YC-1, Evodiamine與Moscatilin在動脈粥狀硬化過程中對於血管平滑肌細胞與巨噬細胞的作用

Abstract

根據衛生署的統計資料顯示，腦血管與心血管疾病一直盤據國人十大死因的前幾名，整體而言約略佔死亡總人數的20%，而這些疾病都和血管細胞 (內皮細胞/平滑肌細胞) 與發炎細胞 (單核球細胞/巨噬細胞) 有關。所以，若是能夠控制發炎反應或是血管血管平滑平滑肌細胞的死亡，在疾病的治療上便會有很大的幫助。因此，本論文以血管平滑肌細胞與巨噬細胞為研究對象，來探討藥物對於細胞的保護以及其抗發炎活性。 論文第一部分主要是要來探討YC-1是否可以有效增加細胞保護蛋白Hsp70的表現以及保護血管平滑肌細胞 (VSMCs)來對抗ox-LDL所造成的細胞毒殺作用。實驗結果發現，給予YC-1後的18-24小時達到最大的Hsp70誘導作用，並且可以觀察到濃度以及時間相關性。進一步的結果顯示，YC-1可以增加熱休克蛋白轉錄因子(HSF-1) 於細胞核內的表現量以及下游基因Hsp70 mRNA的表現。同時，我們也觀察到YC-1對Hsp70的誘導作用並非透過活化已知的cGMP、ROS或是PKC這些路徑而來。最後，我們也發現YC-1對於VSMCs的保護作用也會因為細胞內的殖入siHsp70後而喪失。綜合以上觀察，我們認為在血管性疾病上，YC-1應該可以當成一個Hsp70誘導劑來保護細胞免於受到進一步的傷害。 論文第二部分為evodiamine，是由中草藥吳茱萸Evodia rutaecarpa萃取出來的活性生物鹼，我們發現這一成分可以抑制缺氧 (hypoxia) 引發巨噬細胞產生的發炎反應。根據結果顯示，evodiamine在hypoxia狀態下不但可以抑制COX-2或是iNOS蛋白的表現，也可以降低PGE2的釋放。進一步我們得知evodiamine確實可以抑制hypoxia造成的轉錄因子HIF-1a 的堆積來降低COX-2 mRNA的表現。最後也發現到，evodiamine並不會影響到HIF-1a mRNA或是加速HIF-1a的分解速度，反而是透過影響到HIF-1a的轉譯過程 (translation) 來降低HIF-1a的累積。調控轉譯過程的蛋白，除了4E-BP磷酸化受到抑制外，hypoxia造成的Akt或是p70S6K的磷酸化也都被抑制下來。這些結果顯示出evodiamine對於hypoxia產生的發炎反應主要是透過抑制轉譯過程中蛋白的磷酸化而達到抑制HIF-1a的作用。所以我們認為evodiamine或許在疾病治療上可以用來抑制缺氧所產生的發炎反應。 論文第三部分為moscatilin，是由蘭科植物石斛Dendrobium nobile的莖所得到的萃取成分。我們直接利用LPS來刺激巨噬細胞誘發發炎反應的二大轉錄因子HIF-1a與NF-kB的活化，並且觀察moscatilin對LPS的作用。我們發現moscatilin可以抑制LPS造成的巨噬細胞型態的改變以及發炎蛋白COX-2與iNOS的生成。進一步分析moscatilin對這二個轉錄因子的影響時，可以看到moscatilin主要是透過抑制HIF-1a mRNA，而與蛋白質轉譯過程或是proteasome調控的蛋白分解過程並無直接的關聯性。而moscatilin對於IkBa磷酸化也具有顯著的抑制作用，並且也抑制了p65與p50細胞核的轉移以及阻斷NF-kB與DNA的結合而達到降低NF-kB活性。整體而言，moscatilin抑制COX-2與iNOS的表現主要是透過同時抑制了HIF-1a的生成與NF-kB的活化而來。 在本論文中，我們評估了YC-1、evodiamine與moscatilin 對於動脈粥狀硬化過程中抑制細胞凋亡以及抗發炎的效果。YC-1主要可藉由增加HSF-1轉移到細胞核內以及增加Hsp70的表現量來預防氧化態低密度脂蛋白造成血管平滑肌細胞死亡的現象。此外，evodiamine與moscatilin則是分別透過抑制 HIF-1a與NF-kB來達到降低缺氧環境以及lipopolysaccharide所誘發的發炎反應。其中，evodiamine主要是透過抑制HIF-1a蛋白轉譯過程，而moscatilin則是抑制HIF-1a轉錄以及NF-kB的活性來達到降低發炎蛋白生成的效用。根據我們觀察到的現象， YC-1、evodiamine與moscatilin對於動脈斑的生成具有一定的預防效果。因此，在未來可以試著評估這些藥物在動物模式中的藥效以及用於人類疾病治療的可行性。

