篩選可刺激類升糖素胜肽-1分泌之益生菌及探討其減緩高血糖症之研究

Abstract

第一型糖尿病（type 1 diabetes, T1D）是一種自體免疫疾病，由於胰臟中β細胞逐漸失去功能並死亡，導致胰島素分泌不足及高血糖症狀。類升糖素胜肽-1（glucagon-like peptide-1, GLP-1）是由腸道內泌細胞所分泌之腸泌素，可調節胰臟β細胞之生長、增加胰島素分泌量以調節血糖之恆定。因此本試驗旨在篩選可刺激腸道內泌細胞分泌GLP-1之乳酸菌株。 試驗選用六十株乳酸菌分別與上皮內泌細胞株STC-1以1:100之比例共培養，測定上清液中之GLP-1濃度，並挑選與對照組相比可顯著提高GLP-1分泌量（p< 0.05）之兩株乳酸菌進行定序，分別為Lactobacillus kefiranofaciens 與 Lactobacillus kefiri。為進一步探討此二篩選菌株於動物模型中是否具有刺激GLP-1分泌能力及調節血糖之機能性，動物試驗選用4週齡C57BL/6小鼠進行試驗，以每公斤體重40 mg之鏈脲佐菌素連續腹腔注射4天以誘導T1D，成功誘導T1D之小鼠隨機分組，分別給予磷酸緩衝液之對照組，及每天每隻小鼠給予108 CFU之篩選菌株，一共8週。結果顯示，Lb. kefiranofaciens 與 Lb. kefiri皆能顯著降低T1D小鼠之空腹血糖值，而Lb. kefiri組別經口服糖耐量測定後也顯著改善了小鼠恆定血糖能力。藉由胰臟切片之免疫組織化學染色發現給予Lb. kefiranofaciens 與 Lb. kefiri後，胰臟β細胞對胰島之面積比例顯著高於STZ對照組，同時也觀察到篩選菌株具有調節細胞激素含量及提高血清GLP-1含量之能力。然而在非肥胖糖尿病（non obese diabetes, NOD）小鼠試驗中，給予Lb. kefiri無法顯著預防T1D之發生。本試驗嘗試探討篩選菌株降血糖功效之可能機制，然而結果顯示其降血糖功效似乎與類鐸受體2（Toll-like receptor 2）之訊息傳遞及短鏈脂肪酸（short chain fatty acid）之含量並無關聯。 綜觀上述，本試驗篩選之乳酸菌株Lb. kefiranofaciens與Lb. kefiri於體外試驗及動物試驗皆能刺激GLP-1分泌，且有助於STZ誘導T1D小鼠維持血糖恆定，此二篩選菌株或許具有高度潛力可開發成為具降血糖機能性之益生菌產品，並應用於糖尿病之輔助療法之一。然而其詳細降血糖機制仍有待釐清。

Type 1 diabetes (T1D) is an autoimmune disease, which characterized by progressive dysfunction of pancreatic β cell and loss in β cell mass. The disease generally results in insulin deficiency and hyperglycemia. Glucagon-like peptide-1 (GLP-1) is a gut hormone which secreted by enteroendocrine cell. It has been well documented that GLP-1 could regulate β cell growth and increase insulin secretion to maintain glucose homeostasis. Therefore, our aim was to screen and select lactic acid bacteria (LAB) with abilities to stimulate intestinal GLP-1 secretion. The murine enteroendocrine STC-1 cells were co-cultured with sixty LABs at a ratio of 1: 100 and GLP-1 productions in supernatant were measured. We selected and identified top two strains, Lactobacillus kefiranofaciens and Lactobacillus kefiri, significantly elevating GLP-1 secretion when compared with control group (p< 0.05). We next investigated GLP-1 secretion abilities and improvement of hyperglycemia in vivo. Four-week-old male C57BL/6 mice were induced diabetes by intraperitoneal injection of streptozotocin(STZ) for 4 consecutive days. Diabetic mice were randomly assigned to different groups and were administered PBS or selected two strains 108 CFU per mouse daily for eight weeks. The results exhibited that Lb. kefiranofaciens and Lb. kefiri could significantly lower fasting blood glucose than STZ group after eight week administration. Lb. kefiri also significantly improve glucose homeostasis after oral glucose challenge when compared with the STZ group. We found that Lb. kefiranofaciens and Lb. kefiri group showed significantly higher insulin/pancreas ratio by pancreas immunohistochemistry staining. Modulation effects on cytokines level and an elevation of serum GLP-1 levels were also observed. However, administration of Lb. kefiri did not show significant effect to delay the onset of diabetes in non obese diabetic (NOD) mice. We further tried to address possible mechanism and found that toll-like receptor 2 and short chain fatty acid production might be not involved in blood glucose lowering effect. Taken together, our present study concluded that Lb. kefiranofaciens and Lb. kefiri could stimulate GLP-1 secretion both in vitro and in vivo. Oral administration of Lb. kefiranofaciens and Lb. kefiri provided a benefit to blood glucose homeostasis in STZ-induced T1D model. It suggested that the selected strains might be a potential therapeutic supplementation to T1D. However, the mechanisms need more clarification.