- 學位論文
檜木醇對脂多醣體誘發3T3-L1脂肪細胞發炎反應及胰島素作用受損的抑制功效
The inhibitory effects of hinokitiol on lipopolysaccharide-induced inflammation and impaired insulin action in 3T3-L1 adipocytes
摘要
肥胖是全球性的健康問題，預防肥胖，已成為重要身體保健議題。脂肪組織分泌促發炎細胞激素，包括： interleukin-6（IL-6）、monocyte chemotactic protein-1（MCP-1）和tumor necrosis factor-α（TNF-α）等，可造成胰島素阻抗（insulin resistance），增加糖尿病、心血管疾病、高血壓和癌症等疾病的發生率。檜木醇（Hinokitiol, Hin）為檜木分離的一種天然酚酮化合物，具有抗菌、抗腫瘤和抗發炎等生理功效。Hin是否能改善脂肪細胞發炎和增加胰島素作用及其相關機制尚未清楚，因此本研究利用脂多醣體（lipopolysaccharides, LPS）誘發3T3-L1脂肪細胞發炎反應的研究模式，評估Hin改善脂肪細胞發炎反應和促進胰島素作用的功效。3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay結果顯示脂肪細胞預處理Hin（50、100 µM）24小時後，再處理LPS（1 µg/mL）並不會造成細胞毒性。預處理Hin可降低LPS誘發的IL-6、MCP-1和TNF-α mRNA表現。此外，預處理Hin可減少LPS誘發的Toll-like receptor-4（TLR-4）和meliod differentitation primary response protein 88蛋白質表現，及此兩種蛋白質間的交互作用。預處理Hin亦可抑制LPS誘發的p38和IκB kinase β磷酸化表現、細胞核內p65表現量及轉錄因子Nuclear Factor Kappa B（NF-κB）轉錄活性。利用持續活化型IKK-2 plasmid暫時轉染入脂肪細胞，使NF-κB持續活化，造成預處理Hin不會抑制LPS誘發的NF-κB轉錄活性時發現，Hin抑制LPS誘發的MCP-1 mRNA和蛋白質表現的功效喪失。脂肪細胞給予LPS顯著抑制胰島素誘發磷酸化Protein Kinase B（Akt）蛋白質表現，而預處理Hin可以減輕LPS抑制胰島素誘發磷酸化Akt蛋白質表現，有改善胰島素阻抗的功效。綜合以上結果顯示，Hin在脂肪細胞中，能藉由調控TLR-4/NF-κB的訊息路徑，改善LPS誘發的發炎反應及胰島素阻抗。
並列摘要
Obesity is a global health problem, prevention of obesity, it has become an important health care issues. Pro-inflammatory cytokines including interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1), and tumor necrosis factor-α (TNF-α) secreted by adipose tissue are involved in the development of insulin resistance and its related disease such as diabetes, cardiovascular disease, hypertension and cancer. Hinokitiol (Hin) is a phytochemical isolated from ChamacyparisTaiwanensis, and shows anti-bacterial, anti-tumor, and anti-inflammatory activities. The aim of current study is to investigate the effect of Hin on inflammation and insulin action in 3T3-L1 adipocytes treated with lipopolysaccharide (LPS). Pretreated with Hin (50, 100 μM) for 24 hr and then treated with LPS (1 μg/mL) has no cytotoxic effect on 3T3-L1 adipocytes determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Hin significantly attenuated the LPS-induced mRNA expression of pro-inflammatory mediators, including IL-6, MCP-1, and TNF-α. Additionally, Hin reduced protein expression and interaction of Toll-like receptor-4 (TLR-4) and melioddifferentitation primary response protein 88 in LPS treated 3T3-L1 adipocytes. Pretreaded with Hin inhibited LPS induced phosphorylation of p38 and κB kinase β as well as NF-κB transcriptional activity in 3T3-L1 adipocytes. The Hin did not reverse LPS-induced MCP-1 mRNA and protein expression in 3T3-L1 adipocytes transfected with a constitutively active mutant IκB kinase-β plasmid, which suggests the importance of the inhibition of NF-κB activation by the Hin. Compared with insulin alone treatment, LPS plusinsulin treatment decreased phosphorylated AKT expression, whereas addition of the Hinokitiol reversed the effect of LPS. In summary, Hinokitiolattenuates LPS-induced inflammation and impairment of insulin action through modulating TLR-4/NF-κB pathway in 3T3-L1 adipocytes.