丁酸調控脂肪幹細胞之免疫抑制能力與機轉探討

Functional and Mechanistic Studies of Butyrate Effects on Immunosuppression of Adipose-derived Stem Cells

吳佳蓁(Chia-Chen Wu)

指導教授 ： 徐莞曾

國立臺灣大學/醫學院/藥學研究所/碩士(2020年)

https://doi.org/10.6342/NTU202000151

摘要

研究背景
代謝症候群是指一群容易導致心血管疾病的危險因子聚集，包含腹部肥胖、高血糖、高血壓、高三酸甘油脂及高密度脂蛋白膽固醇偏低。其中，肥胖會誘導促發炎因子產生進而改變脂肪組織的結構和組成，包括脂肪細胞、免疫細胞和脂肪幹細胞 (adipose-derived stem cells, ASC)。ASC已被證實具有調控免疫系統的潛力，但取決於生理環境的條件，在發炎環境下ASC將失去調控功能。短鏈脂肪酸為腸道菌發酵代謝後的主要產物，包含乙酸、丙酸、丁酸和戊酸。這些短鏈脂肪酸除了可以提供腸道能量外，也具有調控免疫系統的作用。許多文獻指出丁酸抑制發炎效果最為顯著，並在脂肪組織中表現出多種調節功能。本論文假設丁酸具有調控免疫系統的能力且可以增加ASC免疫抑制的功能，進而恢復因代謝疾病而失去調控功能的ASC。
實驗方法與結果
利用3H-thymidine incorporation assay測定脾臟細胞經抗體CD3/28刺激之增生反應，結果顯示丁酸增強ASC體外抑制T細胞增生的能力。為了證實丁酸在動物模式具有同樣的作用，本論文將BALB/c小鼠隨機分成3組，分別給予正常飲用水、含100 mM丁酸及200 mM丁酸的飲用水。第21天後，犧牲取各組之ASC分別命名為ASCCtl、ASCBA100mM和ASCBA200mM進行測試，計算免疫抑制百分比；結果顯示ASCBA200Mm具有更強的免疫抑制能力 (ASCBA200mM = 54% vs. ASCCtl = 25%, P < 0.001)。機轉探討方面，以即時定量聚合酶連鎖反應、西方點墨法及酵素沉澱法測定一氧化氮合成酶 (inducible nitric oxide synthase, iNOS) 和雙調蛋白 (amphiregulin, Areg) 基因與蛋白質表現量。綜合實驗結果顯示，有T細胞增生的發炎環境下，ASCCtl加丁酸或ASCBA200mM的組別與ASCCtl相比，iNOS和Areg基因與蛋白質表現量都顯著上升。此外，利用組蛋白乙醯化程度來探討丁酸調控ASC之分生機制。結果顯示ASCCtl加丁酸或ASCBA200mM的組別與ASCCtl相比，組蛋白乙醯化表現量顯著上升，並且能影響組蛋白去乙醯酶之基因。
為了進一步探討丁酸在疾病模式小鼠身上的療效，本論文將可代表人類代謝症候群疾病的 Tsumura, Suzuki, Obese Diabetes (TSOD) 小鼠隨機分成2組：植入丁酸錠劑及無錠劑植入，另以同品系之Tsumura, Suzuki, Non Obese小鼠做為控制組。植入錠劑第42天後分離各組的ASC。3H-thymidine incorporation assay測定顯示，代謝疾病會降低ASC免疫抑制能力，植入丁酸錠劑則恢復TSOD小鼠ASC的免疫抑制能力。
結論
本論文實驗結果顯示丁酸體外加入或是於動物體內投予，增強ASC體外的免疫抑制能力並恢復代謝疾病中ASC免疫抑制的效果，可能是藉由丁酸增加ASC之iNOS及Areg表現。此外，丁酸增加ASC之組蛋白乙醯化的表現，並影響蛋白去乙醯酶的基因變化。在未來發展上建議可利用丁酸增強ASC免疫能力的方式來改善肥胖之脂肪發炎環境。

