Background The cannabinoid receptor-2 （CB2） is important for bone remodeling. In this study, we investigated the effects of CB2 selective antagonist （AM630） on receptor activator of nuclear factor kappa B （RANK）...Background The cannabinoid receptor-2 （CB2） is important for bone remodeling. In this study, we investigated the effects of CB2 selective antagonist （AM630） on receptor activator of nuclear factor kappa B （RANK） ligand （RANKL）induced osteoclast differentiation and the underlying signaling pathway using a monocyte-macrophage cell line-RAW264.7.Methods RAW264.7 was cultured with RANKL for 6 days and then treated with AM630 for 24 hours. Mature osteoclasts were measured by tartrate-resistant acid phosphatase （TRAP） staining using a commercial kit. Total ribonucleic acid （RNA）was isolated and real-time reverse transcriptase-polymerase chain reaction （RT-PCR） was done to examine the expression of RANK, cathepsin K （CPK） and nuclear factor kappa B （NF-κB）. The extracellular signal-regulated kinase （ERK）,phosphorylation of ERK （P-ERK） and NF-κB production were tested by Western blotting. The effect of AM630 on RAW264.7 viability was determined using the 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazoliumbromide （MTT） assay.Results AM630 did not affect the viability of RAW264.7. However, this CB2 selective antagonist markedly inhibited osteoclast formation and the inhibition rate was dose-dependent. The dose of 〉100 nmol/L could reduce TRAP positive cells to the levels that were significantly lower than the control. AM630 suppressed the expression of genes associated with osteoclast differentiation and activation, such as RANK and CPK. An analysis of a signaling pathway showed that AM630 inhibited the RANKL-induced activation of ERK, but not NF-κB.Conclusion AM630 could inhibit the osteoclastogenesis from RAW264.7 induced with RANKL.展开更多
背景:大麻素受体通过与配体结合,调控牙周炎的炎症和骨量,促进牙周组织的愈合,在临床上牙周炎的预防和治疗方面具有重要意义。目的:综述大麻素受体与牙周炎的关系,主要为大麻素Ⅰ型(CB1)受体、大麻素Ⅱ型(CB2)受体与炎症和牙槽骨骨改建...背景:大麻素受体通过与配体结合,调控牙周炎的炎症和骨量,促进牙周组织的愈合,在临床上牙周炎的预防和治疗方面具有重要意义。目的:综述大麻素受体与牙周炎的关系,主要为大麻素Ⅰ型(CB1)受体、大麻素Ⅱ型(CB2)受体与炎症和牙槽骨骨改建的关系,以及涉及的常见细胞信号传导通路,为牙周炎预防和治疗及其在临床其他领域的应用提供思路。方法:检索PubMed、万方数据库、CNKI中国期刊全文数据库1985年7月至2022年7月收录的相关文献。英文检索词为“cannabinoids receptor,CB1 receptor and periodontitis,CB2 receptor and periodontitis,CB1 receptors and bone remodeling,CB2 receptors and bone remodeling,CB1 receptors and signaling pathways,CB2 receptors and signaling pathways”,中文检索词为“大麻素受体,CB1受体和牙周炎,CB2受体和牙周炎,CB1受体和骨改建,CB2受体和骨改建,CB1受体和信号通路、CB2受体和信号通路”,最终纳入107篇文献进行归纳总结。结果与结论:①内源性大麻素系统包含多种受体,其中最具有代表性的为CB1和CB2受体,均属于G蛋白偶联超家族成员;二者在牙周组织中均存在表达;②在天然配体或人工合成激动剂的作用下,大麻素受体可通过不同的代谢通路在体内外产生特定的生理效应,从而调控牙周炎局部的炎症和骨细胞的生成和分化,最终影响炎症和骨量;③进一步研究大麻素受体与牙周炎炎症和牙槽骨骨形成、骨吸收的关系,以及涉及到的常见信号通路——丝裂原活化蛋白激酶(MAPK)信号通路、NF-κB信号通路,为临床上牙周炎的预防和治疗提供新的思路成为目前研究的重点。展开更多
Many researchers employed mammalian expression system to artificially express cannabinoid receptors, but immunoblot data that directly prove efficient protein expression can hardly be seen in related research reports....Many researchers employed mammalian expression system to artificially express cannabinoid receptors, but immunoblot data that directly prove efficient protein expression can hardly be seen in related research reports. In present study, we demonstrated cannabinoid receptor protein was not able to be properly expressed with routine mammalian expression system. This inefficient expression was rescued by endowing an exogenous signal peptide ahead of cannabinoid receptor peptide. In addition, the artificially synthesized cannabinoid receptor was found to aggregate under routine sample denaturing temperatures (i.e.,≥95°C), forming a large molecular weight band when analyzed by immuno-blotting. Only denaturing temperatures ≤75°C yielded a clear band at the predicted molecular weight. Collectively, we showed that efficient mammalian expression of cannabinoid receptors need a signal peptide sequence, and described the requirement for a low sample denaturing temperature in immuno-blot analysis. These findings provide very useful information for efficient mammalian expression and immuno-blotting of membrane receptors.展开更多
