虾青素对大鼠神经病理性痛的影响及脊髓HO-1在其中的作用

Effect of astaxanthin on neuropathic pain in rats and role of spinal heme oxygenase-1

目的评价虾青素对大鼠神经病理性痛的影响及脊髓血红素加氧酶-1(HO-1)在其中的作用。方法SPF级健康成年雄性SD大鼠,体重200~250 g,取鞘内置管成功的大鼠72只,采用随机数字表法分为6组(n=12):空白对照组(C组)、假手术组(Sham组)、神经病理性痛组(NP组)、神经病理性痛+二甲基亚砜组(NP+DMSO组)、神经病理性痛+虾青素组(NP+AST组)和神经病理性痛+锌原卟啉+虾青素组(NP+ZnPP+AST组)。采用坐骨神经结扎法制备大鼠神经病理性痛模型,Sham组只分离坐骨神经,不结扎。造模后5 d时,NP+DMSO组鞘内注射0.5%二甲基亚砜10μl;NP+AST组鞘内注射虾青素1μg(溶于10μl二甲基亚砜中);NP+ZnPP+AST组鞘内注射HO-1抑制剂锌原卟啉24μg(溶于10μl二甲基亚砜中),3 h后鞘内注射虾青素1μg(溶于10μl二甲基亚砜中);3组均每天注射1次,连续注射10 d。分别于造模前1 d和造模后3、7、14 d时测定机械缩足反应阈(MWT)和热缩足潜伏期(TWL)。于造模后14 d时处死大鼠,取L4-6腰段脊髓,采用ELISA法检测TNF-α、IL-1β、SOD和谷胱甘肽过氧化物酶(GHS-PX)含量,采用Western blot法测定HO-1表达。结果与C组和Sham组比较,其余4组造模后各时点MWT降低,TWL缩短,脊髓TNF-α和IL-1β含量升高,HO-1表达上调,NP组、NP+DMSO组和NP+ZnPP+AST组脊髓SOD和GSH-PX含量降低,NP+AST组脊髓SOD和GSH-PX含量升高(P<0.05);与NP组比较,NP+AST组造模后7和14 d时MWT升高,TWL延长,脊髓SOD和GSH-PX含量升高,TNF-α和IL-1β含量降低,脊髓HO-1表达上调,NP+ZnPP+AST组脊髓HO-1表达下调(P<0.05),NP+DMSO组上述各指标差异无统计学意义(P>0.05);与NP+AST组比较,NP+ZnPP+AST组造模后7和14 d时MWT降低,TWL缩短,脊髓SOD和GSH-PX含量降低,TNF-α和IL-1β含量升高,HO-1表达下调(P<0.05)。结论虾青素可减轻大鼠神经病理性痛,其机制与上调脊髓HO-1表达,抑制氧化应激反应和炎症反应有关。

Objective To evaluate the effect of astaxanthin on neuropathic pain in rats and the role of spinal heme oxygenase-1(HO-1).Methods Seventy-two SPF-grade healthy adult male Sprague-Dawley rats,weighing 200-250 g,in which intrathecal catheters were successfully implanted,were divided into 6 groups(n=12 each)by a random number table method:blank control group(group C),sham operation group(Sham group),neuropathic pain(NP)group,NP plus dimethyl sulfoxide(DMSO)group(NP+DMSO group),NP plus astaxanthin group(NP+AST group)and NP plus zinc protoporphyrin plus astaxanthin group(NP+ZnPP+AST group).NP was induced by chronic constriction injury in anesthetized rats.In Sham group,the sciatic nerve was only isolated without ligation.At 5 days after establishing the model,0.5%DMSO 10μl was intrathecally injected in NP+DMSO group,astaxanthin 1μg(dissolved in 10μl DMSO)was intrathecally injected in NP+AST group,HO-1 inhibitor zinc protoporphyrin 24μg(dissolved in 10μl DMSO)was intrathecally injected,and 3 h later astaxanthin 1μg(dissolved in 10μl DMSO)was intrathecally injected in NP+ZnPP+AST group.Injection was given once a day for 10 consecutive days in the 3 groups mentioned above.The mechanical paw withdrawal threshold(MWT)and thermal paw withdrawal latency(TWL)were measured at 1 day before establishing the model and 3,7 and 14 days after establishing the model.The rats were sacrificed at 14 days after establishing the model,and the L4-6 lumbar segments of the spinal cord were removed for determination of the contents of tumor necrosis factor-alpha(TNF-α),interleukin-1beta(IL-1β),superoxide dismutase(SOD)and glutathione peroxidase(GHS-PX)(by enzyme-linked immunosorbent assay)and expression of HO-1(by Western blot).Results Compared with group C and group Sham,the MWT was significantly decreased and TWL was shortened at each time point after establishing the model,the contents of TNF-αand IL-1βwere increased,and the expression of HO-1 was up-regulated in the other four groups,the SOD and GSH-PX contents were significantly decreased in NP group,NP+DMSO group and NP+ZnPP+AST group,and the SOD and GSH-PX contents were significantly increased in NP+AST group(P<0.05).Compared with NP group,the MWT was significantly increased and TWL was prolonged at 7 and 14 days after establishing the model,the contents of TNF-αand IL-1βwere decreased,and the expression of HO-1 was up-regulated in NP+AST group,the expression of HO-1 was down-regulated in NP+ZnPP+AST group(P<0.05),and no significant change was found in the parameters mentioned above in NP+DMSO group(P>0.05).Compared with NP+AST group,the MWT was significantly decreased and TWL was shortened at 7 and 14 days after establishing the model,the contents of SOD and GSH-PX were decreased,the contents of TNF-αand IL-1βwere increased,and the expression of HO-1 was down-regulated in NP+ZnPP+AST group(P<0.05).Conclusion Astaxanthin can reduce NP in rats,and the mechanism is related to up-regulating the expression of HO-1 in the spinal cord and inhibiting oxidative stress and inflammatory responses.