關鍵字

脂肪幹細胞 ； 丁酸 ； 免疫調控 ； 一氧化氮合成酶 ； 雙調蛋白 ； 組蛋白去乙醯酶

並列摘要

Background
Metabolic syndrome (MetS) is a cluster of cardiometabolic risk factors, including visceral obesity, hypertension, insulin resistance, and dyslipidemia. Among them, visceral obesity is the most prevalent form of the MetS. Moreover, obesity ensues a pro-inflammatory environment which changes the structure and composition of the adipose tissue, including adipose-derived stem cells (ASC). ASC have been recognized to exhibit extensive immunomodulatory properties; however, ASC can be beneficial or detrimental to health depending on the environment conditions. Short-chain fatty acids (SCFA) are derived from the microbial fermentation of dietary fibers in the colon. Among the SCFA, butyrate has received remarkable attention for its multiple beneficial effects ranging from the intestinal tract to the peripheral tissues. In addition, butyrate exerts potent anti-inflammatory effects and exhibits multiple regulatory function in the adipose tissue. Thus, we aim to study whether butyrate enhances ASC’s immunosuppressive potency.
Method and Result
For immunosuppressive assay of ASC, the in vitro proliferation study of anti-CD3/28-activated splenocytes, was evaluated by 3H-thymidine incorporation assay. We have demonstrated that butyrate enhanced the immunosuppressive potency of ASC. Next, we investigated whether butyrate could exert the same effects on ASC when it is administered orally to mice. BALB/c mice were divided into three groups randomly: the first group of mice were provided with regular drinking water; the second and the third groups were provided with 100 mM or 200 mM sodium butyrate in drinking water for 21 days, respectively. At the end of the treatment protocol, mice were sacrificed and ASC were isolated from mice in each group, designated as ASCCtl, ASCBA100mM, and ASCBA200mM. In immunosuppressive assay, the results were reported as percent suppression and showed that oral butyrate supplementation significantly enhanced the ex vivo suppressive potency of ASC (ASCBA200mM = 54% vs. ASCCtl = 25%, P < 0.001). Furthermore, western blot and quantitative PCR analysis revealed both ASCCtl with butyrate and ASCBA200mM had a higher gene level of inducible nitric oxide synthase (iNOS) and amphiregulin (Areg) than ASCCtl in inflammatory milieu. Besides, butyrate treatment induced higher level of acetyl histone than ASCCtl and affected different isoforms of histone deacetylase (HDAC) on ASC. To further evaluated the treatment of butyrate in MetS, we used butyrate pellet to treat Tsumura, Suzuki, Obese Diabetes (TSOD) mice for 42 days. The immunosuppressive assay data showed ASC of TSOD had lower suppressive potency than healthy mice. However, the butyrate treatment repaired immunosuppressive function of ASC.
Conclusion
Our findings suggested that butyrate treatment beneficially affected ASC in animal models, partly through enhanced immunosuppressive potency accompanied by up-regulated iNOS and Areg expression. Moreover, our data showed that butyrate affected different types of HDAC on ASC. This work provides underlying information that enhancement of ASC’s immunosuppression by butyrate is a useful tool to improve inflammation of adipose tissue in obesity.

並列關鍵字

adipose-derived stem cells ； butyrate ； immunomodulation ； iNOS ； Areg ； HDAC

參考文獻

1. Apovian C.M., Bigornia S., Mott M., et al., Adipose macrophage infiltration is associated with insulin resistance and vascular endothelial dysfunction in obese subjects. Arterioscler Thromb Vasc Biol, 2008. 28: p. 1654.

2. Shulman G.I., Ectopic fat in insulin resistance, dyslipidemia, and cardiometabolic disease. N Engl J Med, 2014. 371: p. 1131.

3. Patel P. and Abate N., Body fat distribution and insulin resistance. Nutrients, 2013. 5: p. 2019.

4. Friedenstein A.J., Chailakhjan R.K., and Lalykina K.S., The development of fibroblast colonies in monolayer cultures of guinea-pig bone marrow and spleen cells. Cell Tissue Kinet, 1970. 3: p. 393.

5. Blau H.M., Brazelton T.R., and Weimann J.M., The evolving concept of a stem cell: entity or function? Cell, 2001. 105: p. 829.