目的观察大麻素2型受体(CB2R)激动剂JWH-015对阿尔茨海默病(AD)小鼠认知功能下降的改善作用,并探讨其作用机制是否与脑内小胶质细胞激活表型转化密切相关。方法 20只成年雄性C57BL/6J小鼠随机分为溶剂对照组、JWH-015对照组、AD模型组和...目的观察大麻素2型受体(CB2R)激动剂JWH-015对阿尔茨海默病(AD)小鼠认知功能下降的改善作用,并探讨其作用机制是否与脑内小胶质细胞激活表型转化密切相关。方法 20只成年雄性C57BL/6J小鼠随机分为溶剂对照组、JWH-015对照组、AD模型组和JWH-015治疗组。侧脑室注射4μg寡聚态β-淀粉样蛋白1-42(Aβ1-42)和等体积生理盐水构建AD模型和模型对照组后,分别接受连续3周腹腔注射JWH-015 0.5 mg/kg或等量溶剂处理。采用新物体识别实验检测小鼠认知功能;实时定量PCR方法检测皮质、海马脑区M1型小胶质细胞标记分子诱导型一氧化氮合酶(i NOS)和M2型小胶质细胞标志物几丁质酶3样蛋白(Ym1/2)m RNA表达水平。同时,取15只CB2R基因敲除C57BL/6J小鼠(CB2RKO)和5只同窝野生型(CB2RWT)小鼠按基因型和处理随机分为CB2RKO溶剂对照组、CB2RKO AD模型组、JWH-015处理组和CB2RWT小鼠溶剂对照组,用来研究CB2R对认知功能改善作用和小胶质细胞激活表型转化作用的特异性。结果与溶剂对照组相比,C57BL/6J AD模型组小鼠新奇物体识别指数显著降低(P<0.01),同时伴随皮质和海马脑区i NOS m RNA表达显著升高(均为P<0.05)和Ym1/2 m RNA表达显著降低(均为P<0.01);而JWH-015处理可显著提高C57BL/6J AD模型组小鼠新物体识别指数(P<0.05),下调皮质和海马区i NOS m RNA表达水平(均为P<0.05),上调Ym1/2 m RNA表达水平(均为P<0.05);与脑室注射生理盐水的CB2RKO溶剂对照组小鼠相比,CB2RKO AD模型组小鼠识别指数显著降低(P<0.05),皮质和海马脑区i NOS m RNA表达显著升高(均为P<0.05),皮质脑区Ym1/2 m RNA表达显著降低(P<0.05);与CB2RKO AD模型组小鼠相比,JWH-015处理组长时程给药对新奇物体识别指数无显著影响,并对皮质、海马脑区内i NOS及Ym1/2 m RNA的表达水平无显著影响。结论 JWH-015通过特异性激动CB2R改善Aβ诱导的AD模型小鼠的认知能力下降,其机制可能与其直接作�展开更多
基金This work was supported by the grants from Jiangsu Province Key Medical Center （No. ZX200608）, the National Nature Science Foundation of China （No. 30672140, No. 81071451）, the Colleges and Universities Natural Science Foundation in Jiangsu Province （No. 10KJB320019）, the Key Project Surpported by the Medical Science and Technology Department Foundation, Jiangsu Province, Department of Health （No. H201012） and the Social Development Projects in Suzhou （No. SS08020）.
文摘Background The cannabinoid receptor-2 （CB2） is important for bone remodeling. In this study, we investigated the effects of CB2 selective antagonist （AM630） on receptor activator of nuclear factor kappa B （RANK） ligand （RANKL）induced osteoclast differentiation and the underlying signaling pathway using a monocyte-macrophage cell line-RAW264.7.Methods RAW264.7 was cultured with RANKL for 6 days and then treated with AM630 for 24 hours. Mature osteoclasts were measured by tartrate-resistant acid phosphatase （TRAP） staining using a commercial kit. Total ribonucleic acid （RNA）was isolated and real-time reverse transcriptase-polymerase chain reaction （RT-PCR） was done to examine the expression of RANK, cathepsin K （CPK） and nuclear factor kappa B （NF-κB）. The extracellular signal-regulated kinase （ERK）,phosphorylation of ERK （P-ERK） and NF-κB production were tested by Western blotting. The effect of AM630 on RAW264.7 viability was determined using the 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazoliumbromide （MTT） assay.Results AM630 did not affect the viability of RAW264.7. However, this CB2 selective antagonist markedly inhibited osteoclast formation and the inhibition rate was dose-dependent. The dose of 〉100 nmol/L could reduce TRAP positive cells to the levels that were significantly lower than the control. AM630 suppressed the expression of genes associated with osteoclast differentiation and activation, such as RANK and CPK. An analysis of a signaling pathway showed that AM630 inhibited the RANKL-induced activation of ERK, but not NF-κB.Conclusion AM630 could inhibit the osteoclastogenesis from RAW264.7 induced with RANKL.
