基于Wnt通路探讨温阳化浊方含药血清对Ishikawa和JAr细胞胚胎种植模型的影响

The Effect of Wenyang Huazhuo Formula(温阳化浊方)-containing Serum on Ishikawa and JAr Cell Embryo Implantation Model based on Wnt Pathway

目的 探讨温阳化浊方促进胚胎种植的可能作用机制。方法 将20只大鼠随机分为温阳化浊方组和空白组,每组10只。温阳化浊方组大鼠给予温阳化浊方药液21.05 g/kg连续灌胃7天,空白组不予灌胃,制备温阳化浊方含药血清和空白血清。体外培养THP-1单核细胞,佛波酯(PMA)诱导分化成巨噬细胞,再用20 ng/ml脂多糖(LPS)和20 ng/ml γ干扰素(IFN-γ)诱导巨噬细胞M1极化。M1型巨噬细胞分别加入2%、4%、8%、10%、15%、20%的温阳化浊方含药血清和空白血清培养,采用CCK8法检测细胞存活率,选取最大无毒剂量的温阳化浊方含药血清进行后续实验。M1型巨噬细胞分为:模型1组、温阳化浊方含药血清1组、空白血清1组,在诱导M1极化后,温阳化浊方含药血清1组给予筛选浓度的温阳化浊方含药血清,空白血清1组给予同浓度的空白血清,各组均干预24 h和48 h,Real-time PCR法检测M1型巨噬细胞标志物肿瘤坏死因子α(TNF-α)、白细胞介素1β (IL-1β)和M2型巨噬细胞标志物趋化因子配体18 (CCL-18)、甘露糖受体C型1 (MRC-1) mRNA表达。采用Transwell体系共培养M1型巨噬细胞、Ishikawa细胞和JAr细胞,建立体外胚胎种植模型。将胚胎种植模型细胞分为:非容受态组(只包含Ishikawa细胞)、容受态组(包含Ishikawa细胞和JAr细胞)、模型2组(M1型巨噬细胞与Ishikawa细胞、JAr细胞共培养)、温阳化浊方含药血清2组和空白血清2组。温阳化浊方含药血清2组和空白血清2组在M1型巨噬细胞与Ishikawa细胞共培养的同时,分别给予筛选浓度的温阳化浊方含药血清和空白血清干预48 h,再与JAr细胞共培养。Realtime PCR方法检测Wnt通路相关因子Wnt4、Wnt6、Wnt7b、Wnt11、β-连环蛋白(β-catenin)和子宫内膜容受性标志分子整合素αV (ITGαV)、整合素β3 (ITGβ3)、整合素β5 (ITGβ5)、E-钙黏蛋白(E-cadherin)、L-选择素(L-selectin)的mRNA表达;免疫荧光法检测Wnt通路下游分子原癌基因(C-Jun)、间质上皮转换因子(Met)、淋巴细胞增强结合因子1 (LEF1)的表达;因非容受态组无JAr细胞球黏附,观察其余各组的JAr细胞黏附率。结果 根据细胞存活率结果,选择10%温阳化浊方含药血清进行后续实验。干预48 h时,与模型1组比较,温阳化浊方含药血清1组TNF-α和IL-1β mRNA表达显著降低,CCL-18和MRC-1 mRNA表达显著升高(P<0.01),空白血清1组TNF-α mRNA表达显著降低(P<0.05);与温阳化浊方含药血清1组比较,空白血清1组CCL-18和MRC-1 mRNA表达均显著降低(P<0.01)。与非容受态组比较,容受态组细胞中Wnt4、Wnt6、Wnt7b、Wnt11、β-catenin、ITGαV、ITGβ3、ITGβ5、E-cadherin、L-selectin mRNA及C-Jun蛋白表达显著升高(P<0.05或P<0.01);与容受态组比较,模型2组Wnt4、Wnt6、Wnt7b、Wnt11、β-catenin、ITGαV、ITGβ3、ITGβ5、E-cadherin、L-selectin mRNA及C-Jun蛋白表达,JAr细胞的黏附率显著降低(P<0.05或P<0.01);与模型2组比较,温阳化浊方含药血清2组Wnt4、Wnt6、Wnt7b、Wnt11、β-catenin、ITGαV、ITGβ3、ITGβ5 mRNA及C-Jun蛋白表达,JAr细胞的黏附率均显著升高(P<0.01),空白血清2组各指标差异无统计学意义(P>0.05)。结论 温阳化浊方含药血清通过抑制M1型巨噬细胞极化,上调Wnt通路,提高子宫内膜容受性,从而促进慢性子宫内膜炎的胚胎种植。

