蒲公英萃取物對脂多醣體所造成牛乳腺上皮細胞株及周邊血液單核細胞細胞激素表現之影響

The Effect of Taraxacum Mongolicum Extract on the Cytokines Expression in LPS-Stimulated Bovine Mammary Epithelial Cells and Peripheral Blood Mononuclear Cells

許凱程(Kai-Chen Hsu)

指導教授 ： 季昭華

國立臺灣大學/獸醫專業學院/臨床動物醫學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.02058

摘要

蒲公英 ( Taraxacum mongolicum Hand.-Mazz. ) 為著名的傳統中藥，廣泛地被中醫所應用，有清熱解毒、消癰散結、利尿通淋、健胃消炎等功效，通常用來治療炎症性疾病和病毒性傳染病等。乳腺炎 ( Mastitis ) 主要是由細菌或黴菌感染引起乳腺組織的炎症反應。通常使用抗生素治療，但乳品抗生素殘留及抗藥性細菌的問題，日益嚴重，目前首重之課題便是尋找一個替代的治療或預防方法，藉由提高牛隻的免疫能力，增強乳牛抵禦病原菌入侵的能力，為本研究探討的主要目的。
實驗主要分為兩大部分，首先在LPS的刺激實驗中，分別將LPS添加於牛的周邊血液單核細胞 ( PBMCs ) 與乳腺上皮細胞株 ( MAC-T cells ) 中，濃度為 1 μg/mL 與10 μg/mL，以同步定量逆轉錄聚合酶連鎖反應 ( Real time RT-PCR )，測量細胞激素 ( 即IL-1β, IL-6, TNF-α, IL-8, GCP-2 ) 表現量的變化，建立基礎值。再者，進行蒲公英萃取物的添加實驗，以LPS的刺激實驗為基礎，在LPS刺激前的4小時，預先將蒲公英萃取物加入周邊血液單核細胞 ( PBMCs ) 與乳腺上皮細胞株 ( MAC-T cells ) 當中，濃度分別為10 μg/mL 與100 μg/mL，同樣以同步定量逆轉錄聚合酶連鎖反應 ( Real time RT-PCR )方式，進行細胞激素表現量的分析，以評估蒲公英萃取物對細胞激素的影響。
實驗結果顯示，蒲公英萃取物在LPS對於牛隻相關細胞所引起的炎症反應有顯著的抑制效果，主要透過調降 ( Down-regulation ) 細胞內前發炎細胞激素( Pro-inflammatory cytokines ) 與趨化激素 ( Chemokines ) 的產生，以達到抑制炎症反應的效果，避免細胞激素過度產生所造成的傷害，進而達到抑制高體細胞數及仿指乳汁變性的效果。
綜合以上可知，蒲公英透過提高牛隻的免疫能力，增強乳牛抵禦病原菌入侵的能力，抑制過度的炎症反應；本研究結果將有助於了解蒲公英在調控發炎相關方面所扮演的角色，以期未來能應用於臨床的治療用途，降低乳房炎的發生率，進而減少抗生素的使用。

關鍵字

蒲公英 ； 乳腺上皮細胞 ； 周邊血液單核細胞 ； 脂多醣內毒素 ； 前發炎細胞激素 ； 趨化激素 ； 乳房炎

並列摘要

Taraxacum mongolicum Hand.-Mazz. is the famous traditional Chinese medicine, which was commonly used by Chinese local herbal physicians. The herb is frequently used to treat inflammatory disorders and viral infectious diseases. Mastitis is an inflammatory response in mammary tissue mainly caused by bacterial and fungal infections. To prevent increasingly severe problems of drug residues in milk and antibiotic-resistant bacteria, one alternative strategy is to improve an individual’s ability to rid itself of the infective agent by either selecting for or inducing enhanced innate immune capabilities within populations of dairy cattle.
The present study investigated the effect of Taraxacum mongolicum extract on inflammatory responses by bovine mammary epithelial cell line ( MAC-T ) and peripheral blood mononuclear cells ( PBMCs ). Through MTS assay, cells were also exposed to multiple Taraxacum mongolicum extract concentration ( 0, 1000, 100, 10, 1 μg/mL ). Cell viability of MAC-T cells and PBMCs were higher than the control group. It was indicated that Taraxacum mongolicum extract did not inhibit cell proliferation.
A time course ( 0, 2, 4, 8, 16, 24 h ) of the expression of pro-inflammatory cytokines ( including IL-1β, IL-6, TNF-α ) and chemokines ( including IL-8, GCP-2 ) was subsequently performed. Moreover, cells pretreated with Taraxacum mongolicum extract significantly inhibited LPS-stimulated gene expression. It was concluded that the Taraxacum mongolicum extract possessed marked anti-inflammatory effect in down-regulation of pro-inflammatory cytokines and chemokines.
In conclusion, Taraxacum mongolicum can enhance bovine immunity to resist pathogen invasion and inhibit excessive inflammatory responses. Results of our study will be contributive to understand the Taraxacum mongolicum in the regulation of inflammatory responses. Hope it can be an alternative therapy applied in clinical medicine to reduce the incidence of bovine mastitis.

並列關鍵字

Taraxacum mongolicum ； Mammary epithelial cells ； Peripheral blood mononuclear cells ； LPS ； Pro-inflammatory cytokines ； Chemokines ； Mastitis

參考文獻

3. Katrin Schutz, Reinhold Carle, and Andreas Schieber. Taraxacum--a review on its phytochemical and pharmacological profile. J Ethnopharmacol, 2006. 107(3): p. 313-23.

8. Singh, N.K., Sharma N., and Bhadwa M.S., Relationship of Somatic Cell Count and Mastitis: An Overview. Asian-Australasian Journal of Animal Sciences, 2011. 24(3): p. 429-438.

9. Javier Oviedo-Boyso, Juan J. Valdez-Alarcon, Marcos Cajero-Juarez, Alejandra Ochoa-Zarzosa, Joel E. Lopez-Meza, Alejandro Bravo-Patino, and Victor M. Baizabal-Aguirre. Innate immune response of bovine mammary gland to pathogenic bacteria responsible for mastitis. J Infect, 2007. 54(4): p. 399-409.

12. Yu Chi, Shi Zhao-Ru, Chu Chun-Yen, Lee Kuo-Hua, Zhao Xin, and Lee Jai-Wei. Expression of bovine granulocyte chemotactic protein-2 (GCP-2) in neutrophils and a mammary epithelial cell line (MAC-T) in response to various bacterial cell wall components. Veterinary Journal, 2010. 186(1): p. 89-95.

13. Li Cong-jun, Li Robert W., Elsasser Theodore H., and Kahl Stanislaw. Lipopolysaccharide-induced early response genes in bovine peripheral blood mononuclear cells implicate GLG1/E-selectin as a key ligand-receptor interaction. Funct Integr Genomics, 2009. 9(3): p. 335-49.