Ⅱ型大麻素受体抑制剂对纳米级钛颗粒诱导 Ana －1细胞影响的实验研究

Effectso f the CB2 in hibitor AM630 on nanoscale titaniu m particles -induced Ana -1 cell s

目的：观察Ⅱ型大麻素受体（CB2）选择性抑制剂 AM630对纳米级钛（Ti）颗粒诱导下 Ana －1细胞的影响。方法实验分5组，即空白组、颗粒组（Ti 0．5 g／L）、颗粒＋AM63050 nmol／L（AM1）组、颗粒＋AM630100 nmol／L（AM2）组、颗粒＋AM630200 nmol／L（AM3）组。将 Ana －1细胞在不同浓度 AM630（50、100、200 nmol／L）中培养，采用 CCK －8法检测12、24、48 h 后细胞增殖活性，采用酶联免疫吸附试验（ELISA）检测各组细胞培养液中肿瘤坏死因子α（TNF －α）、白细胞介素－1β（IL－1β）含量，采用蛋白质印迹法（Western Blotting）检测 TNF －α、IL －1β、细胞外调节蛋白激酶（ERK）、磷酸化细胞外调节蛋白激酶（p －ERK）的表达水平。结果不同浓度的AM630在各时点对 Ana －1细胞增殖均无影响。颗粒组各时点 Ana－1细胞上清液中 TNF －α、IL －1β的含量与空白组相比明显升高，差异有统计学意义（P ＜0．01），并且随着时间的延长，炎症因子的含量递增；加入不同浓度AM630后，各时间点细胞上清液中 TNF －α、IL －1β的含量较颗粒组呈递减趋势，差异有统计学意义（P ＜0．05）。颗粒组 p －ERK 水平较空白组明显增加，而加入不同浓度 AM630后，p －ERK 水平逐渐降低，差异均具有统计学意义（P＜0．05或 P ＜0．01）。结论CB2选择性抑制剂 AM630能通过抑制 ERK 磷酸化表达水平来降低纳米级钛颗粒诱导的 Ana －1细胞炎性因子的表达。

Objec tive To investigate the effects of AM630, a selective inhibitor of cannabinoid receptor type Ⅱ（CB2） on nanoscale titanium particles （Ti） -induced Ana -1 cells.Meth ods The cells were divided into five groups, according to their exposure to vehicle, particles （Ti 0.5 mg/ml）, particles ＋50 nmol/L AM630, particles ＋100nmol /L AM630, or particles ＋200 nmol/L AM630.Then,their proliferation was measured after 12, 24, and 48 hours of treatment using the cell counting Kit -8 （CCK -8）.The levels of TNF -αand IL -1βin culture media were examined through ELISA.The amounts of TNF -α, IL -1β, ERK and p -ERK were detected by Western Blotting.Results The administration of AM630 did not result in any changes in the proliferation of Ana -1 cells at any concentration discussed. According to ELISA results, the levels of TNF -αand IL -1βwere remarkably increased within the supernatant of cells exposed to the particles alone at all time points, compared with the vehicle group （P 〈0.01）, which further raised as the exposure continued.However, after the addition of various concentrations of AM630, obvious decreases were seen in the quantities of TNF -αand IL -1βat all time points, in comparison with the particle group （P 〈0.05）.The expression of p -ERK in the particle group was significantly higher than that after exposure to the vehicle alone.In contrast, the a-mounts of p -ERK were obviously down -regulated, with the increase of the concentration of AM630 （P 〈0.05 or P 〈0.01）.Conclusion The CB2 selective inhibitor AM630 can reduce the amounts of inflammatory factors within nanoscale titanium particles -induced Ana -1 cells via inhibition of ERK phosphorylation.