清热祛瘀固肾方对anti-β2GPI诱导下的滋养层细胞TLR4/MyD88信号通路的影响

摘要

[目的]以Toll样受体4(Toll-like receptor 4,TLR4)/髓样分化因子(myeloid differentiation factor88，MyD88)通路为切入点，探讨清热祛瘀固肾方（简称清固方）对抗β2糖蛋白I抗体（anti-beta 2 glycoprotein I antibody, anti-β2GPI）诱导的人胎盘滋养层细胞HTR8损害的作用及机理。[方法]将40只雌性Wistar大鼠，随机分为清固方高、中、低剂量组和正常组。分组用药10d后心脏取血,分别制备含不同浓度清固方血清和正常血清。构建MyD88和TLR4真核表达质粒，分别转染HTR8细胞。两组HTR8细胞各分成清固方高、中、低剂量组、肝素组、阴性对照组和空白对照组。阴性对照组及空白对照组给予正常血清，其它组则给予相应的含药血清干预。除空白对照组外，其它各组与小鼠单克隆anti-β2 GPI共孵育后采用流式细胞术检测细胞凋亡情况，酶联免疫吸附试验（enzyme linked immunosorbent assay,ELISA）检测细胞培养上清液中单核细胞趋化因子-1（monocyte hemotactic protein-1，MCP-1）、白细胞介素-1β（interleukin-1beta ，IL-1β）浓度。[结果]经anti-β2GPI诱导的转染TLR4真核表达质粒的HTR8细胞经各组含药血清处理后，清固方高、中、低剂量组IL-1β浓度低于肝素组和阴性对照组，清固方高剂量组及空白对照组MCP-1浓度低于肝素组，差异有统计学意义（P<0.05）；转染MyD88真核表达质粒的HTR8细胞经各组含药血清处理后，清固方高、中、低剂量组及空白组对照IL-1β、MCP-1浓度低于肝素组，阴性对照组IL-1β、MCP-1浓度高于其他各组，差异有统计学意义(P<0.05)。清固方高、中、低剂量组、肝素组总凋亡率均低于阴性对照组，差异有统计学意义（P<0.01），空白对照组总凋亡率低于其他各组，差异有统计学意义（P<0.01）。[结论]清固方通过干预TLR4/MyD88信号转导通路，抑制HTR8细胞表面β2-GPI/anti-β2GPI活化引起的IL-1β、MCP-1分泌和细胞凋亡。%Objective]To explore the associated mechanism of action of Qingre-quyu-gushen decoction (Qinggu decoction for short) on placental trophoblastic cells HTR8 damage induced by anti-β2 glycoproteinⅠantibody(anti-β2 GPI) by using the key point of TLR4/MyD88 signal path. [Methods] 40 female Wistar rats were randomly divided into four groups:high dose group, middle dose group, low dose group and normal blank group. After 10-day gavage via heart blood, the rats were sacrificed and serum was collected to culture the HTR8 cell with different concentration of serum containing traditional Chinese medicine and blank serum. Construction TLR4 and MyD88 eukaryotic expression plasmid and transferred them into human first trimester trophoblast cells HTR8 respectively. HTR8 cells from each of the two groups were divided into Qinggu decoction high-dose group, medium dose group, low-dose group, heparin group, negative control group and blank control group. The negative control group and blank control group were given normal blank group containing serum, and the other group was given corresponding drug intervention. In addition to blank control group, other groups were incubated with mouse monoclonal anti-β2 GPI, then detect apoptosis rate by flow cytometry(FCM) and detect cell culture supernatant of IL-1 beta and MCP-1 concentration by enzyme linked immune sorbent assay(ELISA). [Results]After the treatment of each group of containing serum, the concentrations of IL-1 beta of Qinggu decoction high-dose group, medium dose group and low-dose group in the culture supernatant of trophoblast cells transfection of TLR4 eukaryotic expression plasmid induced by anti-β2 GPI were lower than heparin group and negative control group, and the concentrations of MCP-1 of Qinggu decoction high-dose group was lower than the heparin group(P<0.05). After the treatment of each group of containing serum, the concentration of MCP-1 and IL-1 beta of Qinggu decoction high-dose group, medium dose group, low-dose group and blank group in the culture supernatant of trophoblast cells transfection of MyD88 eukaryotic expression plasmid Induced by anti-β2 GPI were lower than that in the heparin group, and the negative control group was higher than that in other groups(P<0.05). The total apoptosis rate of the high, medium and low dose groups and heparin group were lower than that of the negative control group(P<0.01), and the total apoptosis rate of blank control group was lower than that of other groups(P<0.01). [Conclusion] Qinggu decoction restrain IL-1β, MCP-1 secretion and cell apoptosis induced byβ2 GPI/anti-β2 GPI through the intervention of TLR4/MyD88 signal pathway.