Gamma-aminobutyric acid對VHL基因缺失小鼠腸道菌相及腸道免疫之影響

Abstract

台灣末期腎臟疾病盛行率為全球第一，近年研究指出腎臟病與腸道免疫失衡及腸道菌的產物有關。已知GABA為抑制型神經傳遞物質，具降發炎和調節免疫潛力，但對腸道的影響尚不明確。故本研究欲探討GABA對腎與腸道免疫和腸道菌的影響，以自行繁殖的腎臟Vhlh基因缺失 (Vhlhdel/del) 小鼠會產生自發性腎炎的特性，探討添加GABA是否會影響腎損傷小鼠的腸道菌組成及其腸道免疫。研究分兩部分，實驗一以馬兜鈴酸 (AA) 致Vhlhdel/+小鼠急性腎損傷。將11~13週齡小鼠分別以AIN-93 (Con組、AA組) 或含GABA米 (Gr-AA)、GABA純物質 (G-AA) 的AIN-93飼料餵飼2週後，將AA (4 mg/kg BW/day) 混合於各AA組小鼠飼料，餵飼2週後犧牲。結果顯示GABA可顯著延緩尿蛋白產生，降低血清肌酸酐與尿素氮。Gr-AA組盲腸乳酸菌屬增加，而G-AA組提升盲腸比菲德氏菌屬，且皮耶氏體 (Peyer’s patches, PP) 內調節性T細胞 (Treg) 數目顯著最高。Gr-AA和G-AA組PP細胞在ConA刺激下IL-10的分泌增加。顯示攝取GABA能增加急性腎損傷Vhlhdel/+小鼠腸道有益菌，並促進調節性細胞激素分泌保護腸道。實驗二以自發性腎炎致腎癌的Vhlhdel/del小鼠作為慢性腎臟病的模式，將離乳後5週齡Vhlhdel/del小鼠，分別餵飼AIN-93 (Con組) 或含GABA米 (Gr組)、GABA純物質 (G組) 的AIN-93飼料，10週後犧牲。結果顯示GABA可顯著降低尿液中KIM-1含量，Gr組腸繫膜淋巴結 (MLN) 細胞在ConA刺激下IL-10的分泌增加而TNF-α減少。G組盲腸的乳酸菌屬趨勢提升，PP與MLN內Treg比率下降，且腸道固有層內CD103+樹突細胞也降低，同時，ConA刺激下，PP細胞分泌的IL-10、TGF-β以及MLN細胞分泌的TNF-α、IL-6、IL-10皆降低，顯示GABA米誘使腸道調節性免疫反應，GABA純物質則抑制腸道調節性免疫反應。綜合以上結果，攝食GABA米或GABA純物質後，可能會影響腸道菌相以及腸道免疫反應，是否與延緩腎臟疾病的進程有關，值得進一步探討。

The prevalence of end-stage renal disease in Taiwan is the highest worldwide. Studies have demonstrated that kidney disease is related to imbalance of intestinal immunity and gut microbiota products. GABA is an inhibitory neurotransmitter and suggested great potential for immune-modulatory and anti-inflammatory agents. However, the effects of GABA on intestine is not clear. VHL conditional knockout mice (Vhlhdel/del) in C57BL/6J background spontaneously developed renal inflammation have been studied for renoprotective effects of GABA in our Lab. Therefore, this study is to investigate whether GABA could affect commensal microbiota and intestinal immunity in murine renal injury. Firstly, Vhlhdel/+ mice fed with aristolochic acid (AA) to induce acute kidney injury. Eleven to thirteen-week-old Vhlhdel/+ male mice were fed with AIN-93 diet (Con and AA groups), or GABA containing diets (Gr-AA and G-AA groups). After two weeks, AA (4 mg/kg BW/day) was added in three AA groups for the other two weeks. The results showed that GABA significantly decreased urine protein, serum creatinine and BUN levels. The Gr-AA group increased cecal Lactobacillus. The G-AA group increased the population of cecal Bifidobacterium, and had the highest cell numbers of regulatory T (Treg) cells in Peyer’s patch (PP). Both Gr-AA and G-AA groups increased IL-10 secretion in ConA-stimulated PP cells. These results implied that GABA supplements increased gut beneficial symbionts and regulatory cytokine secretion in AA-induced Vhlhdel/+ AKI mice. Secondly, Vhlhdel/del mice which spontaneously develope renal inflammation and renal carcinoma were used as chronic kidney disease model. Five-week-old weaning Vhlhdel/del mice fed with AIN-93 diet (Con group), or GABA containing diet (Gr and G groups) for ten weeks, and then sacrificed. The results showed that GABA significantly reduced the level of Kim-1 in urine. The Gr group increased the IL-10 secretion in ConA-stimulated mesenteric lymph nodes (MLN) cells, but TNF-α secretion decreased. The G group increased cecal Lactobacillus, decreased the population of Treg cells in both PP and MLN, and the population of CD103+ dendritic cells in lamina propria. Meanwhile, cytokines IL-10 and TGF-β produced by ConA-stimulated PP cells and TNF-α, IL-6 and IL-10 produced by ConA-stimulated MLN cells were decreased. These results indicated that GABA rice promotes regulatory immune response, but GABA pure compound inhibits regulatory immune response in gut. In conclusion, intake of GABA rice or GABA pure compound could affect the intestinal microbiota and intestinal immune responses which might be beneficial for delaying the progress of renal disease in VHL knockout mice.