姜黄素对全脑缺血再灌损伤后大鼠海马区神经元细胞凋亡的影响

The effect of curcumin against cerebral ischemia-induced neuronal apoptosis of SD rats

目的观察姜黄素对大鼠脑缺血再灌注（I／R）后海马神经元凋亡的影响，探讨该药抗神经元凋亡作用的分子机制。方法制作sD大鼠全脑缺血15min再灌注损伤模型。实验分为假手术组（Sham组）、缺血组（I／R组）、姜黄索预处理组；姜黄素预处理组分为30、100、300mg／kg3剂量组（Cur30、Cur100、Cur300），均在缺血前1h腹腔给药。上述实验组按再灌注2h、6h、1d、3d4个时间点再分为4个亚组，每组6只大鼠。分别制备大鼠大脑石蜡标本和活组织冰冻标本。免疫组织化学法检测海马CA1区凋亡相关蛋白B淋巴细胞／白血病-2（bcl-2）、bax及磷酸化c—Jun氨基末端激酶（p-JNK）的表达，原位末端转移酶标记（TUNEL）法检测海马凋亡细胞数。结果与Sham组比较，I／R组及姜黄素预处理组海马CAIbcl＋2和bax均在缺血再灌注后2h表达开始增加，ld达到高峰后开始回落，至3d后基本正常。Cur30组、Curl00组、Cur300组海马CAlbcl-2在再灌注后6h、1d、3d3时点表达水平均明显高于I／R组（P〈0．05），且在1、3d表达水平Curl00组〉Cur30组〉Cur300组（P〈0．05）。I／R组再灌注1、3dbax表达水平最高，其次为Cur300组、Cur30组、Curl00组表达水平最低，Sham组弱表达，各组间两时间点差异均有统计学意义（P〈0．05）。I／R组及姜黄素预处理组海马CAlp-JNK在再灌注2h表达开始增加，1d达峰后下降，至3d恢复；I／R组再灌注6h、1d、3d表达水平明显高于其他各组（P〈0．05），且再灌注6h、1d、3d的表达水平Cur300组〉Cur30组〉Curl00组〉Sham组（P〈0．05）。TUNEL检测发现各缺血组凋亡阳性细胞主要集中在CAl区，再灌注2h有少量出现，后逐渐增多至3d达到高峰后明显减少。Curl00组再灌注6h、1d、3d各点凋亡阳性细胞数明显少于其余三缺血组，且Cur30组〈Cur300组〈I／R组（P〈0．05）。结论姜黄素可显著减少SD大鼠缺血性海马神经元再灌注后凋亡的发生，其机制与减少海马p-JNK及bax表达、增强bcl-2表达有关．且100mz．／kg剂量组姜黄素的保护效果显著优于30m#kg及300mg／kg。

Objective To investigate the effect and molecular mechanisms of curcumin against ischemia reperfusion injury in hippocampus of SD rats, and to provide the experimental and theoretical basis of cureumin in clinical work. Methods One hundred and twenty male SD rats were randomly divided into five groups ： sham group （ sham ） , ischemia-reperfusion group （ L/R）, curcumin group with dosage of 30 mg/kg （Cur30） , 100 mg/kg （Curl00） and 300 mg/kg （Cur300）. Isasteric curcumin solutions or men- struum were intraperitoneally injected at 1st b before ischemia. Each rat was ligated for 15 min and reper- fused except sham group. Rats were killed at 2 h, 6 h, 1 day, and 3 day after reperfusion in each group. Immunohistochemistry was used to observe the expression of B lymphocytes/leukemia-2 （bcl-2） and bax, and the TdT-mediated dUTP nick end labeling （TUNEL） method was used to count the apoptotic neurons. Results As compared with sham group, the expression of bcl-2 and bax in CA1 regions began to increase at 2 h after reperfusion, and reached the peak at 1 days, then decreased, and the expression level de- creased to normal level at 3 day. The expression level of bcl-2 in CA1 regions was higher in Cur30, Curl00 and Cur300 groups at 6 h, 1 day and 3 day after reperfusion than in L/R group （ all P 〈 0. 05 ）. The inten- sity of the expression was Curl00 〉 Cur30 〉 Cur300 at 1 day and 3 day after reperfusion （P 〈0. 05）. The expression of bax was higher in I/R group than in Cur300, Cur30 and Curl00 groups （P 〈 0. 05 ） , and that of bax was lower in Curl00 group at 1 day and 3 day after repertusion （P 〈 0.05 ）. There was no expres- sion of bax in sham group. At 3 day after repeffusion, there was no significance among I/l：l, Curl00, Cur30 and Cm＇300 groups （P 〉0. 05）. In I/R, Cur30, Curl00 and Cur300 groups, the expression of p- JNK in CA1 and CA3 regions began to increase at 2 h after repeffusion, then reached peak at 1 day and de- creased, and the expression level was decreased to the normal level at 7 （lay. At 6 h, 1 day and 3 day after reperfusinn, the expression of p-JNK was higher in I/R group than in Cur300, Cur30, Curl00 and sham groups （ P 〈 0. 05 ） , and the intensity of expression level was Cur300 〉 Cur30 〉 Curl00 〉 sham （P 〈 0. 05 ）. The apoptotic neurons presented mostly in CA1 region in each group. There were only a few apop- tntie neurons at 2 h, the number of apoptntic neurons reached peak at 3 day, and then decreased. The number of apoptotic neurons was less at 6 h, 1 clay, and 3 day in Curl00 group than in I/R, Cur300 and Cur30 groups （ P 〈 0. 05 ）. Conclusion Cureumin can significantly decrease the number of apoptotic neu- rons in hippocampus after ischemia reperiusion in SD rats probably by decreasing the expression of p-JNK and bax, and increasing the expression of bel-2. The effeet of 100 mg/kg eurcumin is better than that of 30 and 300 mg/kg.