熱休克前處置降低內毒素所誘發敗血症老鼠血小板功能的異常:周邊白血球的角色

Previous heat shock treatment attenuates lipopolysaccharide-induced platelet dysfunction in rats: role of peripheral leukocytes

董慧萍(Huei-Ping Dong)

指導教授 ： 楊瑞成

高雄醫學大學/醫學院/醫學研究所/博士(2007年)

https://doi.org/10.6832/KMU.2007.00137

摘要

內毒素是導致敗血症最常見的格蘭氏陰性桿菌細胞壁上面的脂多醣。敗血症會導致血液循環系統障礙，包含血小板去顆粒作用或瀰漫性血管內凝血病變，當產生血管血栓或出血現象時，是導致敗血症的主要死亡原因。雖然已有報告指出血液中的白血球和所接觸的血小板無論在生理狀態或病理狀態下，彼此之間皆互有非常強烈的互動關係。但在敗血症中其相互之間的機制並不十分清楚，為了進一步氂清，所以我們想要利用體外實驗來探究：內毒素所誘發的敗血症中，進行吞噬作用的白血球和血小板凝集能力的相互關係。實驗中我們分別將內毒素置入在富含血小板的血漿、全血以及富含血小板的血漿中加入白血球等三種不同樣品30分鐘、60分鐘、90分鐘，血小板的凝集能力可以藉由血小板刺激劑ADP刺激產生並由血小板凝集检驗儀器測得。實驗結果發現:內毒素置入富含血小板的血漿 PRP 後 30、 60、 90分鐘後均不會影響老鼠血小板的凝集功能反應，然而將內毒素分別置入全血及富含血小板的血漿加入白血球90分鐘後，卻發現老鼠的血小板的凝集功能反應有顯著下降的現象。然而，當單獨外加一氧化氮合成酶抑制劑 L-NAME (NG-nitro-L-arginine methyl ester) 並無法改善敗血症血小板凝集功能下降的現象；但是，當外加過氧化氫酶Catalase (過氧化氫清除者) 時，發現可以顯著改善敗血症血小板凝集功能下降的現象。而且，當一氧化氮合成酶抑制劑L-NAME合併過氧化氫酶一起作用時，發現可以改善敗血症血小板凝集功能下降的現象更為顯著。由這些研究結果中指出:白血球對敗血症血小板凝集功能下降的現象中扮演一個很重要的角色，白血球影響敗血症血小板凝集功能下降的機制很可能主要是經由活化的白血球所釋放至血漿的過氧化氫的作用和過氧化氫影響一氧化氮的作用。
許多報告證實熱休克前處置可以誘發熱休克蛋白家族的表現並可以降低嚴重敗血症實驗動物的器官的傷害和死亡率，然而，熱休克前處置對敗血症血小板功能的保護機制仍然不清楚。因此，我們想要探討的是:熱休克前處置對內毒素所導致的敗血症老鼠中血小板功能反應的影響。實驗動物進行熱休克處置後18小時由股靜脈注射內毒素， 注射90分鐘後，由動物體頸動脈抽出血液樣品，再由血小板凝集检驗儀器測定血小板凝集功能。在這個研究結果中，我們發現內毒素會導致老鼠ex vivo血小板的凝集功能顯著下降的現象，而熱休克前處置可以調節內毒素所導致的血小板功能過低的現象。如果在熱休克處置後7天(同時伴隨著白血球熱休克蛋白的消失)再靜脈注入內毒素，會發現對敗血症中血小板功能的保護作用就隨著消失。同時將正常血小板和敗血症老鼠血漿混合一起作用時，發現敗血症老鼠血漿會抑制正常血小板的凝集功能，而當給予熱休克前處置後並無此現象出現;同時,熱休克前處置敗血症老鼠的血漿會調節敗血症老鼠血小板凝集功能過低的現象;另外，當外加過氧化氫酶時，發現可以顯著改善敗血症血小板凝集功能過低的現象，使血小板活性恢復到快接近正常血小板的凝集能力。相反的，在熱休克前處置的敗血症老鼠過氧化氫酶的加入僅僅提升少許血小板的凝集活性;同時，熱休克前處置也可以顯著調節敗血症血漿中一氧化氮的代謝產物的濃度。這些結果指出:熱休克前處置對敗血症血小板凝集功能過低現象的保護機制中，很可能是經由減弱白血球的活化程度、影響了血漿中過氧化氫酶和一氧化氮的濃度。總結:我們認為白血球在內毒素誘發敗血症中血小板的功能扮演著很重要的角色，那麼對於敗血症病人的出血病徵可以開啟新的治療方向。此外，熱休克處置可能可以藉由影響白血球的活性而對敗血症中血小板功能的產生保護機制。

