丹参酮ⅡA对神经病理性疼痛大鼠的影响
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摘要
目的观察丹参酮ⅡA(TanⅡA)对神经病理性疼痛模型(SNL)大鼠脊髓组织高迁移率族蛋白B1(HMGB1)、Toll样受体4(TLR4)和肿瘤坏死因子-α(TNF-α)、白细胞介素1β(IL-lβ)和白细胞介素10(IL-10)表达的影响,探讨TanⅡA对神经病理性疼痛的作用及其机制。方法成年雄性SD大鼠54只,随机分为3组:假手术组、模型对照组和TanⅡA组(术后腹腔注射TanⅡA 30 mg·kg~(-1)·d~(-1)),分别于术前1 d,术后第3,7,14天测试机械痛阈和热痛阈后,取L_4～L_(6 )脊髓,置于液氮中迅速冷冻。实时荧光定量PCR和Western blotting检测脊髓HMGB1、TLR4 mRNA和蛋白的表达,酶联免疫吸附测定(ELISA)法检测脊髓TNF-α、IL-lβ和IL-10蛋白的表达情况。结果与假手术组比较,模型对照组、TanⅡA组在术后第3,7,14天机械痛阈和热痛阈降低,脊髓HMGB1,TLR4 mRNA和蛋白表达上调(P<0.05)。与模型对照组比较,TanⅡA组在术后第3,7,14天机械痛阈和热痛阈升高,脊髓HMGB1、TLR4 mRNA和蛋白表达下调(P<0.05)。与假手术组比较,模型对照组、TanⅡA组在术后第3,7,14天脊髓TNF-α、IL-1β表达上调(P<0.05),而IL-10表达下调(P<0.05)。与模型对照组比较,TanⅡA组在术后第3,7,14天脊髓TNF-α、IL-1β表达下调(P<0.05),IL-10表达上调,行为学指标改善(P<0.05)。结论丹参酮ⅡA能减轻大鼠神经病理性疼痛,可能机制是在分子水平抑制HMGB1-TLR4信号通路及其下游的细胞因子,HMGB1-TLR4信号通路可能与神经病理性疼痛有关,是治疗神经病理性疼痛的靶点。
Objective Detect the expression level of HMGB1,TLR4,TNF-α,IL-1β and IL-10 in spinal cord tissue of neuropathic pain model rat after tanshinone ⅡA treated to explore its effect on neuropathic pain and the mechanisms. Methods A total of 54 males Sprague-Dawley(SD) rats were randomly divided into three groups:sham-operated group, model control group, and Tan ⅡA group.Tan ⅡA was administered intraperitoneally to rats in Tan ⅡA group at a dose of 30 mg·kg~(-1) daily for 14 days after surgery.The pain threshold was measured 1 day before SNL(baseline) and 3, 7, and 14 days after surgery.The expressions of HMGB1, TLR4 mRNA and protein in lumbar spinal cord 4-6(L_4-L_6) were assessed by RT-PCR and Western blotting, respectively.The levels of TNF-α, IL-1β and IL-10 in the spinal cord were detected by ELISA. Results The paw withdrawl threshold(PWT) and paw withdrawl latency(PWL) were significantly decreased after SNL(P<0.05).The expressions of TLR4, HMGB1 mRNA and protein were significantly increased(P<0.05),and the levels of TNF-α, IL-1β were significantly increased after SNL compared with those in the sham-operated group(P<0.05).After Tan ⅡA treatment, HMGB1 and TLR4 mRNA and protein levels were reduced significantly(P<0.05).TNF-α and IL-1β were downregulated, but IL-10 was upregulated in the spinal cords of SNL-induced rats(P<0.05),which was accompanied by improvement of pain behaviours in the Tan ⅡA group(P<0.05). Conclusion These results indicate Tanshinone ⅡA inhibited SNL-induced neuropathic pain via multiple effects, the possible mechanism is that HMGB1-TLR4 signal transduction pathway and its downstream cytokines are inhibited at the molecular level.It also suggesting that HMGB1-TLR4 signal transduction pathway may be related to neuropathic pain and is a target for the treatment of neuropathic pain.
