高油飲食與葉酸營養狀況對免疫調節的影響

Abstract

本研究為探討高油飼料長期餵食下與較低LPS劑量致急性發炎，是否肥胖會降低急性發炎小鼠的存活率，6週齡C57BL/6J公鼠給予高油飲食22週，測定血清細胞激素與血中調節型T細胞作為觀察發炎前的免疫指標，於28週齡時腹腔注射10 mg/kg BW LPS誘發小鼠急性發炎，觀察小鼠生命期，並於小鼠死亡時測定肝臟mRNA表現量。結果顯示，肥胖增加小鼠血清IL-6的分泌，降低血中CD4+ T細胞比例，但延長急性發炎小鼠的生命期，降低肝臟中IL-1β的表現，肝臟中發炎基因表現量未與生命期有相關性，顯示肥胖加劇的發炎可能不影響LPS致急性發炎鼠的存活率。 葉酸缺乏為另一可能促發炎因子，葉酸缺乏於動物模式下是否會加劇急性發炎反應有待確認，因此本研究給予C57BL/6J公鼠葉酸缺乏飲食12週，以15 mg/kg BW LPS致急性發炎，觀察小鼠生命期並測定小鼠發炎後各時間點的體溫，取小鼠死亡時肝臟測定發炎基因表現量。結果顯示缺乏葉酸不影響C57BL/6J小鼠的存活率，且致發炎11小時後小鼠體溫與存活時數呈顯著正相關。 此外，已知國人攝食葉酸的主要食物來源為蔬菜及水果，而今國人偏向高油飲食型態下，蔬果類攝取不足時葉酸缺乏的影響為何，有待研究。為探討高油飲食的情況下，葉酸缺乏或補充對小鼠免疫細胞與脂質代謝的影響，將高油飲食小鼠分為葉酸缺乏組Hf-f0/a (添加1 %抗生素)、HF-f0、葉酸1倍組HF-f1與葉酸10倍組HF-f10，餵食20週。結果顯示HF-f0組脂肪組織有較重的趨勢，脂肪組織內F4/80+細胞浸潤比例高於HF-f10組，血清總膽固醇、HDL-C與LDL-C較少，肝臟中有較多膽固醇含量，血清中磷脂醯膽鹼含量較少，但血清與肝臟中三酸甘油酯各組間並無差異。HF-f0組脾臟細胞增生能力顯著較低，脾臟naive T細胞比例顯著高於HF-f10組。經裂殖素活化後，HF-f0組的脾臟細胞分泌較多IL-2與較少IL-6，HF-f10組脾臟細胞分泌較少IFN-γ；未受活化的狀態下，HF-f0組的脾臟細胞分泌較多IL-6，腹腔細胞亦有較高的IL-6分泌量；HF-f0組肝臟IL-6與IL-1β mRNA表現量增加，F4/80 mRNA表現量減少，血清IgA與IgG分泌量減少；HF-f10組血清IgA分泌增加。葉酸缺乏飲食添加抗生素不影響小鼠的生長，但與HF-f0組比較，添加抗生素會增加血清膽固醇、降低肝臟膽固醇含量，降低脾臟細胞IL-2與IL-6的分泌，增加LPS活化時IL-10分泌量，降低腹腔細胞分泌IL-6，亦降低肝臟IL-6與IL-1β mRNA表現量，增加IgA的分泌。 綜合上述結果，高油飲食與葉酸缺乏不影響 LPS 致急性發炎小鼠的存活率。在高油飲食狀況下，缺乏葉酸會增加肝臟膽固醇含量，降低血清膽固醇，影響膽固醇代謝，並抑制脾臟免疫細胞的分化與活化能力，同時會促進腹腔細胞分泌發炎細胞激素，降低血清IgA與IgG分泌量；葉酸補充增加白色脂肪組織重量，但可降低脂肪組織的巨噬細胞浸潤程度，降低脾臟細胞分泌 IFNγ並增加血清 IgA 的分泌。缺乏葉酸可加劇發炎，降低抗體分泌與免疫細胞活性，補充葉酸則有利於調節發炎反應與促進抗體產生。

It is controversial that obesity increase the mortality of acute inflammation. In this study, we prolonged feeding time of high-fat diet and lowered the dose of LPS to investigate whether obesity reduced the lifespan of mice in acute inflammation. C57BL/6J male mice were fed high-fat diet for 22 weeks, serum cytokines and blood T cell subtype were examined, and mice were intraperitoneally injected with a 10 mg/kg body weight (BW) LPS to induce acute inflammation, and lifespan was observed. Results indicated that obese mice had higher serum IL-6 and lower CD4+ T cells proportion, however obese mice had longer lifespan in acute inflammation, and lower hapatic IL-1β mRNA expression. Correlation between hepatic inflammatory cytokines expression and lifespan was not observed, indicating that obesity may not reduce the lifespan in acute inflammation. Furthermore, we used LPS-induced acute inflammation murine model to study if folate deficiency could shorten the lifespan. C57BL/6J male mice fed folate-deficient diet for 12 weeks were injected with 15 mg/kg BW LPS, lifespan and body temperature were observed. Results showed that folate deficiency didn’t influence the lifespan of C57BL/6J mice in acute inflammation, but there was a positive correlation between lifespan and body temperature at 11 hr after LPS-induced inflammation. Our study also investigated whether folate status may affect fat deposit and immune cells activation in high-fat diet feeding. C57BL/6J male mice were fed high-fat diet containing different content of folate: HF-f0/a (0 mg, 1% antibiotics added), HF-f0 (0 mg), HF-f1 (2 mg) and HF-f10 (20 mg) per kg diet. Mice fed for 20 weeks were sacrificed at 26 weeks of age. Blood triglyceride, total cholesterol, HDL-C, LDL-C, antibody level, and hepatic lipid concentration were analyzed. Immune cells activation was examined by spleen T lymphocyte proliferation, spleen naïve T cells population, and cytokines produced by splenocytes and primary macrophages. Hepatic inflammatory-related cytokines mRNA expression was examined by qPCR. Results showed that HF-f0 had higher adipose tissue weight and higher F4/80+ macrophage infiltration but lower serum total cholesterol, HDL-C and LDL-C concentration, serum triglyceride wasn’t observed significant differences among groups. On the contrary, HF-f0 had higher hepatic cholesterol content. In addition, spleen T lymphocyte proliferation was significantly decreased, but the percentage of spleen naïve T cells was increased in the HF-f0 group. IL-2 was increased when cells were activated, while IL-6 was decreased in splenocytes of the HF-f0 group. Primary macrophages in peritoneal fluid in the HF-f0 group also had higher IL-6 secretion, and hepatic IL-6 and IL-1β mRNA expressions were elevated, but total IgA and IgG in the serum were reduced. HF-f10 significantly increased serum IgA. On the other hand, antibiotics could not affect mice growth but increased serum cholesterol, reduced hepatic cholesterol, reduced IL-2 and IL-6 secretion of splenocytes, increased IL-10 secretion of splenocytes in LPS stimulation, also reduced hepatic IL-6 and IL-1β mRNA expressions, and elevated serum IgA production compared to the HF-f0 group. To summarize, high-fat diet and folate deficiency could not affect the lifespan of mice in LPS-induced inflammation but may block cholesterol transport and cause lipid accumulate in liver, inhibit T cell differentiation/activation, and may promote inflammatory response because of higher IL-6 production of primary macrophages and elevated hepatic IL-6 and IL-1β mRNA expressions. Folate deficiency may also depress humoral immunity by reduced IgA and IgG level. In contrast, folate supplementation could benefit immune regulation and induce antibodies production.