文摘背景:大麻素受体通过与配体结合,调控牙周炎的炎症和骨量,促进牙周组织的愈合,在临床上牙周炎的预防和治疗方面具有重要意义。目的:综述大麻素受体与牙周炎的关系,主要为大麻素Ⅰ型(CB1)受体、大麻素Ⅱ型(CB2)受体与炎症和牙槽骨骨改建的关系,以及涉及的常见细胞信号传导通路,为牙周炎预防和治疗及其在临床其他领域的应用提供思路。方法:检索PubMed、万方数据库、CNKI中国期刊全文数据库1985年7月至2022年7月收录的相关文献。英文检索词为“cannabinoids receptor,CB1 receptor and periodontitis,CB2 receptor and periodontitis,CB1 receptors and bone remodeling,CB2 receptors and bone remodeling,CB1 receptors and signaling pathways,CB2 receptors and signaling pathways”,中文检索词为“大麻素受体,CB1受体和牙周炎,CB2受体和牙周炎,CB1受体和骨改建,CB2受体和骨改建,CB1受体和信号通路、CB2受体和信号通路”,最终纳入107篇文献进行归纳总结。结果与结论:①内源性大麻素系统包含多种受体,其中最具有代表性的为CB1和CB2受体,均属于G蛋白偶联超家族成员;二者在牙周组织中均存在表达;②在天然配体或人工合成激动剂的作用下,大麻素受体可通过不同的代谢通路在体内外产生特定的生理效应,从而调控牙周炎局部的炎症和骨细胞的生成和分化,最终影响炎症和骨量;③进一步研究大麻素受体与牙周炎炎症和牙槽骨骨形成、骨吸收的关系,以及涉及到的常见信号通路——丝裂原活化蛋白激酶(MAPK)信号通路、NF-κB信号通路,为临床上牙周炎的预防和治疗提供新的思路成为目前研究的重点。
基金supported by a grant from Army Medical Research Program of China(No.08G168)
文摘Many researchers employed mammalian expression system to artificially express cannabinoid receptors, but immunoblot data that directly prove efficient protein expression can hardly be seen in related research reports. In present study, we demonstrated cannabinoid receptor protein was not able to be properly expressed with routine mammalian expression system. This inefficient expression was rescued by endowing an exogenous signal peptide ahead of cannabinoid receptor peptide. In addition, the artificially synthesized cannabinoid receptor was found to aggregate under routine sample denaturing temperatures (i.e.,≥95°C), forming a large molecular weight band when analyzed by immuno-blotting. Only denaturing temperatures ≤75°C yielded a clear band at the predicted molecular weight. Collectively, we showed that efficient mammalian expression of cannabinoid receptors need a signal peptide sequence, and described the requirement for a low sample denaturing temperature in immuno-blot analysis. These findings provide very useful information for efficient mammalian expression and immuno-blotting of membrane receptors.
文摘目的观察大麻素2型受体(CB2R)激动剂JWH-015对阿尔茨海默病(AD)小鼠认知功能下降的改善作用,并探讨其作用机制是否与脑内小胶质细胞激活表型转化密切相关。方法 20只成年雄性C57BL/6J小鼠随机分为溶剂对照组、JWH-015对照组、AD模型组和JWH-015治疗组。侧脑室注射4μg寡聚态β-淀粉样蛋白1-42(Aβ1-42)和等体积生理盐水构建AD模型和模型对照组后,分别接受连续3周腹腔注射JWH-015 0.5 mg/kg或等量溶剂处理。采用新物体识别实验检测小鼠认知功能;实时定量PCR方法检测皮质、海马脑区M1型小胶质细胞标记分子诱导型一氧化氮合酶(i NOS)和M2型小胶质细胞标志物几丁质酶3样蛋白(Ym1/2)m RNA表达水平。同时,取15只CB2R基因敲除C57BL/6J小鼠(CB2RKO)和5只同窝野生型(CB2RWT)小鼠按基因型和处理随机分为CB2RKO溶剂对照组、CB2RKO AD模型组、JWH-015处理组和CB2RWT小鼠溶剂对照组,用来研究CB2R对认知功能改善作用和小胶质细胞激活表型转化作用的特异性。结果与溶剂对照组相比,C57BL/6J AD模型组小鼠新奇物体识别指数显著降低(P<0.01),同时伴随皮质和海马脑区i NOS m RNA表达显著升高(均为P<0.05)和Ym1/2 m RNA表达显著降低(均为P<0.01);而JWH-015处理可显著提高C57BL/6J AD模型组小鼠新物体识别指数(P<0.05),下调皮质和海马区i NOS m RNA表达水平(均为P<0.05),上调Ym1/2 m RNA表达水平(均为P<0.05);与脑室注射生理盐水的CB2RKO溶剂对照组小鼠相比,CB2RKO AD模型组小鼠识别指数显著降低(P<0.05),皮质和海马脑区i NOS m RNA表达显著升高(均为P<0.05),皮质脑区Ym1/2 m RNA表达显著降低(P<0.05);与CB2RKO AD模型组小鼠相比,JWH-015处理组长时程给药对新奇物体识别指数无显著影响,并对皮质、海马脑区内i NOS及Ym1/2 m RNA的表达水平无显著影响。结论 JWH-015通过特异性激动CB2R改善Aβ诱导的AD模型小鼠的认知能力下降,其机制可能与其直接作�
