白对照组显著提高;而寡霉素使正常组和缺氧4h组Cx40 mRNA表达显著下降。与空白对照组相比,EGb761使Cx43 mRNA的表达不同程度下降,以正常组和缺氧1h组较为明显;而寡霉素使缺氧4h组Cx43 mRNA表达较空白对照组显著升高。HIF-1α的免疫沉淀可以杂交出各组的Cx43条带,而只能杂交出正常对照组和缺氧1h组微弱的Cx40条带。结果说明:HIF-1α可通过上调Cx40的表达和下调Cx43的表达调节血管舒张反应性的变化;HIF-1α与Cx40、Cx43可能存在直接的结合作用,对Cx43的作用较强,对Cx40的作用相对较弱,其具体结合特性及有无直接作用尚需进一步研究确认。
3. HIF-1α对失血性休克钙敏感性的调节作用
失血性休克后,SMA血管环钙敏感性呈现先升高后下降趋势。血管对钙浓度的量-效曲线在休克0.5 h明显左移,Emax显著增加。在休克2h至4h,血管环对Ca2+敏感性明显下降,Ca2+的量效曲线明显右移,Emax显著降低。使用寡霉素后,SMA血管环对钙浓度的量-效曲线均右移,以休克1h和休克2h组较为显著。寡霉素降低了休克早期的钙敏感性,并使其在休克后期上升;其中,以休克0.5h组和休克1h组下降较为明显。休克后,MLC20磷酸化水平出现先升高后下降的变化趋势。与正常组相比,休克即刻升高明显;随休克时相延长逐步下降,休克3h组和休克4h组明显低于正常组。使用寡霉素抑制HIF-1α后, MLC20磷酸化水平没有出现先升高后下降的趋势,各组间均无显著差异。而与休克组相比,寡霉素处理组在休克即刻的高磷酸化水平受到显著抑制,而休克3h组和休克4h组的磷酸化水平显著升高。结果说明:HIF-1α参与了失血性休克后血管钙敏感性的调节,在休克早期有正性调节作用,而在休克晚期呈负向调节。
结论:
1. HIF-1α参与了失血性休克后血管舒张反应性的调节。对于非内皮依赖的血管舒张反应性仅在休克早期存在正性调节作用;对内皮依赖的血管舒张反应性,在休克早期存在正性调节作用,而在休克晚期存在负向调节作用。
2. MEGJ Cx40可能参与了休克晚期血管舒张反应的下降,Cx43可能参与了休克早期血管舒张反应的升高。HIF-1α可通过上调Cx40和下调Cx43的表达调节血管的舒张反应性。
3. HIF-1α参与了失血性休克后血管钙敏感性的调节,在休克早期有正性调节作用,而在休克晚期呈负向调节。
After sever trauma or shock, vascular reactivity to vasoconstrictors and vasodilators is greatly reduced, which results in hypotension, disorder of perfusion and damage of cell and tissue. Previous studies showed that it may be related to the increased level of nitric oxide (NO), the functional disorder of the K+ and Ca+ channels on the vascular smooth muscle cell (VSMC) membrane and the hyperpolarization of cell membrane. Furthermore, our previous study showed that calcium desensitization (the decrease of force/Ca2+ ratio) existed in the myocardium cell following severe trauma or shock.
Hypoxia-inducible factor 1α(HIF-1α) is the oxygen-regulated subunit of the transcriptional activator HIF-1, which mediates the changes of gene expression in response to the cellular oxygen concentrations. Our early study revealed that HIF-1αplayed an important role in hemorrhagic shock-induced vasoconstrictive hyporeactivity. And the possible mechanism of HIF-1αregulating vascular reactivity includes the pathway of eNOS/iNOS—NO, HO-1—CO, COX-2—PGI. But it’s not clear whether HIF-1αtakes part in the regulation of vasodilative reactivity following hemorrhagic shock and its mechanism. However, we had found that MEGJ played very important role in the regulation of vasodilative reactivity following hemorrhagic shock before.Thus, using oligomycin as the tool drug and rat hemorrhagic shock model, we obversed the regulatory effect of HIF-1αon vasodilative reactivity and the relationship to MEGJ following hemorrhagic shock in the present study.