Objective To explore the possible mechanism of Wenyang Huazhuo Formula(温阳化浊方,WHF)in promoting embryo implantation.Methods Twenty rats were randomly divided into WHF group and blank group,with 10 rats in each group.Rats in the WHF group were given 21.05 g/kg of WHF liquid by gavage for 7 days,while those in the blank group was not given intragastric administration,so as to prepare WHF-containing serum and blank serum.THP-1 monocytes were cultured in vitro and were differentiated into macrophages induced by phorbol ester(PMA).Furthermore,20 ng·ml^(-1) lipopolysaccharide(LPS)and 20 ng·ml^(-1) gamma Interferon(IFN)-γ)were used to induce M1 polarization of macrophages.M1 macrophages were cultured in the 2%,4%,8%,10%,15%,and 20%WHF-containing serum and blank serum.CCK8 method was used to detect cell viability,and WHF-containing serum having the nontoxic maximum content were selected for follow-up experiment.M1 macrophages were divided into three groups:model 1 group,WHF-containing serum 1 group,and blank serum 1 group.After M1 macrophages polarization,WHF-containing serum 1 group was given selected concentration of WHF-containing serum,while blank serum 1 group was given same concentration of blank serum for 24 and 48 hours.Real-time fluorescence quantitative PCR(Real-time PCR)was used to detect mRNA expression of M1 macrophage markers including tumor necrosis factor-α(TNF-α)and interleukin 1β(IL-1β)and M2 macrophage markers including chemokine ligand 18(CCL-18)and mannose receptor type C-l(MRC-1).Transwell system was used to co-culture M1 macrophages,Ishikawa cells and JAr cells to establish an in vitro embryo implantation model.These cells were divided into five groups:non-receptive group(only Ishikawa cells),receptive group(including Ishikawa cells and JAr cells),model 2 group(M1 macrophages co-cultured with Ishikawa cells and JAr cells),WHF-containing serum 2 group and blank serum 2 group.While M1 macrophages were co-cultured with Ishikawa cells,the latter two groups were administered with selected concentration of WHF-containing serum and blank serum respectively for 48 hours,and then were co-cultured with JAr cells.Real-time PCR was used to detect mRNA expression of Wnt pathway related factors including Wnt4,Wnt6,Wnt7 b,Wnt11 andβ-catenin and endometrial receptive markers including integrinαV(ITGαV),integrinβ3(ITGβ3),integrinβ5(ITGβ5),E-cadherin and L-selectin.Immunofluorescence assay was used to detect the expression of downstream molecules of Wnt pathway including proto-oncogene(C-Jun),mesenchymal transition factor(Met)and lymphocyte enhancer binding factor 1(LEF1).JAr cell adhesion rate was measured in all the groups except for non-receptive group where there was no JAr cell spheroid adhesion.Results According to the results of cell viability,10%WHF-containing serum was used for subsequent experiments.After 48 hours,compared to those in the model 1 group,the mRNA expression of TNF-αand IL-1βwere reduced significantly,while CCL-18 and MRC-1 mRNA expression increased in the WHF-containing serum 1 group(P<0.01),and TNF-αmRNA expression significantly decreased in the blank serum 1 group(P<0.05).The mRNA expression of CCL-18 and MRC-l were significantly lower in the blank serum 1 group than the WHF-containing serum 1 group(P<0.01).Compared to the nonreceptive group,the receptive group had significantly increased mRNA expressions of Wnt4,Wnt6,Wnt7 b,Wnt11,β-catenin,ITGαV,ITGβ3,ITGβ5,E-cadherin and L-selectin and protein expression of C-Jun(P<0.05 or P<0.01).Compared to the receptive group,the model 2 group had significantly decreased mRNA expressions of Wnt4,Wnt6,Wnt7 b,Wnt11,β-catenin,ITGαV,ITGβ3,ITGβ5,E-cadherin and L-selectin,protein expression of C-Jun,and the adhesion rate of JAr cells(P<0.05 or P<0.01).Compared to those in the model 2 group,the mRNA expressions of Wnt4,Wnt6,Wnt7 b,Wnt11,β-catenin,ITGαV,ITGβ3 and ITGβ5,protein expression of C-Jun,and the adhesion rate of JAr cells increased significantly in the WHF-containing serum 2 group(P<0.01),while there was no significant differences with blank serum 2 group(P>0.05).Conclusion WHF-containing serum can inhibit the polarization of M1 macrophages,up-regulate Wnt pathway,and improve endometrial receptivity,thereby promoting embryo implantation in chronic endometritis.