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目的观察姜黄素(curcumin,Cur)对慢性坐骨神经结扎损伤大鼠脊髓背角神经元中程序细胞死亡因子5(programmed cell death 5,PDCD5)表达的影响。方法建立慢性坐骨神经结扎损伤(chronic construction injury,CCI)模型;健康雄性SD大鼠84只,随机分为6组:空白对照组(n=4)、假手术组(Sham组)(n=16)、神经结扎组(CCI组)(n=16)和不同浓度姜黄素组[Cur 50 mg/kg组(n=16)、Cur 100 mg/kg组(n=16)及Cur 200 mg/kg组(n=16)]。Cur组行坐骨神经结扎,并腹腔注射姜黄素50、100、200 mg/(kg·d),持续至术后21 d。Sham组,CCI组和Cur组按取材时间不同分为4个亚组(n=4),即术后3 d亚组、术后7 d亚组、术后14 d亚组和术后21 d亚组。分别于术前2 d及3 d、7 d、14 d和21 d测定行为学;采用末端生脱氧核糖核苷酸转移酶介导生物素标记(terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling,TUNEL)标记法标记脊髓背角浅层凋亡细胞;免疫印迹法(Western blot,WB)法检测PDCD5蛋白的表达。结果空白组与Sham组相比,MTW和TWL差异无统计学意义(P>0.05)。CCI组术后MTW和TWL逐渐降低,在第14 d时降至最低值。给予Cur 100 mg/(kg·d)处理后,MTW和TWL逐渐增加,与CCI组在3 d、7 d、14 d及21 d比较,差异有统计学意义(P<0.05)。空白组与Sham组相比,差异无统计学意义(P>0.05)。CCI组在术后3 d即出现凋亡细胞增多,细胞凋亡高峰期出现在术后7~14 d,与Sham组相比,差异有统计学意义(P<0.05);给予不同浓度Cur处理后,凋亡细胞在术后3 d开始减少,Cur 100 mg/(kg·d)组与其他Cur组相比,差异有统计学意义(P<0.05);另可见Cur 100 mg/(kg·d)组在术后7 d、14 d及21 d,与其他Cur组、CCI组相比,差异有统计学意义(P<0.05)。空白对照组与Sham组在3 d、7 d、14 d及21 d时的PDCD5蛋白均为低水平表达,差异无统计学意义(P>0.05)。而CCI组PDCD5蛋白的表达水平,在3 d时已开始增高,在7 d及14 d时,PDCD5蛋白表达水平明显增高,14 d为PDCD5蛋白表达的高峰期,与Sham组比较,二者差异有统计学意义(P<0.05);在21 d时,PDCD5蛋白表达较7 d及14 d时降低,但仍高于3 d时的表达水平。给予低浓度Cur处理后,PDCD5蛋白表达水平较CCI组开始降低,在Cur 100 mg/(kg·d)组,PDCD5蛋白表达明显减低,与CCI组比较,二者差异有统计学意义(P<0.05)。在Cur200 mg/(kg·d)组,PDCD5蛋白表达较Cur100mg/(kg·d)组升高,二者差异有统计学意义(P<0.05)。结论姜黄素可以减轻CCI诱发的神经病理性痛行为,减少脊髓背角神经元细胞的凋亡,其机制可能与抑制PDCD5蛋白的表达有关。
Objective To observe the effect of curcumin (Cur) on the expression of programmed cell death 5 (PDCD5) in spinal dorsal horn neurons of rats with chronic lumbar spinal cord injury. Methods Chronic construction injury (CCI) model was established in rats. 84 healthy male SD rats were randomly divided into 6 groups: control group (n = 4), sham operation group (n = 16) (N = 16), Cur 100 mg / kg group (n = 16) and Cur 200 mg / kg group (n = 16) (n = 16)]. Curdling the sciatic nerve and curcumin 50, 100 and 200 mg / (kg · d) intraperitoneally in Group Cur were continued for 21 days. Sham group, CCI group and Cur group were divided into 4 subgroups (n = 4) according to the different time of drawing, namely subgroup at 3 days, subgroup at 7 days after operation, subgroup at 14 days after operation and postoperative day 21 Subgroup. Behavioral tests were performed at 2 days and 3 days, 7 days, 14 days and 21 days before surgery respectively. Terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL) ) Were used to label the apoptotic cells in the dorsal horn of spinal cord. The expression of PDCD5 protein was detected by Western blot (WB). Results Compared with Sham group, there was no significant difference between MTW and TWL in blank group (P> 0.05). After operation, the MTW and TWL in CCI group decreased gradually and reached the lowest level on the 14th day. After treatment with Cur 100 mg / (kg · d), the MTW and TWL increased gradually. Compared with CCI group, the difference was statistically significant (P <0.05) at 3 d, 7 d, 14 d and 21 d. Compared with Sham group, there was no significant difference in blank group (P> 0.05). The number of apoptotic cells in CCI group increased on the 3rd day after operation, and the peak of apoptotic cells appeared on the 7th to 14th day postoperatively. Compared with Sham group, the difference was statistically significant (P <0.05) (P <0.05). Cur also showed that Cur 100 mg / (kg · d) group showed a significant difference compared with other Cur groups (P <0.05) Compared with other Cur group and CCI group, the difference between group d and group C was statistically significant (P <0.05) at 7 d, 14 d and 21 d after operation. The PDCD5 protein expression in the blank control group and the Sham group at 3 d, 7 d, 14 d and 21 d was low, with no significant difference (P> 0.05). The expression of PDCD5 protein in CCI group began to increase at day 3, and the expression of PDCD5 protein increased significantly at day 7 and day 14, and peaked at 14 days. Compared with Sham group, the difference of the expression of PDCD5 protein (P <0.05). At 21 d, PDCD5 protein expression was lower than that at 7 d and 14 d, but still higher than that at 3 d. After treatment with low concentration of Cur, PDCD5 protein expression began to decrease compared with CCI group, PDCD5 protein expression was significantly reduced in Cur 100 mg / (kg · d) group, compared with CCI group, the difference was statistically significant (P < 0.05). The PDCD5 protein expression in Cur200 mg / (kg · d) group was higher than that in Cur100 mg / (kg · d) group, the difference was statistically significant (P <0.05). Conclusion Curcumin can alleviate the neuropathic pain induced by CCI and decrease the apoptosis of neurons in spinal dorsal horn. Its mechanism may be related to the inhibition of PDCD5 protein expression.