富氢液通过增加自噬治疗大鼠神经病理性疼痛

Hydrogen-rich saline treated neuropathic pain in rats by increasing autophagy

目的评价自噬在富氢液治疗神经病理性疼痛中的作用。方法将鞘内置管成功的40只成年雄性SD大鼠随机分为5组:假手术组(S组)、神经病理性疼痛组(C组)、富氢液组(H组)、自噬抑制剂组(M组)和富氢液+自噬抑制剂组(HM组),各8只。采用坐骨神经慢性压迫法(CCI)制备大鼠神经病理性疼痛模型。M组和HM组于术后鞘内注射自噬抑制剂3-甲基腺嘌呤(3-MA)30μg/kg,H组和HM组于术后腹腔注射富氢液(0.6 mmol/L)10 mL/kg,其他组鞘内/腹腔给予等量生理盐水,2次/d,连续7 d。于造模前1 d和造模后1、3、5、7和14 d(T0—T5)测定大鼠机械刺激缩足阈值(MWT)和热刺激缩足潜伏期(TWL)。取脊髓L4—L6节段,采用Western blot法检测自噬相关蛋白微管相关蛋白轻链3(LC3)Ⅱ、Beclin-1和p62蛋白的表达;并测定脊髓组织中超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量。结果与S组比较,C组T2—T5时MWT和TWL均降低,T5时脊髓LC3Ⅱ、Beclin-1和p62蛋白表达水平升高,SOD活性降低,MDA含量增加(P<0.05)。与C组比较,H组T2—T5时MWT和TWL均升高,T5时脊髓LC3Ⅱ和Beclin-1蛋白表达水平升高,p62蛋白表达水平减少,SOD活性增强,MDA含量减少(P<0.05);M组T2—T5时MWT和TWL均降低,T5时脊髓LC3Ⅱ和Beclin-1蛋白表达水平减少,p62蛋白表达水平升高,SOD活性降低,MDA含量增加(P<0.05)。与M组比较,HM组T2—T5时MWT和TWL均升高,T5时脊髓LC3Ⅱ和Beclin-1蛋白表达水平升高,p62蛋白表达水平减少,SOD活性增高,MDA含量减少(P<0.05)。结论富氢液可减轻大鼠神经病理性痛,抑制脊髓氧化应激,其机制可能与增加自噬有关。

Objective To evaluate the role of autophagy in the treatment of neuropathic pain(NP)with hydrogen-rich saline.Methods Forty adult male Sprague-Dawley rats with successful intubation were randomly divided into 5 groups(n=8)using a random number table:the sham operation group(group S),the neuropathic pain group(group C),the hydrogen-rich saline group(group H),the autophagy inhibitor group(group M)and the hydrogen-rich saline+autophagy inhibitor group(group HM).There were 8 rats in each group.The NP model was established by chronic constriction of the sciatic nerve(CCI)in rats.The autophagy inhibitor 3-methyladenine(3-MA)was intraperitoneally injected with 30μg/kg in the group M and the group HM.The hydrogen-rich saline(0.6 mmol/L)was intraperitoneally injected with 10 mL/kg in the group H and the group HM.The other groups were intraperitoneally injected with the same amount of normal saline twice a day for 7 consecutive days.Paw withdrawal threshold to mechanical stimulation(MWT)and paw withdrawal latency to thermal stimulation(TWL)were measured at 1 day before and 1,3,5,7 and 14 days after modeling(T0-T5).After the last measurement of pain threshold,the L4-L6 segment of spinal cord was removed for determination of the expression of autophagy-related proteins microtubule-associated protein light chain 3(LC3)Ⅱ,Beclin-1 and p62 proteins by Western blot assay.The expression levels of superoxide dismutase(SOD)and malondialdehyde(MDA)in spinal cord tissue were detected.Results Compared with the group S,MWT and TWL were decreased in the group C at T2-5,the expression levels of LC3Ⅱ,Beclin-1 and p62 were increased,SOD activity was decreased,and MDA content was increased at T5(P<0.05).Ⅱlevels were increased,p62 protein expression levels were decreased,SOD activity was increased,and MDA content was decreased at T5(P<0.05).MWT and TWL were decreased in the group M at T2-5,LC3Ⅱand Beclin-1 protein expression levels were decreased,p62 protein expression levels were increased,SOD activity was decreased,and MDA content was increased at T5(P<0.05).Compared with the group M,MWT and TWL were increased in the group HM at T2-5,LC3Ⅱand Beclin-1 protein expression levels were increased,p62 protein expression levels were decreased,SOD activity was increased,and MDA content was decreased at T5(P<0.05).Conclusion Hydrogen-rich saline can alleviate neuropathic pain and inhibit oxidative stress in spinal cord in rats,and the mechanism may be related to the increase of autophagy.