漸進式熱前置處理與熱休克蛋白在心肌保護作用之角色

Abstract

目標：比起全身熱療，漸進式熱前置處理（PTP）因引起較少的血行動力變化、內質網（ER）壓力和氧化刺激而對血管提供較佳之保護。我們認為PTP亦可能可有效地減少因心肌缺血/再灌注所誘導的細胞凋亡和自噬。 方法：將67隻雄性Wistar老鼠隨機分為非PTP控制組，和在一個週期或是連續3個週期進行42℃水浴的PTP 後24及72小時（1-24，1-72，3-24和3-72組）共5組。我們測量在左冠狀動脈前降支結紮3小時與再灌注2小時後體內產生的心肌負氧離子含量。另外由微循環、心電圖和心肌梗塞面積來決定心肌功能和受損程度。PTP因結抗煙酰胺腺嘌呤二核苷酸磷酸氧化酶gp91媒介的氧化刺激，內質網壓力和細胞凋亡及自噬產生的保護作用，則採用Western blot和免疫組化來檢驗。 結果：冠狀動脈缺血/再灌注在控制組及PTP的老鼠上均能抑制心肌的微循環，誘發心電圖ST段部位升高，並增加心肌梗塞之面積。缺血/再灌注會透過加強煙酰胺腺嘌呤二核苷酸磷酸氧化酶gp91的表現、胞漿細胞色素C釋放和降低粒腺體Bcl-2的含量而增加心肌負氧離子。因心肌受傷而被激活的ER壓力-78-kDa的葡萄糖調節蛋白的表現增加了Bax/Bcl-2比值，切割後caspase3的表現和聚（ADP-核糖）聚合酶的片段則導致細胞凋亡的形成﹔並藉由促進LC3-II的表達，進而導致細胞自噬。PTP治療會在老鼠的心臟中升高熱休克蛋白70、熱休克蛋白32和Bcl-2、Bcl-xL與錳超氧化物歧化酶，尤以3-72組量最高。PTP治療能顯著恢復心肌微循環、降低氧化刺激、ER壓力、細胞凋亡、細胞自噬和梗塞面積。 結論：PTP能透過加強抗氧化、抗凋亡和抗細胞自噬機制顯著降低心臟缺血/再灌注之損傷。

Objectives: Progressive thermal preconditioning (PTP) provides vascular protection with less hemodynamic fluctuations, endoplasmic reticulum (ER), and oxidative stress compared with whole body hyperthermia. We suggest PTP might efficiently diminish cardiac ischemia/reperfusion-induced apoptosis and autophagy injury. Methods: A total of 67 male Wistar rats were divided into a non-PTP control group, 24 or 72 hours after a single cycle or 3 consecutive cycles of PTP in a 42℃ water bath (1-24, 1-72, 3-24, and 3-72 groups). We measured the cardiac O2‾ amount in vivo in response to left anterior descending coronary artery ligation for 2 hours and reperfusion for 3 hours. Cardiac function and injury were determined by microcirculation, electrocardiography, and infarct size. The PTP-induced protective effects on nicotinamide adenine dinucleotide phosphate oxidase gp91-mediated oxidative stress, ER stress, and apoptosis- and autophagy-related mechanisms were examined using Western blot and immunohistochemistry. Results: Coronary arterial ischemia/reperfusion depressed cardiac microcirculation, induced ST-segment elevation and increased infarct size in non-PTP and PTP rats. Ischemia/reperfusion enhanced the cardiac O2‾ levels by enhanced nicotinamide adenine dinucleotide phosphate oxidase gp91 expression, cytosolic cytochrome C release, and decreased mitochondrial Bcl-2 expression. Cardiac injury activated ER stress–78-kDa glucose-regulated protein expression, increased the Bax/Bcl-2 ratio, cleaved caspase 3 expression and poly-(ADP-ribose)-polymerase fragments, leading to apoptosis formation, and promoted LC3-II expression, resulting in autophagy formation. PTP treatment elevated heat shock protein 70, heat shock protein 32, Bcl-2, Bcl-xL, and manganese superoxide dismutase in the rat heart, especially in the 3-72 group. PTP treatment significantly restored cardiac microcirculation, decreased oxidative stress, ER stress, apoptosis, autophagy, and infarct size. Conclusions: PTP significantly reduced cardiac ischemia/reperfusion injury by upregulating antioxidant, anti-apoptotic, and anti-autophagic mechanisms.