全反式维甲酸对小鼠缺血性脑损伤的保护作用及调节性T细胞数量的影响

Effect of all-trans retinoic acid on cerebral ischemia injury and regulatory T cell accounts in stroke mice

目的通过检测全反式维甲酸(ATRA)处理对小鼠短暂性大脑中动脉缺血(tMCAO)后24 h的脑梗死容积以及不同时期脾脏内CD4+CD25+Foxp3+调节性T细胞(Treg)的变化,探讨ATRA是否具有通过干预Treg分化实现对小鼠缺血性脑损伤的保护作用。方法 60只昆明小鼠随机分为预处理组(n=40)和后处理组(n=20)。每组再分为tMCAO联合ATRA处理组,tMCAO联合DMSO对照组。预处理组在小鼠tMCAO前1周开始每天腹腔注射含100 mL/L DMSO的ATRA(10 mg/kg,1次/d)或100 mL/L DMSO(n=20/组)。7 d后处死一部分小鼠行流式细胞术(FCM)测定Treg在脾细胞中的百分率(n=10/组),另一部分小鼠通过腔内线栓法建立tMCAO模型(n=10/组),24 h后进行神经行为学评分(NDS),其后行氯化三苯基四氮唑(TTC)染色观察脑梗死容积。后处理组在小鼠tMCAO模型建立后,即刻腹腔注射ATRA(10 mg/kg)或相等体积的100 mL/L DMSO(n=10/组),24 h后行NDS,测定脑梗死容积和Treg在脾细胞内的百分率。结果 ATRA预处理7 d未能改善小鼠tMCAO后24 h的神经功能障碍(P>0.05),未能降低的脑梗死容积(P>0.05)。ATRA后处理可以明显改善小鼠tMCAO后24 h的神经功能障碍(P<0.05),显著降低脑梗死容积(P<0.05)。然而,ATRA预处理和后处理对小鼠脾脏内的Treg均无影响(P>0.05)。结论在小鼠tMCAO前给予ATRA处理7 d对脑缺血损伤无保护作用,在小鼠tMCAO后及时给予ATRA治疗后24 h有显著的保护作用,但与调节Treg分化无明显关系。

Objective To investigate the cerebral infarct volume 24 hours after transient middle cerebral artery occlusion （tMCAO） and the proportion of CD4＋ CD25＋ Foxp3＋ regulatory T cells （Tregs） in splenocytes in diverse periods after all-trans retinoic acid （ATRA） treatment in mice, so as to explore whether ATRA have the protection against cerebral ischemia damage in mice through intervening Treg differentiation. Methods Sixty male Kunming mice were randomly divided into two groups, i.e. pretreatment （ n =40） and post-treatment （ n =20） groups. Each group was against divided into two subgroups, i.e. tMCAO combined with ATRA treatment group, tMCAO combined with DMSO control group. Pretreatment groups： mice were treated intraperitoneally with ATRA （10 mg/kg） dissolved in 100 mL/L DMSO or equivalent volume of 100 mL/L DMSO daily for 7 days （n=20/group）. Ten mice in each group were sacrificed and the proportion of Tregs in splenocytes was analyzed by flow cytometry （FCM） after 7-day pretreatment. The other 10 mice in each group were subjected to tMCAO by modified monofilament method. Neurologic deficit score （NDS） was recorded and the infarct volume was assessed by 2, 3, 5- triphenyltetrazolium chloride （TTC） staining 24 hours after tMCAO. The mice in post-treatment groups were treated intraperitoneally with ATRA （10 mg/kg） or equivalent volume of 100 mL/L DMSO immediately after the reperfusion of tMCAO modeling （ n = 10/group）. NDS and infarct volume were assessed and the proportion of Tregs in splenocytes was analyzed 24 hours after tMCAO. Results ATRA pretreatment for 7 days failed to improve neurologic function deficit （ P 〉 0.05） and to reduce the cerebral infarct volume （P〉0.05） 24 hours after tMCAO in mice. ATRA post-treatment could markedly improve neurologic function （ P 〈 0.05） and reduce the cerebral infarct volume （ P 〈 0.05） 24 hours after tMCAO. However, neither ATRA pretreatment nor post-treatment had effect on the proportion of Trags in the splenocytes of mice （P 〉 0. 05）. Conclusion ATRA administered before tMCAO for 7 days failed to protect brain against ischemic damage. ATRA administered immediately following tMCAO induced cerebral protective effect 24 hours after tMCAO. The results suggest that Tregs change is not involved in the neuroprotection mechanism of ATRA.