Cardiovascular disease is one of major issues in human health. Atheroclerosis is a chronic and complex disease, and leads to a lot of cardiovascular disease. In the progress of atheroclerosis, we can find endothelial dysfunction, monocytes infiltration and cell apoptosis, which result in thrombosis and stroke. If we can have a good solution to repressing inflammation or decreasing cell death, it seems to be useful in the treatment of atheroclerosis. In this thesis, we study if YC-1 can protect vascular smooth muscle cells from death and explore if two natural products, evodiamine and moscatilin, can repress inflammation in macrophages. Hsp70 functioning as molecular chaperon in physiological conditions is induced under stress environment, which affords a defensive mechanism for cells to escape cellular damage. Several lines of evidence have shown that YC-1 provided protection against balloon injury-induced vascular injury and neurological disorders. Therefore, the first study was conducted to evaluate whether YC-1 could effectively induce Hsp70 expression and protect vascular smooth muscle cells (VSMCs) against oxidized low-density lipoprotein-induced cytotoxicity. We showed that YC-1 enhanced Hsp70 expression in VSMCs through a concentration- and time-dependent manner with maximum expression at 18 and 24 hours without involving the cGMP, ROS, PKC signals in the pathway. We demonstrated that the nuclear level of HSF-1 increased at 2 hour after YC-1 treatment, and Hsp70 expression was directed by the upregulation of Hsp70 mRNA which peaked at 6 hours and followed by a decline. Finally, we found that YC-1 protects VSMCs from oxidized low-density lipoprotein-inducing apoptosis, and the effect would be reversed in siHSP70-transfected cells. According to our observations, YC-1 would be an effectively pharmacological Hsp70 inducer that can be used as a cytoprotective agent in vascular diseases. Microenvironmental conditions found in injured tissues are characterized low level of oxygen and by infiltration of monocytes/ macrophages. In a previous study, extracts of Evodia rutaecarpa exhibited anti-inflammation activity through inhibition of reactive oxygen species production and inducible nitric oxide synthase expression in activated inflammatory cells. Therefore, the object of the second study was to investigate whether evodiamine, extracted from the traditional Chinese herb, Evodia rutaecarpa, could repress hypoxia-induced inflammatory response. We showed that evodiamine repressed not only COX-2 and iNOS expression but also PGE2 release under hypoxic conditions. Furthermore, our studies indicated that COX-2 mRNA and upstream HIF-1a were inhibited by evodiamine. In addition, we found that evodiamine inhibited hypoxia-evoked phosphorylation of Akt, p70S6K, and 4E-BP without affecting the level of HIF-1a mRNA and the degradation rate of HIF-1a. These results suggest that the mechanism of repression of hypoxia-induced COX-2 expression by evodiamine is through the inhibition of HIF-1a at the translational level, and is primarily mediated via dephosphorylation of Akt, p70S6K, and 4E-BP. These results indicated that evodiamine could be an effective therapeutic agent against inflammatory diseases involving hypoxia. Inflammation plays a key role in all stages of atherosclerosis, and infection was reported to contribute to atherosclerosis. In traditional medicine, Dendrobium nobile was used to cure bacteria-induced fever, but there is no clear action mechanism to elucidate it. Therefore, in the third study, we investigated the signaling pathways involved in the inhibition of HIF-1a and NF-kB by moscatilin, one extract fraction of Dendrobium nobile, under LPS challenge. The results showed that moscatilin had a significant inhibition on pro-inflammatory enzymes (COX-2 and iNOS) expression and macrophage activation under LPS stimulation. We also showed that moscatilin had a dominant repression on HIF-1a expression via down-regulating HIF-1a mRNA without inhibiting cell viability, translation machinery or proteasome-mediated degradation of HIF-1a. Moreover, the results showed that moscatilin suppressed nuclear translocation of NF-kB subunits, p65 and p50, and NF-kB activity by inhibiting phosphorylation of IkBa. Taken together, we suggested that moscatilin inhibited both COX-2 and iNOS expressions through down-regulating HIF-1a and NF-kB in RAW264.7. In this study, YC-1, evodiamine, moscatilin were used to evaluate the effect of anti-apoptosis and anti-inflammation in atherosclerosis. YC-1 increased HSF-1 nuclear translocation and Hsp70 expression to prevent vascular smooth muscle cells from ox-LDL-induced apoptosis. Additionally, evodiamine and moscatilin repressed hypoxia- or LPS-stimulated inflammatory responses through down-regulating HIF-1a and NF-kB, respectively. Among them, evodiamine inhibited HIF-1a translation, and moscatilin inhibited HIF-1a transcription and NF-kB nuclear transclocation to repressed pro-inflammatory protein expression. According to above, YC-1, evodiamine, and moscatilin play a positive role in preventing atherogenesis. Therefore, it is useful to evaluate the potency in animal atherosclerotic model and the possibility for human being.