Analysis of T cell apoptosis induced by volatile fatty acids

(1)牙周病细菌的代谢副产物--挥发性脂肪酸，尤其是丁酸，可诱导小鼠胸腺细胞、脾T细胞、人外周血单核细胞和Jurkat细胞的凋亡。细胞对丁酸的敏感性依次为胸腺细胞、Jurkat细胞、脾T细胞、PBMC。(2)Western blotting检测参与DNA损伤反应和诱导细胞凋亡的bcl2、bax、Fas和FasL的表达，发现丁酸处理的细胞bcl2表达减少，磷酸化的bcl2消失。BAX、Fas和Fas-L表达无明显变化。流式细胞仪分析显示丁酸可抑制bcl2细胞的比例，但不抑制Fas细胞的比例。(3)RT-PCR和Western blotting分析表明丁酸诱导的T细胞凋亡中P53的表达没有增加。DNA片段化分析表明，含有p53抑癌基因胚系缺陷小鼠的T细胞对丁酸诱导的T细胞凋亡的敏感性与野生型T细胞相同。(4)流式细胞仪Annexin V结合实验表明，丁酸可增加早期和晚期凋亡细胞的数量。(5)丁酸诱导的T细胞凋亡伴随着Caspase 3和Caspase 6的活性，而不是Caspase 1和Caspase 8的活性。

(1) Volatile fatty acids, especially butyric acid, metabolic by-products of periodontopathic bacteria, induced apoptosis in murine thymocytes, splenic T cells, human peripheral blood mononuclear cells (PBMC), and Jurkat cells. The apoptosis sensitivity of these cells to butyric acid was thymocytes > Jurkat cells> splenic T cells> PBMC.(2) Western blotting analysis which was used to study the expression of bcl-2, Bax, Fas and FasL, involved in the response to DNA damage and the induction of apoptosis, indicated that expression of bcl-2 was reduced and phosphorylated bcl-2 was also disappeared in butyric acid-treated cells. No changes in Bax, Fas and Fas-L was demonstrated. FACS analysis revealed that butyric acid suppressed the ratio of bcl-2^+ cells but not of Fas^+ cells.(3) RT-PCR and Western blotting analysis indicated that there was no increase in p53 expression in butyric acid-induced T cell apoptosis. The DNA fragmentation assay indicated that T cell blasts derived from mice containing a germ-line deficiency of the p53 tumor suppressor gene were susceptible to butyric acid-induced apoptosis to the same degree as wild-type T cells.(4) Annexin V binding experiments in flow cytometly revealed that butyric acid increased the number of early- and late-stage apoptotic cells.(5) Butyric acid-induced T cell apoptosis was accompanied by Caspase 3 and Caspase 6 protease activities but not by Caspase 1 and Caspase 8 protease activities.