Methods:
The experiments were conducted in three parts. In the first part, we observed the role of HIF-1αin the development of vasodilative hyporeactivity following hemorrhagic shock in rats. Superior mesenteric artery (SMA) from rats at different time after shock (shock 0h, 0.5h, 1h, 2h, 3h and 4h) was adopted to assay the vasodilative reactivity via observing the dilatation initiated by sodium nitroprusside (SNP) and acetylcholine (Ach) with isolated organ perfusion system with or without the HIF-1αinhibitor oligomycin. Meanwhile, the changes of HIF-1αmRNA were measured by RT-PCR.
In the second part, we observed the relationship between the role of HIF-1αin the development of vasodilative hyporeactivity and myoendothelial gap junction (MEGJ).We imitated the ischemic SMA of hemorrhagic shock in vitro by using the treatment of oxygen-glucose deprivation. And SMA at different time after oxygen-glucose deprivation (0h, 0.5h, 1h, 2h, 3h and 4h) was adopted to assay the vasodilative reactivity by Ach with isolated organ perfusion system with or without oligomycin.Then we treated the SMA rings with connexin 40 and connexin 43 antisense oligodeoxyuncleotide (Cxs AODN) and measured the vasodilative reactivity by Ach.After using extract of ginkgo biloba leaf EGb761 and oligomycin to induce and inhibit HIF-1αexpression, we semi-quantitated Cx40 and Cx43 mRNA expression of SMA by RT-PCR and observed their relationship to HIF-1αvia co-immunoprecipitaion and immunoblot analysis.
In the third part, we observed the role of HIF-1αin the development of calcium desensitization following hemorrhagic shock in rats. Superior mesenteric artery (SMA) from rats at different time after shock (shock 0h, 0.5h, 1h, 2h, 3h and 4h) was adopted to assay the calcium sensitivity via observing the contraction initiated by Ca2+ with isolated organ perfusion system with or without the HIF-1αinhibitor oligomycin. Meanwhile, the phosphorylation level of MLC20 was measured by western blot.
Results:
1. The HIF-1αwas responsible for the regulation of the vasodilative reactivity following hemorrhagic shock: The vascular reactivity of SMA to SNP in each shock group was significantly decreased at lower concentration (10-9, 10-8, 10-7mol/L) of SNP (P<0.05 or P<0.01); oligomycin further depressed the response of SMA to SNP. HIF-1αplayed a positive role to the non-endothelium-dependent vasodilation reactivity in the earlier stage of shock.The response of SMA to Ach in shock groups was transiently decreased after shock, and then began to increase during the compensatory period of shock, which reached the peak level at 2h after shock, and then decreased again. Oligomycin decreased the increase trend of Ach induced relaxing response in the earlier period of hemorrhagic shock, and increased the decrease trend in the late period of shock. HIF-1αmRNA exhibited a time-dependent increase following shock, and peaked at 4h, which was positively correlated with the endothelium-dependent vascular relaxation reactivity in the earlier period of hemorrhagic shock, and negatively correlated with in the late period of shock. HIF-1αplayed a positive role to the endothelium-dependent vasodilation reactivity in the earlier stage of sock, but show negative effect in the late period.
2. The relationship between MEGJ and HIF-1αas regulators of the vasodilative reactivity:
(1) The vascular reactivity of SMA to Ach in oxygen-glucose deprivation has similar changes to that after shock. The response of SMA to Ach reached the peak level at 1h after hypoxia, and then decreased. Oligomycin decreased the increase trend of Ach induced relaxing response in the short time of hypoxia, and increased the decrease trend in long time of hypoxia. The vasodilative reactivity of oligomycin group was lower than those of hypoxia-control group immediately after hypoxia (P<0.01), and higher in 3h and 4h after hypoxia (P<0.05). HIF-1αplayed a positive role to the endothelium-dependent vasodilatation reactivity in the earlier stage of hypoxia, but showed negative effect in the late period.
