芹菜素对Con A诱导的免疫性肝炎模型小鼠的保护作用
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摘要
目的:研究芹菜素对T细胞和RAW264.7细胞生物学行为的影响作用,以及其对自身免疫性肝炎模型小鼠的治疗效果,为新型免疫抑制药物的开发奠定理论基础。方法:利用荧光标志抗体双染色、CFDA-SE染色、PI染色和Annexin V-FITC/PI双染色结合流式细胞术检测芹菜素对多克隆刺激剂刀豆蛋白A(ConA)诱导的T细胞活化、增殖、细胞周期和地塞米松(DEX)诱导的T细胞凋亡的影响;利用MTT法、Griess试剂盒检测法和荧光微球检测法检测芹菜素对LPS刺激的RAW264.7细胞增殖、NO分泌和吞噬功能的影响;以外周血丙氨酸氨基转移酶(ALT)和肝脏石蜡切片为指标建立稳定的自身免疫性肝炎小鼠模型,并以上2个指标摸索芹菜素对自身免疫性肝炎模型小鼠的治疗效果;利用尾静脉注射CFDA-SE染色的T细胞检测芹菜素对体内T细胞增殖的影响。结果:芹菜素(25～200μmol/L)显著抑制Con A诱导的T细胞表达CD69、CD25和CD71,同时显著性抑制T细胞的增殖,使细胞周期阻滞于G_0/G_1期,并明显抑制T细胞凋亡(P＜0.01),且呈剂量依赖关系。芹菜素(25～200μmol/L)显著抑制LPS诱导的RAW264.7细胞的增殖、NO分泌和吞噬功能(P＜0.01),且呈剂量依赖关系。一次性尾静脉注射20mg/Kg Con A,2 h ALT开始升高,8 h达到峰值,显著性高于正常范围(P＜0.01)。肝脏石蜡切片显示,随着时间的延长,肝细胞的坏死程度和炎症细胞的浸润程度逐渐严重,8 h最为显著,并与Control组具有统计学差异(P＜0.01)。腹腔注射8次50 mg/Kg芹菜素,ALT均大于正常值;腹腔注射6～8次100 mg/Kg芹菜素,ALT小于正常值,显著性小于Con A组(P＜0.01),与CsA组无统计学意义(P＞0.05);腹腔注射4～6次200 mg/Kg芹菜素,ALT小于正常值,显著性小于Con A组(P＜0.01),与CsA组无统计学意义(P＞0.05)。在各浓度芹菜素治疗组中,随着ALT的降低,肝细胞的坏死程度和炎症细胞的浸润程度相应地减轻,与CsA组无统计学意义(P＞0.05)。48 h,Control组流式图只有一个亲代峰,体内T细胞不增殖;Con A组流式图呈现4个子代峰,体内T细胞分裂4代,与Control组具有统计学意义(P＜0.01);CsA显著性抑制Con A诱导的体内T细胞增殖(P＜0.01),流式图仅呈现一个小的子代峰;100 mg/Kg和200 mg/Kg芹菜素组流式图呈现2～3个子代峰,与Con A组具有统计学意义(P＜0.01)。结论:芹菜素不但能够抑制ConA诱导的T细胞体外活化、增殖和凋亡,阻滞细胞周期于G_0/G_1期,并同时抑制LPS刺激的RAW264.7细胞的增殖、NO分泌和吞噬功能,从而暗示了芹菜素对适应性免疫应答和固有性免疫应答具有抑制作用。此外,芹菜素能够治疗Con A诱导的模型小鼠自身免疫性肝炎疾病,并同时抑制T细胞体内增殖,但效果无CsA显著。
Aim:To study the influence of Apigenin(AP) on the biological behaviour of both T cells and RAW264.7 cells,and the therapeutic efficacy of AP to autoimmune hepatitis(AIH),in order to supply theory for developing new immunosuppressive drugs.Methods:The effects of AP on the activation, proliferation and cell cycle of T cells in response to Con A were measured by two-color fluorescent antibody,CFDA-SE and PI combined with flow cytometry;the apoptosis of T cells induced by DEX was measured by Annexin V-FITC/PI combined with flow cytometry.The effects of AP on the proliferation,NO secretion and phagocytosis of RAW264.7 cells in response to LPS were measured by MTT method, Griess kit and fluorescent microbeads.To found a steady mouse model of AIH on the base on ALT and liver histological section,and to investigate the therapeutic efficacy of AP on AIH according to above indexes.The effects of AP on the proliferation ofT cells in vivo was measured by injecting T cells dyed by CFDA-SE.Rusults:AP(25～200μmol/L) inhibits the expression of CD69,CD25 and CD71 on T cells in response to Con A,and inhibits the proliferation arresting cell cycle at G_0/G_1 and apoptosis of T cells significantly in a dose-dependent manner(P＜0.01).AP(25～200μmol/L) inhibits the proliferation,NO secretion and phagocytosis of RAW264.7 cells in response to LPS significantly in a dose-dependent manner(P＜0.01).ALT starts to heighten after 2 h injecting Con A by i.v.,and reaches peak value at 8 h exceeding the normal value significantly(P＜0.01).The liver histological section shows that the apoptosis and inflammatory cells infiltration get worse in a time-dependent manner, and significant at 8h.ALT value is higher than normal value after injecting 50mg/Kg AP for 8 times,but 100mg/Kg AP for 6～8 times or 200mg/Kg AP for 4～6 times can decrease ALT value to normal value without statistical difference with CsA ground(P＜0.01).Control ground has only one parent peak at 48 h showing T cells are still in vivo,Con A ground has 4 off-spring peaks showing T cells generate 4 times in vivo. CsA inhibits T cells proliferation in vivo significantly showing only one off-spring peak.100mg/Kg and 200mg/Kg AP ground have 2～3 off-spring peaks,and have statistical difference with Con A ground(P＜0.01).Conclusion:AP not only inhibits the activation,proliferation arresting cell cycle at G_0/G_1 in response to Con A and apoptosis of T cells in response to DEX,but also inhibits the proliferation,NO secretion and phagocytosis of RAW264.7 cells in response to LPS,showing AP has the potency of inhibition of adaptive immunological system and innative immunological system.Even,AP can cute AIH and inhibit the proliferation ofT cells in vivo.
