黃芩素對於脂多醣及溶血磷脂醯膽鹼所誘導之心肌細胞損傷的保護機制

The Protecting Mechanisms of Baicalein in Lipopolysaccharide and Lysophosphatidylcholine-Induced Cardiomyocytes Injury

陳懷民(Huai-Min Chen)

指導教授 ： 葉竹來

高雄醫學大學/醫學院/臨床醫學研究所博士班/博士(2015年)

https://doi.org/10.6832/KMU.2015.00114

摘要

Chinese Abstract:
心臟衰竭是個體在遭受心肌缺血或是疾病感染時，常出現的併發症或後遺症，而在心臟收縮功能異常的情況下，個體就更容易因為多重器官衰竭及組織灌流不足而導致死亡。而在臨床上最常造成心因性死亡的原因則是以敗血症及冠狀動脈阻塞性心臟病為兩大病因。目前已有許多實驗證實脂多醣 (lipopolysaccharide-LPS)會對生物造成嚴重的敗血性反應，敗血病之後常見的併發症包括了心肌機能失調，會進而明顯導致病患敗血性休克而死亡。另一方面在心肌梗塞導致心肌細胞缺血性壞死後，會造成細胞膜上磷脂層代謝產物-溶血磷脂醯膽鹼(lysophosphatidylcholine- LysoPC)的堆積，並誘發一系列惡性反應的進行。
在探討具有潛在性可以有效減少因為LPS以及LysoPC所引起的心肌細胞受損而導致心臟損害而死亡的藥物過程中，作者研究了黃芩素 (Baicalein) 對心肌保護的作用。黃芩是傳統中草藥可增加免疫力、抗氧化及抗感染的作用。黃芩目前也被証實對於降低自由基 (reactive oxygen species - ROS) 的生成與預防缺血再灌流引起之損傷 (ischemia-reperfusion injury) 有相當大的功效。黃芩素是一種存在於Scutellaria baicalensis根部的黃酮類化合物。黃芩素是黃芩組成中的重要成份，對於肺臟細胞及肝臟細胞的保護作用已有相當多的實驗証實，但在心肌細胞的作用機轉的探討仍處在起步的階段。
此次的研究分成兩個部分來探討黃芩素對心肌細胞的保護作用的機制，分別針對脂多醣 (LPS) 引起的敗血症和溶血磷脂醯膽鹼 (LysoPC) 引起的心肌細胞毒性這兩個模型，來探討黃芩素對兩種不同刺激的保護機轉。因此在第一部分裡，作者首先研究暴露在LPS刺激下的新生Wistar大鼠心肌細胞裡黃芩素的抗發炎作用和其機制。在以LPS作用的實驗，探討初期調節物質：如tumor necrosis factor-α (TNF-α)及 interleukin-6，以及後期調節物質：如high mobility group box 1 (HMGB1)、inducible nitric oxide synthase (iNOS) 及 NO的生成，而基質金屬蛋白酵素-2和-9 (matrix metalloproteinases-MMP-2/-9)的表現也是作者關注的重點。
實驗結果發現黃芩素會減少ROS的形成，而且會顯著地降低LPS所誘發的心肌細胞肥大。此外，以黃芩素預先處理對LPS引起的初期調節物質以及後期調節物質及iNOS和NO的釋放和生成也有明顯抑制的作用。而黃芩素也明顯地下調 MMP-2/-9對刺激的反應，並且會減少HMGB1從細胞核到細胞質的轉移 (translocation)。這些結果都表明黃芩素能透過抑制ROS和致炎性cytokines的生成來保護心肌細胞以防止LPS引起的心臟傷害。而這些心肌保護的作用也許是藉由抑制HMGB1和MMP-2/-9的傳遞路徑來完成。
而LysoPC 則是心肌細胞發生缺氧後在細胞膜磷脂層上所堆積的代謝產物，是導致心臟失能的主要物質，它會造成心肌細胞內鈣離子濃度上升而導致嚴重的心律不整及心室頻脈。此外LysoPC也是活化ROS形成與誘導cells apoptosis/necrosis 的重要物質，而這些的反應也都促使宿主因心臟衰竭而死亡。因此在第2個實驗裡，作者要探討的是老鼠H9C2胚胎心肌細胞以黃芩素預處理是否能防止LysoPC堆積而引起的心臟毒性的惡化。而在這實驗，作者則探討mitogen-activated protein kinases (MAPK) pathway，包括了ERK、JNK及p38; 以及 apoptotic mechanisms 包括 Bcl-2/Bax、caspase-3、caspase-9 及 cytochrome C pathways等。研究結果發現黃芩素能減少LysoPC所引起的細胞死亡、ROS的生產以及細胞內鈣濃度的增加。而且黃芩素也能抑制 LysoPC所引起的細胞凋亡，並伴隨著pro-apoptotic信號因子Bax 減少及抗凋亡信號因子Bcl-2的增加，進而導致Bcl-2 / Bax 比值的上升。實驗也發現黃芩素也能抑制LysoPC所誘導的 cytochrome C，casapase-3，casapase-9等的活化，並抑制 ERK 1/2，JNK 和 p38的磷酸化作用。最後作者結論黃芩素能透過降低ROS的生成，抑制鈣離子的超載以及去活化MAPK的傳遞途徑來達到保護心肌細胞以防止LysoPC 所誘導的細胞凋亡。
這些重要的結果都顯示出黃芩素能保護心肌細胞免於因LPS及LysoPC所誘導而產生細胞傷害。而這些保護作用是透過降低ROS的生成，抗發炎及抗凋亡的機制而達成。基於實驗的結果，作者認為黃芩素可以用來治療因外因性的LPS刺激或者是因內因性膜磷脂LysoPC異常的累積所導致的惡性反應，並做為心臟保護的藥物。而作者未來也希望透過動物實驗來更進一步了解，黃芩素對心肌保護的機制及其作用機轉，並能進而引伸至臨床實驗而達到治療病人的目的。