(2) Cx40 AODN improved the vascular response of SMA to Ach in 4h hypoxia groups (P<0.01), and Cx43 AODN decreased the reactivity in non-hypoxia groups (P<0.05).These results showed that Cx40 and Cx43 took part in the regulation of vascular reactivity after hemorrhagic shock.Cx40 had a suppressing effect on vasodilator reactivity, while Cx43 had an enhancing effect on vasodilator reactivity.
(3) EGb761 incresad the expression of HIF-1αmRNA at non-hypoxia groups and 4h hypoxia groups (P<0.05 or P<0.01). And expression of Cx40 mRNA increased by the effect of EGb761, while expression of Cx43 mRNA decreased. As compared with the control groups, oligomycin made the expression of Cx40 mRNA in non-hypoxia groups and 4h hypoxia groups decrease significantly (P<0.01), and made the expression of Cx43 mRNA in 4h hypoxia groups increase significantly(P<0.01). HIF-1αwas present in the immunoprecipitates of Cx43 in all groups,and HIF-1αwas present weakly in the immunoprecipitates of Cx40 in control groups and 1h hypoxia groups. HIF-1αcan up-regulate the expression of Cx40 mRNA, and down-regulate the expression of Cx43 mRNA. HIF-1αmight directly act on Cx43 and might act on Cx40 weakly following shock.
3. The HIF-1αwas responsible for the regulation of the calcium sensitivity following hemorrhagic shock.As compared with the control group, the cumulative dose-response curves of SMA to Ca2+at 0.5h after shock shifted to the left, the maximal contractions (Emax) of Ca2+ were increased significantly (P<0.05 or P<0.01). But the cumulative dose-response curves of SMA to Ca2+ at late shock(2h to 4h after shock) shifted to the right, Emax of Ca2+ were significantly decreased (P<0.05 or P<0.01). Oligomycin made the cumulative dose-response curves of SMA to Ca2+ at 1h and 2h after shock shift to the right, and the calcium sensitivity was decreased in the period of early shock (0.5h and 1h after shock) (P<0.05),and increased in the period of late shock. The phosphorylation of MLC20 was increased during the compensatory period of shock, and then decreased. As compared with the shock group, oligomycin inhibited the high phosphorylation level of MLC20 at early shock (immediate after shock) significantly (P<0.05), and the phosphorylation of MLC20 was increased at late shock (3h and 4h after shock) significantly (P<0.01) by the treatment of oligomycin. HIF-1αup-regulates the calcium sensitivity in the earlier period of hemorrhagic shock, and down-regulates it in the late stage.
Conclusions:
1. The endothelium-dependent vascular relaxation reactivity in the earlier period of hemorrhagic shock is increased, and decreased in the late stage. Endothelium-independent vascular relaxation reactivity is markedly decreased following hemorrhagic shock. HIF-1αplays a positive role to the non-endothelium-dependent vasodilatation reactivity in the earlier stage of sock, and plays a positive role to the endothelium-dependent vasodilatation reactivity in the earlier stage of sock, but show negative effect in the late period.
2. Cx40 can depress endothelium-dependent vasodilator in late hemorrhagic shock, Cx43 can enhance it in earlier stage. HIF-1αhas an obivious effect on Cx40 and Cx43, which can up-regulate the expression of Cx40 mRNA, and down-regulate the expression of Cx43 mRNA. HIF-1αmight directly act on Cx43 and might act on Cx40 weakly following shock.
3. HIF-1αup-regulates the calcium sensitivity in the earlier period of hemorrhagic shock, and down-regulates it in the late stage through regulating the phosphorylation level of MLC20.
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