Cannabinoid CB_(2) receptors and spinal microglia are implicated in tingenone-mediated antinociception in mice

Objective:To investigate the antinociceptive effect of tingenone on inflammatory pain,as well as and the involvement of the cannabinoid receptors type 2(CB2)and spinal microglia in this process.Methods:Male Swiss mice were subjected to inflammatory pain induced by intraplantar injection of carrageenan.The nociceptive threshold was measured by von Frey filaments test.Tingenone was administered orally 60 min before carrageenan injection.To evaluate the involvement of CB2 receptor,endocannabinoids,and microglia,AM630(a CB2 receptor antagonist),MAFP(an inhibitor of an enzyme that hydrolyses endocannabinoids),and minocycline(a microglial inhibitor)were given intrathecally 20 min before tingenone administration.In addition,an immunofluorescence assay was used to evaluate CB2 receptor and CD11 B(a microglial marker)expression in the spinal cord dorsal horn.Results:Tingenone significantly reduced carrageenan-induced hyperalgesia,which was reversed by pretreatment with AM630.MAFP and minocycline potentiated and prolonged the tingenoneinduced antinociception.CD11 B expression was increased in the spinal cord dorsal horn of mice with inflammatory pain pretreated with tingenone,which was reduced by AM630,MAFP,and minocycline.Conclusions:CB2 receptors and endocannabinoids participate in the tingenone-induced antinociception which may involve the inhibition of microglia at spinal level.

Activation of cannabinoid receptor CB2 regulates LPS-induced pro-inflammatory cytokine production and osteoclastogenic gene expression in human periodontal ligament cells

Background and Objective: It has been found that human periodontal ligament (hPDL) cells express cannabinoid receptor CB2. However, the functional importance of CB2 in hPDL cells exposed to bacterial endotoxins is not known. Here we investigate if the inflammation promoter lipopolysaccharide (LPS) affects CB2 expression and if activation of CB2 regulates LPS-induced pro-inflammatory cytokine production and osteoclastogenic gene expression in hPDL cells. Methods: The hPDL cells were obtained from extracted teeth of periodontally healthy subjects. CB2 expression in hPDL cells exposed to LPS was deter- mined by quantitative real-time PCR analysis. Then, the cells were incubated with or without CB2-specific agonist HU-308 before further stimulation with LPS. In some experiments, the cells were pre-treated with CB2-specific antagonist SR144528. The production of pro-inflammatory cytokines interleukin-1 beta (IL- 1β), interleukin-6 (IL-6) and tumor necrosis factoralpha (TNF-α) was assessed by enzyme-linked immunosorbent assay (ELISA). The mRNA expression of osteoclastogenic genes osteoprotegerin (OPG) and receptor activator of NF-κB ligand (RANKL) was examined using quantitative real-time PCR analysis. Results: CB2 expression in hPDL cells was markedly enhanced by LPS. HU-308 significantly suppressed the production of IL-1β, IL-6 and TNF-α exposed to LPS, whereas SR144528 attenuated this effect. The OPG/RANKL ratio decreased when exposed to LPS, furthermore increased significantly with the addition of HU-308 and finally decreased markedly after pretreatment with SR144528. Conclusion: Our study demonstrated that activation of CB2 had anti-inflammatory and anti-resorptive effects on LPS-stimulated hPDL cells. These findings suggest that activation of CB2 might be an effective therapeutic strategy for the treatment of inflammation and alveolar bone resorption in periodontitis.

