- 學位論文
探討金絲桃素抑制C型肝炎病毒表觀遺傳之調控機制
The mechanisms of epigenetic regulation of hypericin inhibiting HCV replication
摘要
C型肝炎病毒（Hepatitis C virus, HCV）為一種正向單股RNA病毒，歸類為黃熱病毒科（Flaviviridae）肝炎病毒屬（Hepacivirus）。根據美國疾管署的統計約有四分之三的C型肝炎患者因注射藥物及性行為而感染C型肝炎病毒。所以C型肝炎躍昇為傳染性極高的疾病，根據世界衛生組織（World Health Organization, WHO）發佈的資料統計，目前每年約有39.9萬人死於C型肝炎病毒感染，主要是肝硬化和肝细胞癌。由於直接抗病毒药物（Direct acting antivirals）可以有效治療C型肝炎病毒感染但因價格昂貴，目前治療覆蓋率仍偏低，故尋求有效的替代方案仍為當務之急。金絲桃素(hypericin)為貫木連翹(Hypericum perforatum L.)的主要成分，具有抗C型肝炎病毒的效果，但是其作用機轉未明，本研究目的希望能確認金絲桃素(hypericin)的作用機轉。 我們在此選用帶有HCV1b replicon的Ava.5細胞株做為抗C型肝炎病毒藥物之研究平台，並進一步探討藥物作用機制。首先，我們以數個藥物處理Ava.5細胞株後，觀察細胞型態，及利用MTS assay觀察藥物對細胞存活的影響。在藥物不影響細胞生長存活的狀況下，以反轉錄酶鏈聚合酶連鎖反應(RT-PCR)及西方墨點(Western blot) 分別分析藥物抑制C型肝炎病毒(HCV)複製NS5A RNA 及NS3 protein的能力；在確定金絲桃素(hypericin)能明顯抑制HCV複製能力後，我們以數個蛋白抑制劑處理Ava.5細胞株，藉以探討其抑制C型肝炎病毒複製能力的可能訊息傳導路徑；為了研究金絲桃素抑制 C型肝炎病毒複製機轉，我們添加5-aza-2’-deoxycytidine (5-Aza-dC)和chidamide 來分析組蛋白修飾(histone modification)的機制。添加shRNA以確定血紅素加氧酶-1(heme oxygenase-1, HO-1)在C型肝炎病毒複製遭抑制時所扮演的角色。研究結果顯示：金絲桃素及其他藥物試劑對Ava.5細胞株皆無細胞毒性，金絲桃素使血紅素加氧酶-1(heme oxygenase-1, HO-1)和NS5A下調(down regulation)與作用時間、劑量呈現正相關。由金絲桃素所導致的NS3活性下降和提升HO-1 的表現皆可因Chidamide 而非5-Aza-dc的加入而發生。LY294002抑制C型肝炎病毒複製藉由抑制HO-1的表現。金絲桃素 抑制C型肝炎病毒複製未透過p-AKT的作用。實驗中發現shHO-1亦可抑制C型肝炎病毒複製。 以上總結金絲桃素(hypericin) 抑制C型肝炎病毒複製透過降低血紅素加氧酶-1(heme oxygenase-1, HO-1)活性與去已醯化(deacetylation)反應。
並列摘要
Hepatitis C virus (HCV) is a positive-sensed, single-stranded RNA virus that belong to the Flaviviridae family and Hepacivirus. According to World Health Organization (WHO), acute HCV infection is usually asymptomatic, and is only very rarely (if ever) associated with life-threatening disease. About 15–45% of infected persons spontaneously clear the virus within 6 months of infection without any treatment. The remaining 60–80% of persons will develop chronic HCV infection. Of those with chronic HCV infection, the risk of cirrhosis of the liver is between 15–30% within 20 years. The combination therapy of peginterferon alpha-2a and ribavirin is recommended as a standard care for chronic HCV infections in clinically before 2015. Due to the discovery of the direct acting antivirals (DAAs), the standard of care of HCV infections is changing rapidly. Sofosbuvir, daclatasvir and the sofosbuvir/ledipasvir combination are part of the preferred regimens in the WHO guidelines. Although the production cost of DAAs is low, these medicines remain very expensive in many high- and upper middle-income countries. Regards to complications of peginterferon alpha-2a, ribavirin and cost of DAAs, access to treat HCV infection is valuable. The aim of this study is to elucidate the mode of action of hypericin in Ava5 human hepatoma cell line (Huh7 derivative) harboring HCV subgenomic replicon RNA. First, we used MTS assay to measure cytotoxicity after the drugs treated cells. The treatment with the drugs at concentrations that did not induce cell death, and then we examined HCV subgenomic RNA by reverse transcription polymerase chain reaction and western blot to check the drugs whether inhibit HCV replication. Finally, we confirmed Hypericin can inhibit HCV1b NS5A RNA and NS3 protein, then we treated several protein inhibitor for Ava.5 cells to clarify the biochemical mechanism that inhibit HCV replication. To investigate how hypericin inhibits HCV replication, 5-aza-2’-deoxycytidine (5-Aza-dC) and chidamide were used for determining histone modification. Furthermore, shRNA was applied to confirm the role of heme oxygenase (HO-1) in HCV repression. As a result, hypericin in experiment were tested and showed no cytotoxicity. Hypericin reduced HO-1 and NS5A in a time- and dose- dependent manner. Chidamide, but not 5-Aza-dc, restored hypericin-induced reduction in HCV NS3 expression and reversed HO-1 expression in Ava5 cells. LY294002 inhibited HCV replication via HO-1 down-regulation. Constitutive expressed p-AKT was not involved in hypericin-induced reduction in HCV replication. In addition, shHO-1 inhibited HCV replication. In conclusion, hypericin inhibits HCV replication via down-regulation of HO-1 expression and deacetylation.
參考文獻
延伸閱讀
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