第一部分：HMG-CoA還原酶抑制劑調控NLRP3發炎體活化之機制探討 第二部分：探討葛根芩連湯在動脈硬化疾病上的應用潛力及作用機制

Abstract

第一部分: 在臨床上，HMG-CoA還原酶抑制劑 (statins) 已被廣泛作為降血脂的藥物，用於治療心血管疾病。除了可以治療高血脂病患，近年來也有研究報導指出，statins具有多重治療效果，例如：抑制發炎反應、抗腫瘤、甚至於具有免疫調節作用。而這些多重性的治療潛力的作用機轉主要是由於statins會造成異戊二烯 (isoprenoid)生合成路徑的受損，進而抑制Ras、RhoA、Cdc42或Rac1小分子G蛋白質相關的訊息傳遞路徑，進而調節細胞功能達到多元性的治療效果。近年研究報導發現在單核白血球（monocytes）或是人類週邊血液單核球細胞（peripheral blood mononuclear cells），給予statins的刺激會造成capsase-1的活化而誘導IL-1β的釋出。同時也指出異戊二烯對於caspase-1活性的抑制佔有重要的角色。由於inflammasome對於caspase-1活化及過度的IL-1β釋出與許多發炎疾病有關，故inflammasome複合體的調控機制在醫學上的研究與臨床上的應用也變得極為重要。因此，在這研究中我們深入探討statins刺激caspase-1活性的詳細作用機制。就目前報導已知，NLRP3 inflammasome的活化至少可受控於三大主要路徑，分別是 （1）外生性ATP鍵結P2X7受體(P2X7R)促使細胞內鉀離子流失，（2）細胞內活性氧化物（ROS）堆積及（3）溶酶體破裂（lysosomal rupture）釋出蛋白酶（如cathepsin B）。在我們的研究裡，將於人類單核球細胞株（THP-1）針對此三條路徑進行statins誘導的caspase-1活化的機制探討。首先發現Fluvastatin 及Lovastatin的處理的確可與脂多醣體（lipopolysaccharide，LPS）具有協同作用而達到誘發NLRP3活化。根據我們的結果，在LPS的處理下，statins活化caspase-1及誘導IL-1

PartI: The HMG-CoA reductase inhibitors, namely statins, are widely used for treatment of lowering cholesterol in clinical. However, statins are not only therapeutically administered in hypercholesterolemia but also have multiple therapeutic potentials, such as in anti-inflammation, anti-tumor, and immunomodulation. Most of these pleiotropic and atypical actions are resulting from the impairment of isoprenoid biosynthesis and interference with signal cascades mediated by small G proteins. Recent studies further unexpectedly observe the stimulating effects of statins on caspase-1 activation and IL-1β secretion in monocytes and peripheral blood mononuclear cells, and suggest the crucial roles played by isoprenoids in the inhibition of caspase-1 activity. Since inflammasome-linked caspase-1 activation and overproduction of IL-1β are highly associated with many inflammatory diseases, understanding the cellular regulation of inflammasome complex becomes important in medical research and therapeutic prospect. Therefore, in this study we like to dissect the molecular mechanism in detail that underlies the stimulating effects of statins on caspase-1. In this respect, three commonly recognized mechanistic models for NLRP3 inflammasome activation (i.e. ATP/P2X7R/K+ efflux, ROS and lysosomal upture) were investigated in statin-induced caspase-1 activation in human THP-1 monocytes. We found fluvastatin or lovastatin treatment can synergize with LPS to trigger inflammasome NLRP3 activation. Moreover, statins-stimulated caspase-1 activation and IL-1β production in LPS-primed THP-1 cells are related to GGPP deficiency and P2X7R activation, but not to ROCK activity. The increases in endogenous ATP release and P2X7R gene expression account for the activation of P2X7R. We also provided evidence that statin-induced moderate ROS elevation is involved in the inflammasome activation. Moreover cathepsin B inhibitor was shown to reduce statin-induced events, suggesting the involvement of lysosomal instability in the action of statins. Lastly we showed the ability of statins to increase NLRP3 gene and protein expression in LPS-treated condition. Taken together, statin-induced enhancement of NLRP3 inflammasome activation in THP-1 monocytes covers multiple mechanisms, i.e. increases in ATP release, P2X7R expression and activation, ROS production, lysosomal rupture and NLRP3 expression. These data not only shed new insight into isoprenylation-dependent regulation of NLRP3 inflammasome, but also unmask mechanisms for statin-elicited inflammasome activation. PartII: The Ger-Gen-Chyn-Lian-Tang (GGCLT) is a mixture of Chinese herbal medicines, which consists of Puerariae radix, Scutellariae radix, Coptidis rhizoma and Glycyrrhizae radix. Even though individual herbal medicine has been proved to prevent inflammation, cell proliferation, and/or cholesterol overload most in cellular studies, it remains unclear whether the medicine herbals in combination could achieve beneficial efficacy in atherosclerosis. In this study, we used animal and cellular models to evaluate the combination benefits in atherosclerotic progression. In animal model, GGCLT dosage (2g/kg/day) could decrease the serum levels of total cholesterol, LDL and TG in ApoEKO mice fed with high cholesterol diet. GGCLT and PDL-05 (serum metabolite of Pueraria lobat, also as the major metabolite constituent of GGCLT), however, did not prevent vascular smooth muscle cells and bone marrow-derived macrophages from proliferation and inflammation. Instead activation of IKK, p38, JNK, Akt and/or ERK, and increased expression of iNOS and/or COX-2 are induced with different extents by GGCLT and PDL-05. Therefore, our in vitro data rule out the involvement of anti-proliferation and anti-inflammation in GGCLT-induced in vivo benefits in atherosclerosis. Further studies in the biosynthesis, clearance and uptake of cholesterol are hopeful to unravel the action mechanism of GGCLT in atherosclerosis treatment.