關鍵字

敗血症 ； 內毒素 ； 血小板凝集 ； 一氧化氮合成酶抑制劑 ； 過氧化氫酶熱休克

並列摘要

Endotoxemia causes several hematological dysfunctions, including platelet degranulation or disseminated intravascular coagulation, leading to thrombotic and hemorrhagic events which are the main cause of death in patients with sepsis. The aim of this first study was to investigate the mechanisms involved in the platelet aggregation and in their interaction with leukocytes in whole blood after stimulation with endotoxic lipopolysaccharide (LPS) in vitro. Platelet aggregation ability was induced by ADP and measured by an aggregometer. Platelet-rich plasma (PRP) and normal whole blood were incubated with LPS for 30 min, 60 min and 90 min. The results showed that LPS did not directly induce platelet aggregation in PRP. On the contrary, LPS caused a decrease in platelet aggregation that could be detected 90 min after LPS was incubated with the whole blood. Moreoever, both platelet-leukocyte interaction and the platelet hyporesponsiveness can be significantly attenuated by the addition of catalase (H2O2 scavenger) and catalase combined with L-NAME (NG-nitro-L-arginine methyl ester, nitric oxide synthase inhibitor), but not only L-NAME. In the in vitro experimental, these results indicated that LPS-induced phagocytosing leukocytes might influence platelet function suggested a potential role in sepsis and the leukocyte-platelet interaction was attenuated by the H2O2 generation of reactive oxygen radical.
Several studies demonstrated that previous heat shock treatment caused expression of heat-shock proteins (HSPs) and reduced both organ dysfunction and mortality in experimentally induced severe sepsis. However, the protective mechanism of heat shock treatment on platelet function of sepsis remains unclear. The aim of this second study was to investigate the effect of heat shock treatment on platelet aggregation ex vivo in endotoxin-induced septic rats. Rats of the heated group were heated by whole-body hyperthermia 18h before LPS injection. Blood samples were obtained from the carotid artery 90 min after LPS injection. Platelet aggregation ability was measured by an aggregometer. Results revealed that platelet aggregation ex vivo was significantly inhibited in LPS-induced rats in a manner of dose-dependence. Previous heat shock treatment caused overexpression of HSPs and significantly attenuated the LPS-induced platelet hyporesponsiveness. This attenuation disappeared in accordance with absence of leukocyte HSP72 at 7 days after heat shock treatment. Aggregation of normal platelets was also inhibited by incubating with plasma obtained from endotoxemic rats but not from preheated endotoxemic rats. Furthermore, no significant hyporesponsiveness was found in endotoxemic platetlets in addition of preheated endotoxemic plasma. Addition of H2O2 scavenger catalase diminished the platelet hypo-responsiveness significantly only in non-heated endotoxemic rats. Moreover, the plasma nitrite and nitrate level was significantly attenuated in preheated endotoxemic rats. These results revealed that previous heat shock treatment might modulate LPS-induced hypo-responsiveness of platelets by attenuating leukocyte activation and changing the plasma components, possibly through altering the H2O2 and NO concentration.

並列關鍵字

sepsis ； lipopolysaccharide ； platelet aggregation ； L-NAME, catalase, heat shock

參考文獻

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