探討杜莎藻之類胡蘿蔔素萃取物抑制病毒感染後誘發的炎症反應

The suppressive effect of carotenoid extract of Dunaliella salina algae on virus-induced inflammatory response in RAW264.7 cells

陳威志(Wei-Zhi Chen)

指導教授 ： 張元衍

中山醫學大學/醫學院/微生物免疫研究所/碩士(2013年)

https://doi.org/10.6834/CSMU.2013.00072

摘要

疱疹病毒 (Herpes simplex virus; HSV)感染對於人類的健康具有相當程度的危害，當免疫力低下的患者受到此病毒感染可能會引起許多嚴重的疾病並可能會導致較高的致死率。由於HSV對人具有高度的傳染性，因此常以同為阿爾發疱疹病毒亞科之假性狂犬病毒(Pseudorabies virus, PRV)來模擬HSV的感染。杜莎藻(Dunaliella salina)是一種嗜鹽性、微小單細胞生物(無細胞壁)其包含豐富的類胡蘿蔔素(特別是all-trans β-carotene 和 9- 或 9‘-cis-β-carotene)，目前已經被拿來應用在保健食品。在過去的研究中已經發現杜莎藻具有良好的抗細菌性內毒素(LPS)所誘導巨噬細胞(RAW264.7)之發炎反應。本論文的研究，利用假性狂犬病毒感染RAW264.7細胞所誘發炎症反應的模式，探討杜莎藻是否具有調節病毒感染所引起的炎症反應。我們將RAW264.7細胞預先添加杜莎藻(100 μM)1.5小時後，在感染0.1 MOI 的PRV，並於不同時間點收集上清液及細胞萃取物，再利用ELISA及western blot等方法來檢測NO與發炎相關細胞激素的作用機制。由ELISA的實驗結果發現杜莎藻確實能抑制由PRV感染後所誘發的NO且能降低IL-1β、IL-6、TNF-α和MCP-1等發炎相關細胞激素的表現。經western blot的實驗結果，發現杜莎藻可藉由NF-κB pathway (如: NF-κB p50、NF-κB p65、IKK、JNK以及P38)與 IL-6 pathway(如: JAK-2, p-JAK-2, STAT-1/3, p-STAT-1/3) 來抑制NF-κB的活化，降低iNOS與COX-2表現，進而抑制炎症反應。因此我們也利用上述路徑的各種蛋白抑制劑(P38、ERK、JNK、STAT-3、JAK)來確認PRV所誘導炎症反應的路徑，結果顯示除了ERK抑制劑之外，其他的抑制劑(P38、JNK、STAT-3、JAK)確實能夠抑制PRV所誘導的NF-κB、iNOS與COX-2表現，達到抗發炎的作用。綜合上述的實驗結果可知，杜莎藻不只能抑制細菌性的發炎反應，同時也具有抑制病毒感染後所造成的發炎反應。

關鍵字

杜莎藻 ； 類胡蘿蔔素

並列摘要

Herpes simplex virus type (HSV) infection has a considerable degree of human health hazards, immunocompromised patients infected by this virus, it may cause more severe disease and higher mortality. Pseudorabies virus (PRV) is always used to imitate human HSV due to both belonging to the DNA virus and not infecting human.
Dunaliella salina (Chlorophyceae) is a halophilic unicellular micro-alga with a mucus surface coat (no cell wall). The alga has plenty of carotenoids (especially all-trans s-carotene and 9- or 9‘-cis-s-carotene), and has been exploited as a health food product, a pro-vitamin A supplement, a food coloring agent, and an additive to food and cosmetics. Our previous report has been demonstrated that D. salina had good anti-inflammatory activities for LPS stimulated macrophages (RAW264.7 cells). In the present study, we used a model of PRV-infected RAW264.7 cells to investigate the D. salina against virus induced inflammation and possible mechanism. Therefore, we used the ELISA and western blot to investigate the signaling pathway of the anti- inflammatory effect of D. salina on RAW264.7 cells. The results of ELISA and western blot indicated that D. salina significantly inhibited the levels of NO and inflammatory cytokines (contains: IL-1s、IL-6、TNF-α and MCP-1) in RAW264.7 cells infected by PRV. Further studies revealed that D. salina also down-regulated of iNOS and COX-2 protein expressions, include NF-κB pathway (contains: NF-κB (p50 and p65), IKK, p-IKK), MAPK pathway of JNK, p-JNK, P38, p-P38 and JAK/STAT pathway (contains: JAK-2, p-JAK-2, STAT-1/3, p-STAT-1/3). In addition, we also used variety of protein inhibitors (P38, ERK, JNK, STAT-3 and JAK) to confirm whether the anti-inflammatory effects of PRV-stimulated pro-inflammatory mediators in RAW264.7 cells via MAPK Pathway and JAK/STAT pathway. Interestingly, P38, JNK inhibitors, treatment strongly blocked the MAPK pathway activation; STAT-3 and JAK inhibitors treatment strongly blocked the JAK/STAT pathway activation, as evidenced by the decrease in NF-κB, iNOS and COX-2 expression level by PRV infection. Taken together, our data indicated that D. salina suppressesd the generation of pro-inflammatory mediators, such as iNOS, COX-2 and inflammatory cytokines as well as their regulatory genes in PRV-infected RAW264.7 cells by inhibiting NF-κB/MAPK and JAK/STAT signaling pathway.

並列關鍵字

Dunaliella salina ； carotenoid

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