Objective Detect the expression level of HMGB1,TLR4,TNF-α,IL-1β and IL-10 in spinal cord tissue of neuropathic pain model rat after tanshinone ⅡA treated to explore its effect on neuropathic pain and the mechanisms. Methods A total of 54 males Sprague-Dawley(SD) rats were randomly divided into three groups:sham-operated group, model control group, and Tan ⅡA group.Tan ⅡA was administered intraperitoneally to rats in Tan ⅡA group at a dose of 30 mg·kg~(-1) daily for 14 days after surgery.The pain threshold was measured 1 day before SNL(baseline) and 3, 7, and 14 days after surgery.The expressions of HMGB1, TLR4 mRNA and protein in lumbar spinal cord 4-6(L_4-L_6) were assessed by RT-PCR and Western blotting, respectively.The levels of TNF-α, IL-1β and IL-10 in the spinal cord were detected by ELISA. Results The paw withdrawl threshold(PWT) and paw withdrawl latency(PWL) were significantly decreased after SNL(P<0.05).The expressions of TLR4, HMGB1 mRNA and protein were significantly increased(P<0.05),and the levels of TNF-α, IL-1β were significantly increased after SNL compared with those in the sham-operated group(P<0.05).After Tan ⅡA treatment, HMGB1 and TLR4 mRNA and protein levels were reduced significantly(P<0.05).TNF-α and IL-1β were downregulated, but IL-10 was upregulated in the spinal cords of SNL-induced rats(P<0.05),which was accompanied by improvement of pain behaviours in the Tan ⅡA group(P<0.05). Conclusion These results indicate Tanshinone ⅡA inhibited SNL-induced neuropathic pain via multiple effects, the possible mechanism is that HMGB1-TLR4 signal transduction pathway and its downstream cytokines are inhibited at the molecular level.It also suggesting that HMGB1-TLR4 signal transduction pathway may be related to neuropathic pain and is a target for the treatment of neuropathic pain.
引文
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[2] 李阳,刘开祥,俸军林,等.丹参酮ⅡA对脑缺血再灌注损伤大鼠的保护作用及机制 [J].时珍国医国药,2008,19(7):1648-1649.
[3] KIM S H,CHUNG J M.An experimental model for peri-pheral neuropathy produced by segmental spinal nerve ligation in the rat [J].Pain,1992,50(3):355-363.
[4] LI H,XIE W,STRONG J A,et al.Systemic anti-infla-mmatory corticosteroid reduces mechanical pain behavior,sympathetics prouting,and elevation of proinflammatory cytokines in a rat model of neuropathic pain [J].Anesthesiolog,2007,107(3):469-477.
[5] HARGREAVES K,DUBNER R,BROWN F,et al.A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia [J].Pain,1988,32(1):77-88.
[6] LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the method [J].Methods,2001,25(4) :402-408.
[7] NAKAMURA Y,MORIOKA N,ABE H,et al.Neuropathic pain in rats with a partial sciatic nerve ligation is alleviated by intravenous injection of monoclonalantibody to high mobility group box-1[J].PLoS One,2013,18(8):e73640.
[8] MAEDA T,OZAKI M,KOBAYASHI Y,et al.HMGB1as a potential therapeutic target for neuropathic pain [J].J Pharmacol Sci,2013,123(4):301-305.
[9] KUMAR H,KAWAI T,AKIRA S.Toll-like receptors and innate immunity[J].Biochem Bio Phys Res Comm,2009,388(4):621-625.
[10] KAWAI T,AKIRA S.TLR signaling[J].Cell Death Differ,2006,13(10):816-825.
[11] LACAGNINA M J,WATKINS L R,GRACE P M.Toll-like receptors and their role in persistent pain [J].Pharmacol Ther,2018,184:145-158.
[12] ZHOU L J,YANG T,WEI X,et al.Brain-derived neuro-trophic factor contributes to spinal long-term potentiation and mechanical hypersensitivity by activation of spinal microglia in rat[J].Brain Behav Immun,2011,25(2):322-334.
[13] MILLIGAN E D,PENZKOVER K R,SODERQUIST R G,et al.Spinal interleukin-10 therapy to treat peripheral neuropathic pain[J].Neuromodulation,2012,15(6):520-526.
[14] LIU K,MORI S,TAKAHASHI H K,et al.Anti-high mobi-lity group box 1 monoclonal antibody ameliorates brain infarction induced by transient ischemia in rats[J].FASEB J,2007,21(14):3904-3916.
[15] SU X,WANG H,ZHAO J,et al.Beneficial effects of ethyl pyruvate through inhibiting high-mobility group box 1 expression and TLR4/NF-κB pathway after traumatic brain injury in the rat[J].Mediators Inflamm,2011,2011:807142.