大麻素受体CB1对子宫腺肌病子宫内膜-肌层交界区平滑肌细胞增殖和凋亡的影响及其作用机制

The effect of cannabinoid receptor CB1 on the proliferation and apoptosis of smooth muscle cells in the endometrial-myometrial interface of adenomyosis and its mechanism of action

目的观察大麻素受体CB1在子宫腺肌病子宫内膜-肌层交界区(endometrial-myometrial interface,EMI)平滑肌细胞的表达及对细胞增殖和凋亡功能的影响。方法选择2017年7月至2018年8月于首都医科大学附属北京妇产医院因子宫腺肌病行子宫切除术的患者16例为腺肌病组,选择同期因宫颈上皮内瘤变Ⅲ级、宫颈癌ⅠA期以及上皮性卵巢肿瘤行子宫切除术的患者11例为对照组。对两组患者EMI平滑肌细胞进行分离及原代培养。采用蛋白印迹法检测两组细胞CB1表达及通路相关蛋白AKT、MAPK/Erk1表达情况;采用CB1受体拮抗剂AM251和激动剂ACEA处理腺肌病组EMI平滑肌细胞,蛋白印迹法检测各组细胞中CB1表达及通路相关蛋白AKT、MAPK/Erk1蛋白表达情况,采用CCK8检测各组细胞增殖情况。结果(1)腺肌病组CB1(0.64±0.16 vs 0.50±0.11)、p-AKT(0.48±0.08 vs 0.27±0.08)以及p-Erk1/2(2.17±0.82 vs 1.18±0.35)的表达量明显高于对照组,差异有统计学意义(P<0.05)。(2)ACEA与AM251分别单独作用腺肌病EMI平滑肌细胞后存活率分别为(169.9±18.3)%、(41.5±3.5)%,与对照组(将对照组设为100%)相比,差异均有统计学意义(P<0.001)。而联合用药组细胞存活率为(103.4±11.5)%,与对照组相比,差异无统计学意义(P>0.05)。(3)AM251作用前后,细胞凋亡率分别为(32.75±13.00)%及(12.89±4.16)%,而ACEA作用后则为(8.08±1.86)%,差异均有统计学意义(P<0.05),而联合用药前后比较,差异无统计学意义(P>0.05)。(4)ACEA能提高ADS组子宫EMI区平滑肌细胞CB1蛋白、磷酸化AKT及MAPKs家族中ERKl/2的磷酸化水平,而AM251使其下调,与对照组相比,差异均有统计学意义(P<0.05)。结论大麻素受体CB1可通过激活AKT和MAPK/Erk信号通路促进腺肌病EMI平滑肌细胞的体外增殖并抑制其凋亡,从而参与子宫腺肌病的发生发展。

Objective To observe the expression of cannabinoid receptor CB1 in endometrial-myometrial interface(EMI) smooth muscle cells of adenomyosis and its effect on cell proliferation and apoptosis.Methods From July 2017 to August 2018,16 patients who underwent hysterectomy for adenomyosis at Beijing Obstetrics and Gynecology Hospital,Capital Medical University were selected as the ADS group.And 11 patients with cervical intraepithelial neoplasia grade III,cervical cancer stage IA and epithelial ovarian tumors who underwent hysterectomy were selected as the control group.EMI smooth muscle cells from the uterus of two groups were isolated and primary cultured.Western blotting was used to detect the expression of CB1,mitogen-activated protein kinase(MAPK) signaling related-proteins AKT and MAPK/Erk1 in the EMI smooth muscle cells of two groups;CB1 receptor antagonist AM251 and agonist ACEA were used to treat uterine EMI smooth muscle cells in ADS group, and western blotting was used to detect CB1 expression and MAPK signaling-related proteins AKT,MAPK/Erk1 protein expression, CCK8 was used to detect cell proliferation in each group.Results(1) The expression level of CB1,p-AKT and p-Erk1/2 in ADS group were significantly higher than those in control group(0.64±0.16 vs 0.50±0.11,0.48±0.08 vs 0.27±0.08,2.17±0.82 vs 1.18±0.35,respectively),the differences were statistically significant(P<0.05).(2) After treating the cells of adenomyosis group with ACEA alone and AM251 alone, the cell survival rate was(169.9±18.3)% and(41.5±3.5)%.Compared with the control group(cell survival rate as 100%),the differences were statistically significant(P<0.001).While treating the cells of adenomyosis group with combined ACEA and AM251,the cell survival rate was(103.4±11.5)%,which was not statistically significant compared with the control group(P>0.05).(3) Before and after AM251 treatment, the apoptosis rate was(32.75±13.00)% and(12.89±4.16)%.After ACEA,the results showed that was(8.08±1.86)%.The differences were statistically significant(P<0.05),but there was no statistical significance between before and after the combined treatment(P>0.05).(4) ACEA can increase the expression of CB1 protein, phosphorylation of AKT and ERK1/2 in MAPKs family, while AM251 downregulated them in ADS group.Compared with the control group, the differences were statistically significant(P<0.05).Conclusion The cannabinoid receptor CB1 play a key role in the development of ADS,which can promote the proliferation of EMI smooth muscle cells from adenomyosis in vitro and inhibit their apoptosis by activating the AKT and MAPK/Erk signaling pathways.