Aim:To study the influence of Apigenin(AP) on the biological behaviour of both T cells and RAW264.7 cells,and the therapeutic efficacy of AP to autoimmune hepatitis(AIH),in order to supply theory for developing new immunosuppressive drugs.Methods:The effects of AP on the activation, proliferation and cell cycle of T cells in response to Con A were measured by two-color fluorescent antibody,CFDA-SE and PI combined with flow cytometry;the apoptosis of T cells induced by DEX was measured by Annexin V-FITC/PI combined with flow cytometry.The effects of AP on the proliferation,NO secretion and phagocytosis of RAW264.7 cells in response to LPS were measured by MTT method, Griess kit and fluorescent microbeads.To found a steady mouse model of AIH on the base on ALT and liver histological section,and to investigate the therapeutic efficacy of AP on AIH according to above indexes.The effects of AP on the proliferation ofT cells in vivo was measured by injecting T cells dyed by CFDA-SE.Rusults:AP(25～200μmol/L) inhibits the expression of CD69,CD25 and CD71 on T cells in response to Con A,and inhibits the proliferation arresting cell cycle at G_0/G_1 and apoptosis of T cells significantly in a dose-dependent manner(P＜0.01).AP(25～200μmol/L) inhibits the proliferation,NO secretion and phagocytosis of RAW264.7 cells in response to LPS significantly in a dose-dependent manner(P＜0.01).ALT starts to heighten after 2 h injecting Con A by i.v.,and reaches peak value at 8 h exceeding the normal value significantly(P＜0.01).The liver histological section shows that the apoptosis and inflammatory cells infiltration get worse in a time-dependent manner, and significant at 8h.ALT value is higher than normal value after injecting 50mg/Kg AP for 8 times,but 100mg/Kg AP for 6～8 times or 200mg/Kg AP for 4～6 times can decrease ALT value to normal value without statistical difference with CsA ground(P＜0.01).Control ground has only one parent peak at 48 h showing T cells are still in vivo,Con A ground has 4 off-spring peaks showing T cells generate 4 times in vivo. CsA inhibits T cells proliferation in vivo significantly showing only one off-spring peak.100mg/Kg and 200mg/Kg AP ground have 2～3 off-spring peaks,and have statistical difference with Con A ground(P＜0.01).Conclusion:AP not only inhibits the activation,proliferation arresting cell cycle at G_0/G_1 in response to Con A and apoptosis of T cells in response to DEX,but also inhibits the proliferation,NO secretion and phagocytosis of RAW264.7 cells in response to LPS,showing AP has the potency of inhibition of adaptive immunological system and innative immunological system.Even,AP can cute AIH and inhibit the proliferation ofT cells in vivo.
引文
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[1] Hougee S, Sanders A, Faber J, et al. Decreased pro-inflammatory cytokine production by LPS-stimulated PBMC upon in vitro incubation with the flavonoids apigenin, luteolin or chrysin, due to selective elimination of monocytes/macrophages[J]. Biochem Pharmacol., 2005, 69(2):241-248.
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