- 學位論文
探討荔枝花萃取物抗第一型單純疱疹病毒之機制
Mechanism of Lychee Flower Extract against Type I Herpes Simplex Virus (HSV-1)
摘要
HSV-1為疱疹病毒科雙股DNA病毒，可導致人類產生許多眼部疾病。疱疹性角膜炎會導致角膜細胞病變，其所誘發的慢性免疫發炎反應會造成角膜基質產生傷疤、變薄以及新生血管的形成，使視力永久損傷。荔枝花萃取物是目前已知具有抗氧化以及抗發炎功效之天然藥物。本研究利用單純疱疹病毒感染兔子角膜上皮細胞SIRC細胞探討其反應機制，並且探討荔枝花萃取物是否具有抗病毒的功效。我們將SIRC細胞預先前處理荔枝花萃取物(0.2 μg/ml、0.1 μg/ml、0.05 μg/ml) 1.5小時後，再感染1 MOI的HSV-1，並於24小時後收取上清液及細胞萃取物，再利用乳酸脫氫酶檢測細胞毒性、Cell Counting Kit-8檢測細胞存活率、病毒斑試驗檢測病毒力價以及ELISA與western blot來檢測發炎相關激素的作用機制。由本研究得知預先前處理荔枝花萃取物的細胞可降低細胞毒性與細胞增生，並有效抑制病毒力價，以及提升修復角膜組織之相關因子IL-6的表現，並透過抑制mTOR pathway (pmTOR、pp70s6k)降低細胞增生表現，進而增加細胞吞噬蛋白Beclin-1、LC3之表現，此外透過抑制JAK-2表現可減少血管新生。綜合本研究的結果可知荔枝花萃取物是一種相當有潛力應用在抑制病毒複製的天然藥物，未來可再進一步探討它與抗病毒藥物的合併使用是否可以增加藥物的吸收以達到更有效率的抗病毒功用。
並列摘要
Herpes simplex virus Type I (HSV-1) belonging to the Herpesviridae family of large enveloped DNA viruses is capable of causing a wide array of human ocular diseases. Herpes simplex keratitis (HSK)-induced cytopathogenicity together with the chronic immune-inflammatory reaction can trigger stromal scarring, thinning, and neovascularization which may lead to permanent vision impairment. Lychee flower extract is known for antioxidant and anti-inflammatory effects. Therefore, this study investigated the mechanism of SIRC cells infected by HSV-1 and examined the antiviral capabilities of lychee flower. SIRC cells were pretreated with Lychee flower extract (0.2 μg/ml、0.1 μg/ml、0.05 μg/ml) for 1.5 hours then infected with 1 M.O.I of HSV-1 for 24 hours. The supernatants and cell extracts were collected for Lactic dehydrogenase (LDH) assay, Cell Counting Kit-8 (CCK), plaque assay, Enzyme-linked immunosorbent assay (ELISA) and western blotting. The results indicate lychee flower extract decreases the cell cytotoxicity, cell viability, and virus titers; it also increases the IL-6 production for healing wounded cornea. In addition, lychee flower extract decreased JAK-2 protein expression causing anti-angiogenesis; mTOR pathway (pmTOR and pp70s6k) was inhibited the cell proliferation and enhanced the autophagy protein expression (Beclin-1 and LC3). The results showed the antiviral capabilities and the protective effects of lychee flower. According to our results suggests that lychee flower extract is potential an anti-HSK (herpes simplex keratitis) for HSV-1 infection.