骨癌痛大鼠中脑导水管周围灰质腹外侧区P2X7受体激活对D?丝氨酸释放的影响

Effect of the activation of P2X7 receptor on D-serine release in ventrolateral periaqueductal gray in rats with bone cancer pain

目的中脑导水管周围灰质腹外侧区(vlPAG)P2X7受体激活介入骨癌痛的形成和维持机制。文章拟建立骨癌痛大鼠模型,通过脑微透析结合ELISA技术观察vlPAG中P2X7受体激活对D-丝氨酸水平的影响。方法雌性SD大鼠42只,随机数字表法分成4组:正常对照组(n=12)、假手术组(n=12)、骨癌痛组(n=12)和P2X7受体特异性拮抗剂(A-438079)组(n=6)。骨癌痛组大鼠建立大鼠胫骨转移性骨癌痛模型,注入20μL含SHZ-88乳腺癌细胞的RPMI-1640培养基细胞悬液(癌细胞1×10^7个/0.5 mL);假手术组注入等体积的经过处理的癌细胞(SHZ-88乳腺癌细胞在90℃沸水中持续20 min),其余操作同骨癌痛组;正常对照组不进行任何处理。P2X7受体拮抗剂组前面操作同骨癌痛组,但灌流液中加入P2X7受体特异性拮抗剂A-438079。正常对照组、假手术组和骨癌痛组分别于同一时间点检测大鼠建模侧后足热痛阈值和机械痛阈值;免疫组化法检测各组21 d时间点大鼠vlPAG中P2X7受体阳性表达;正常对照组、假手术组、骨癌痛组和P2X7受体拮抗剂组使用ELISA法检测各组大鼠vlPAG透析液中D-丝氨酸变化。结果骨癌痛组大鼠机械痛阈值和热痛阈值在第5、7、10、14、18、21天均低于正常对照组和假手术组(P<0.01)正常对照组和假手术组大鼠vlPAG中有散在P2X7阳性表达,骨癌痛组大鼠P2X7受体阳性细胞数较正常对照组和假手术组显著升高(P<0.01)。骨癌痛组大鼠vlPAG中D-丝氨酸含量[(220.28±63.38)ng/mL]较正常对照组大鼠[(148.09±46.89)ng/mL]和假手术组[(147.32±51.44)ng/mL]大鼠明显升高(P<0.05)。P2X7受体拮抗剂组大鼠vlPAG中D-丝氨酸含量[(134.20±41.77)ng/mL]较骨癌痛组明显降低(P<0.05)。结论vlPAG P2X7受体激活后促进D-丝氨酸释放进而参与大鼠骨癌痛机制的调节。

Objective The activation of P2X7 receptor in ventrolateral periaqueductal gray(vlPAG)is involved in the formation and maintenance of bone cancer pain(BCP).This study will establish a rat model of BCP and observe the effect of the activation of P2X7 receptor in vlPAG on D-serine level through brain microdialysis combined with ELISA.Methods Forty-two female SD rats were divided into four groups by random number table:normal control group(n=12),sham group(n=12),BCP group(n=12)and P2X7 receptor antagonist group(n=6).The model of metastatic BCP in the tibias of the rats was established in the BCP group,and 20μL of RPMI-1640 medium cell suspension containing SHZ-88 breast cancer cells was injected(1×10^7 cancer cells/0.5 mL).The sham group was injected with treated cancer cells of the same volume(SHZ-88 breast cancer cells were kept in boiling water at 90℃for 20 min),and the rest of the operation was the same as the BCP group.The normal control group received no treatment.The P2X7 receptor antagonist group was treated the same as the BCP group,except that the P2X7 receptor-specific antagonist A-438079 was added to the perfusion solution.The thermal pain threshold and mechanical pain threshold were detected at the same time in the normal control group,the sham group and the BCP group.The positive expression of P2X7 receptor in vlPAG of rats was detected by immunohistochemistry in each group in 21 days.The changes of D-serine in vlPAG dialysate were detected by ELISA in each group.Results The mechanical pain threshold and thermal pain threshold of the rats in BCP group on Day 5,7,10,14,18 and 21 were lower than those of the normal control group and sham group(P<0.01).The positive expression of P2X7 was scattered in vlPAG in normal control group and sham group.The number of P2X7 receptor positive cells in the BCP group was significantly higher than that in the control group and sham group(P<0.01).The content of D-serine in vlPAG of the rats in BCP group[(220.28±63.38)ng/mL]was significantly higher than that in the control group[(148.09±46.89)ng/mL]and the sham group[(147.32±51.44)ng/mL](P<0.05).The content of D-serine in vlPAG[(134.20±41.77)ng/mL]in P2X7 receptor antagonist group was significantly lower than that in BCP group(P<0.05).Conclusion The activation of the P2X7 receptor in ventrolateral periaqueductal gray promotes D-serine release and participates in the mechanisms of BCP in rats.