探討胞外熱休克蛋白在大鼠敗血性心肌病變的作用

Effects of Extracellular Heat Shock Protein on Septic Cardiomyopathy in Rats

林士仁(Shih-Jen Lin)

指導教授 ： 葉竹來

高雄醫學大學/醫學院/藥理學科研究所/碩士(2011年)

摘要

熱休克蛋白 (heat shock protein, HSP)以往被認為其作用僅侷限於細胞內，近年研究發現其也可被分泌至細胞外，且有許多生理功能。雖然在敗血性休克之病人，血中HSP會升高，然而，在敗血性心肌病變 (septic cardiomyopathy)時，胞外熱休克蛋白 (extracelluar heat shock protein, eHSP)在其致病機轉中扮演的角色仍有待了解。因此我們將進一步調查eHSP 在敗血性心肌病變發病機制中所扮演的角色。
在敗血性心肌病變的動物模式中，我們使用Wistar品系老鼠，麻醉後靜脈注射給予內毒素引發敗血症，經由股動脈及左心室插管監測其血行動力學的變化。
由實驗結果得知，內毒素靜脈注射後可明顯地導致敗血性休克與心肌功能下降之變化:包含平均動脈壓下降、左心室收縮壓下降、心跳變快、Max dp/dt以及Min dp/dt之下降等。此外也測量到TNF-α、NO、GOT/GPT、UA及LDH之血漿濃度迅速上升，且引起glucose下降。在心臟及肝臟組織中iNOS、COX-2、ERK1/2、p38、IκBα、p65及MMP-9之蛋白表現有顯著的上升。
若於LPS注射前10分鐘給予20 μg/kg的rHSP73於大鼠之靜脈，可發現其平均動脈壓、左心室收縮壓、Max dp/dt以及Min dp/dt 皆有抑制惡化之現象，並且對LPS導致血中之TNF-α、NO、GOT/GPT、UA及LDH濃度上升，與心臟及肝臟組織中iNOS、COX-2、ERK1/2、p38、IκBα、p65及MMP-9之活化，皆有抑制之作用。
由以上成果發現rHSP73 可減少LPS所造成的心肌功能失調，同時也發現rHSP73可以藉由抑制上游的p38及ERK的MAPK路徑，並抑制IκBα磷酸化及NF-κB轉入細胞核，使得下游iNOS、COX-2及TNF-α的蛋白減少，而iNOS的減少也使得NO無法大量增加，TNF-α的減少也使MMP-9的分泌減少。因此由這些成果得知，胞外熱休克蛋白在敗血性心肌病變的可能具有增進心肌功能與抗發炎反應之重要角色。

關鍵字

胞外熱休克蛋白 ； 內毒素 ； 敗血性心肌病變

並列摘要

Heat shock proteins (HSP) had been regarded as only with intracellular functions. Recent studies suggest that they can also be released to extracellular space and have physiologic functions. Given the facts that there are elevated plasma HSP levels in patients with septic shock, however, the role of extracellular HSP (eHSP) in the mechanisms in septic cardiomyopathy is still unknown. We therefore further investigated the role of eHSP in the pathogenesis of septic cardiomyopathy.
In the animal model of septic cardiomyopathy, we anesthetized the Wistar rats and injected lipopolysaccharide (LPS). Then the hemodynamic changes were monitored via catheters through femoral artery and catheters into the left ventricle via the carotid artery.
We found that intravenous injection of LPS induced a profound myocardial depression indicated by decreased mean arterial pressure, left ventricular systolic pressure, Max dp/dt and Min dp/dt, with compensatory increase of heart rates. In addition, plasma levels of TNF-α, nitric oxide, GOT/GPT, uric acid and LDH were increased but the plasma level of glucose was decreased. The protein levels of iNOS, COX-2, ERK1/2, p38, IκBα, p65 and MMP-9 were increased by LPS in heart and liver tissue.
However, these changes were significantly attenuated when HSP73 recombinant protein (rHSP73; 20 μg/kg, iv) were injected 10 minutes before LPS injection. Our results showed that after pretreatment with rHSP73, the myocardial suppression was attenuated with higher mean blood pressure, left ventricular systolic pressure and Max dp/dt and Min dp/dt. In addition, the increased plasma levels of TNF-α, NO, GOT/GPT, glucose, UA and LDH induced by LPS were suppressed by rHSP73, and the increased protein levels of iNOS, COX-2, ERK1/2, p38, IκBα, p65 and MMP-9 were also suppressed by rHSP73.
In conclusion, we found that rHSP73 inhibited LPS-induced myocardial dysfunction through suppression of cytokine production such as TNF-α and expression of iNOS and COX-2. This effect might mediated by the inhibition of MAPK and IκBα/NF-κB signaling pathway. It’s also down-regulated the plasma level of NO, and the protein and activation level of MMP-9. Taken together, these results indicate that eHSP has both anti-inflammatory effects and cardioprotective effects in septic cardiomyopathy.

並列關鍵字

eHSP ； LPS ； septic cardiomyopathy ； MAPK

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