大麻素受体CB2在机械牵张力介导的人牙周膜细胞成骨分化中的作用

目的:研究大麻素Ⅱ型受体(cannabinoi dreceptor Ⅱ,CB2)在机械牵张力介导的人牙周膜细胞中的表达以及成骨分化中的作用。方法:体外培养人牙周膜细胞,构建细胞-机械牵张力加载模型,施加不同大小的机械牵张力,采用Real-timePCR和细胞免疫荧光化学技术检测CB2在人牙周膜细胞中mRNA和蛋白的表达。用碱性磷酸酶(ALP)试剂盒检测机械牵张力介导的细胞ALP活性。结果:对人牙周膜细胞施加不同大小的机械牵张力24h,CB2 mRNA的表达随机械牵张力的力值增大而显著性增加(P<0.05),在18%拉伸应变率作用下表达量最高(P<0.05),此时CB2蛋白的表达显著增加。加入CB2激动剂HU-308后,施加18%拉伸应变率的机械牵张力作用于人牙周膜细胞24h,ALP活性显著性增加(P<0.05)。结论:CB2在人牙周膜细胞中的表达与机械牵张力的力值具有相关性。在机械牵张力作用下,大麻素受体CB2与其配体结合能够促进人牙周膜细胞的成骨分化,从而在正畸牙槽骨改建中发挥重要作用。

大麻素受体2激动剂在谷氨酸氧化应激损伤中保护作用实验研究

目的:评价大麻素受体2(CB 2受体)通过抗氧化应激机制在谷氨酸对共培养小胶质细胞和神经元损伤中的保护作用。方法:采用随机数字表法将培养的N9小胶质细胞和HT22神经元分为四组(n=24):对照组(Con组)、谷氨酸组(Glu组)、CB 2受体激动剂AM1241+谷氨酸组(AM1241+Glu组)和AM1241+CB 2受体拮抗剂AM630+谷氨酸组(AM1241+AM630+Glu组)。Con组正常培养24 h;Glu组用含10 mmol/L谷氨酸的培养基孵育24 h;AM1241+Glu组用含2μmol/L AM1241和10 mmol/L谷氨酸的培养基孵育24 h;AM1241+AM630+Glu组含2μmol/L AM1241、6μmol/L AM630及10 mmol/L谷氨酸的培养基孵育24 h。采用MTT法测定细胞活力,采用化学比色法测定乳酸脱氢酶(LDH)、超氧化物歧化酶(SOD)活性及丙二醛(MDA)的含量。结果:与Con组比较,Glu组和AM1241+AM630+Glu组细胞活力和SOD活性降低,LDH活性和MDA含量升高(均P<0.05),AM1241+Glu组细胞活力和SOD活性降低,LDH含量升高(均P<0.05);与Glu组比较,AM1241+Glu组细胞活力和SOD活性升高,LDH活性和MDA含量降低(均P<0.05);与AM1241+Glu组比较,AM1241+AM630+Glu组细胞活力和SOD活性降低,LDH活性和MDA含量升高(均P<0.05)。结论:CB 2受体激活减轻谷氨酸诱发小胶质细胞和神经元损伤的机制与抗氧化作用有关。

大麻素CB1受体对大鼠视网膜神经节细胞诱发动作电位的作用(英文)

激活大麻素CB1受体(CB1Rs)通过调控多种离子通道,从而调节脊椎动物视网膜的功能。本文旨在利用膜片钳全细胞记录技术,在大鼠视网膜薄片上研究CB1Rs对神经节细胞兴奋性的作用。结果显示,在电流钳制状态下,灌流CB1R激动剂WIN55212-2(WIN,5μmol/L)对神经节细胞的自发动作电位发放频率和静息膜电位均没有显著影响。在灌流液中加入CNQX,D-APV,bicuculline和strychnine以阻断神经节细胞的兴奋性和抑制性输入,灌流5μmol/L WIN对正向电流注入(+10pA到+100pA)诱发的动作电位的频率也没有显著影响。位相分析结果显示,触发动作电位的阈值电位和触发第一个动作电位的延迟时间在加入WIN前后也没有显著改变;然而,WIN显著降低动作电位的上升和下降相速率(±dV/dtmax),而且该作用可被CB1R拮抗剂SR141716所阻断。此外,在阻断突触输入的情况下,WIN对神经节细胞的膜电位也没有显著影响。以上结果提示,激活大麻素CB1Rs通过调控诱发动作电位,从而调节大鼠视网膜神经节细胞的兴奋性。