關鍵字

Baicalein ； Lipopolysaccharide ； Lysophosphatidylcholine ； Matrix metalloproteinase ； High mobility group box 1 (HMGB1) ； membrane phospholipids ； MAPK ； Reactive oxygen species ； Bcl-2/Bax

並列摘要

English Abstarct:
Heart failure is an usual complication or sequel when the cardiomyocytes were suffered from the myoischemia or infection. In the situation of abnormal cardiac contractility, the individuals are tent to death because of the multiple organs’ failure and the poor tissue perfusion. In the clinical conditions the most two causes related with cardiac death are the septicemia and coronary arterial occlusive disease. Myocardial dysfunction, a common complication after sepsis and myocardial infarction, significantly contributes to the death of patients with septic shock and ischemic heart failure. In the search for potentially effective drugs to decrease mortality from sepsis, author investigated the cardioprotective effects of baicalein on lipopolysaccharide (LPS) and lysophosphatidylcholine (LysoPC) induced cardiac damages.
The Huang-Qin is a traditional Chinese herbal medicine which can increase the immunity, and possesses the properties of the anti-oxidant function, and the anti-infection. It is also proven to be able to significantly decrease the production of reactive oxygen species (ROS) and to prevent the ischemia-reperfusion injury (Woo et al., 2005). The baicalein is a flavonoid present in the root of Scutellaria Baicalensis. Baicalein is an important composition in the root of Huang-Qin, it already had quite a lot of experiments to have verified the protective effects to lung cell injuries as well as liver injury, but the protective mechanisms to cardiomyocytes are still at the preliminaries. LPS is the glycolipid of the cell membrane of Gram-negative bacteria and it functions as the endotoxin which can cause a local or systemic inflammatory reaction in the host after bacterial infection. The stimulation of LPS will result in the activations of macrophages, inducible nitric oxide synthase (iNOS), NO, prostaglandins, and ROS in the hosts; and eventually lead to the death of hosts because of septicemia and cardiac failure. This study is divided into two sections to survey the effects of the signaling mechanisms of baicalein in cardiomyocytes in LPS-induced septicemia and lysoPC-induced cytotoxic models.
Therefore, in the first section, we investigated the anti-inflammatory effects and mechanisms of baicalein in neonatal Wistar rat cardiomyocytes with exposure to LPS. The release of early pro-inflammatory cytokine (e.g., TNF-α and interleukin-6) and late pro-inflammatory cytokine (e.g., HMGB1 as well as the production of iNOS and NO were examinated. The expression of MMP-2/-9 is also under investigated. Author found that baicalein significantly attenuated LPS-induced cardiac hypertrophy and counteracted the ROS generation. In addition, pretreatment with baicalein inhibited both LPS-induced early and late pro-inflammatory cytokine releases, iNOS expression and NO production. Finally, baicalein also significantly down-regulated the expression of MMP-2/-9 and attenuated the HMGB1 translocation from nucleus to cytoplasm. These results suggest that baicalein can protect cardiomyocytes from LPS-induced cardiac injuries via inhibition of ROS and inflammatory cytokine production. These cardio-protective effects are possibly mediated through inhibition of the HMGB1 and MMP-2/-9 signaling pathways.
LysoPC, a metabolite from membrane phospholipids, accumulates in the ischemic myocardium and plays an important role in the development of myocardial ventricular arrhythmia. In addition, LysoPC also activates the production of ROS and induces the cells’ apoptosis and necrosis. Such reactions will promote the cardiac death of hosts. Therefore, in the second section, author investigated if baicalein could protect against LysoPC-induced cytotoxicity in rat H9C2 embryonic cardiomyocytes. In this study, the author also investigated the MAPK pathway, which includes the ERK, JNK and p38; and the apoptotic mechanisms, which include the Bcl-2/Bax, caspase-3, caspase-9 and cytochrome C pathways. Author found that baicalein could prevent LysoPC-induced cell death, production of ROS, and increase the intracellular calcium concentration. Besides, baicalein also inhibited LysoPC-induced apoptosis, with the decreased pro-apoptotic Bax protein, and the increased anti-apoptotic Bcl-2 protein, which result in an increase in the Bcl-2/Bax ratio. Author also found that baicalein attenuated LysoPC-induced activation of cytochrome C, casapase-3, casapase-9, and the phosphorylation of ERK 1/2, JNK, and p38. Finally, author concluded that baicalein protected cardiomyocytes from LysoPC-induced apoptosis by reducing ROS production, inhibition of calcium overload, and deactivation of MAPK signaling pathways.
In summary, these novel results indicate that baicalein can protect cardiomyocytes from LPS and LysoPC-induced cells’ damages via reducing ROS production, anti-inflammation, and anti-apoptosis. Based on these findings, baicalein could be applied in the treatment of the septic reactions caused by the outer stimulation of LPS or by the inner stimulation of abnormal accumulation of LysoPC from membrane phospholipids, and it is suggested to be developed as a cardioprotective agent. In the final the author hope to take a further step to understand the cardiac protective mechanisms of baicalein and to apply the baicalein to patients in the clinical situations.

並列關鍵字

Baicalein ； Lipopolysaccharide ； Lysophosphatidylcholine ； Matrix metalloproteinase ； High mobility group box 1 (HMGB1) ； membrane phospholipids ； MAPK ； Reactive oxygen species ； Bcl-2/Bax

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