探討種子胜肽Lunasin對於肥胖引起的發炎模式之免疫調節作用

Abstract

肥胖伴隨著脂肪組織中數種免疫細胞的浸潤及促發炎細胞激素生成，形成低度慢性發炎環境，與許多慢性疾病的發生相關，更是癌症發生的危險因子之ㄧ。Lunasin為一天然種子胜肽已被證實具有抗腫瘤、抗發炎、抗氧化、降低膽固醇及免疫調節等功用，但對於肥胖相關的發炎反應與其可能的作用機制仍不甚清楚。因此，本研究欲探討lunasin是否具有降低肥胖相關的發炎介質生成，並調節其免疫作用。實驗分為兩部分，實驗主題I：探討lunasin對肥胖相關發炎之免疫細胞的調節。此部分建立三個體外肥胖模式，模式一，以瘦體素 (leptin)刺激RAW264.7細胞之發炎模式；模式二，使用脂肪細胞條件培養液(Adipocyte-conditioned medium, Ad-CM)培養RAW264.7細胞；模式三，RAW264.7巨噬細胞與3T3成熟脂肪細胞共同培養環境，分別同時處理Lunasin。結果發現在LPS+leptin以及LPS+Ad-CM組別，lunasin處理分別顯著抑制促發炎細胞激素IL-6、TNF-α及IL-1生成。在動物實驗部分，為探討lunasin對肥胖小鼠免疫細胞分泌的發炎介質之影響，使用C57BL/6J Narl母鼠，分別餵食低脂 (LF)及高脂 (HF)飲食，收集初代免疫細胞同時加入lunasin處理，發現在LPS刺激之HF組腹腔巨噬細胞，lunasin顯著降低IL-6分泌；而脾臟細胞以PHA刺激培養並加入lunasin的處理，其HF組中的IL-4分泌，顯著降低。進一步探討使用lunasin 低劑量 (HF-LL)與高劑量 (HF-LH) 腹腔注射，對於肥胖小鼠免疫細胞的發炎介質的影響。實驗主題II：探討飲食誘導的肥胖對小鼠乳癌生成之影響。給予飲食誘發肥胖小鼠化學致癌劑DMBA (7,12-Dimethylbenz[a]anthracene)以誘發乳腺癌生成，結果發現在高脂飲食組，給予DMBA腫瘤發生率為20% (n=1/5)及而低脂組為14.3% (n=1/7)。由以上結果可知，在體內與體外實驗中，lunasin處理具有降低肥胖相關的促發炎細胞激素的生成。Obese progression accompanies chronic inflammation mainly due to infiltrate multiple immune cells in adipose tissue and then produce pro-inflammatory cytokines. This low-grade chronic inflammatorymicro-environment is associated with many chronic diseases, such as type II diabetes mellitus and cancer. Lunasin, is a natural seed peptide, has been shown many bioactivities such as anti-tumoranti-inflammatory, antioxidant, reduce cholesterol, and immune regulation properties. However, there is still a little information described the mechanisms of lunasin acts on obesity-related inflammation, even obesity-related tumor development. Therefore, the present study is to investigate whether lunasin reduced obesity-related inflammatory mediator, and affect immune function. There are two parts of experiments. In the Experiment Ι, to investigate the effects of lunasin on RAW264.7 macrophages in obese physiological model, the RAW264.7 cells were cultured in mediacontaining leptin, adipocye conditioned medium (Ad -CM), or co-culture of adipocyte and macrophage in transwell system, and then treated with lunasin plus LPS stimulation, respectively. The results were showed that the secretions of MCP-1 and IL-1β were decreased by lunasin treatment in leptin and Ad-CM conditions plus LPS stimulation at the same time. Subsequently, in vivo studies were carried on to investigate the effect of lunasin regulate immune cells ofmice fed the experimental diets. The C57BL/ 6J Narl female mice fed the experiment diets containing low-fat (LF) and high-fat (HF) for 10 weeks. The results were showed that the secretion of IL-6 in peritoneal cells stimulated by LPS of HF group was decreased by lunasin treatment. To confirm the effect of lunasin, intraperitoneal injection (IP) of lunasin, at 20 mg/kg and 4 mg/kg body weight was used in dietary induced obese mice (HF) and control mice (LF). In Experiment II, to investigate the effects of breast tumorigenesis in obese mice, a model of oral gavage chemical carcinogen 7,12-Dimethylbenz [a] anthracene (DMBA) induced breast cancer in C57BL/ 6J Narl female mice fed the experiment diet LF and HF for 10 weeks was built. The mice were divided into a LF, HF, LF+DMBA (LFD) and HF+DMBA (HFD), weekly monitoring tumorigenesis situation. The results indicated that incidence of breast tumors is about 20% in HFD group and 14.3% in LFD group. In summary, the results indicated that lunasin reduced proinflammatory cytokine productions in both in vitro and in vivo models, suggesting lunasin executes potentially anti-inflammatory actions in the obesity-related conditions. More studies are needed to explore the mechanisms of lunasin’s action in the obesity related inflammation and tumorigenesis.