滋肾活血法联合低分子肝素改善小鼠复发性流产及对VEGF/VEGFR-2/p-ERK/ERK信号通路的影响

目的 通过观察滋肾活血法联合低分子肝素对复发性流产小鼠流产率及蜕膜中血管内皮生长因子/血管内皮生长因子受体2/磷酸化的细胞外调节蛋白激酶/细胞外调节蛋白激酶（VEGF/VEGFR-2/p-ERK/ERK）信号通路的影响，以探讨其作用机制。方法 68只未孕CBA/J雌鼠，26只DBA/2雄鼠，8只BALB/c雄鼠，按雌：雄=2：1，建立CBA/J×DBA/2习惯性流产小鼠模型50只，随机数字法分为模型组（蒸馏水）、LMWH组（1000U/kg）、滋肾活血方低剂量（3.9g/kg）+LMWH（1000U/kg）组、滋肾活血方中剂量（11.7g/kg）+LMWH（1000U/kg）组、滋肾活血方高剂量（35.1g/kg）+LMWH（1000U/kg）组，每组10只；建立CBA/J×BALB/c正常妊娠小鼠模型10只作为正常对照组（蒸馏水）。按照人鼠体表面积换算每日滋肾活血方灌胃药量和LMWH注射量，给药干预2周。计算各组小鼠胚胎丢失率；免疫组化法测定小鼠蜕膜中VEGF蛋白的表达；蛋白质印迹法（Western blot）测定小鼠蜕膜中VEGF、VEGFR-2、p-ERK、ERK表达。结果 与正常对照组比较，模型组胚胎丢失率上升（38.38%比7.29%，P<0.05），该组免疫组化VEGF蛋白平均光密度值降低（0.22±0.07）比（0.23±0.01），P<0.01]，该组WB结果显示VEGF、VEGFR-2蛋白表达下降VEGF：（2.01±0.08）比（2.90±0.06）；VEGFR-2：（2.62±0.01）比（3.15±0.08），P均<0.05]。与模型组比较，LMWH组、滋肾活血方低剂量+LMWH组、滋肾活血方中剂量+LMWH组和滋肾活血方高剂量+LMWH组胚胎丢失率下降（23.76%、16.83%、12.75%、10.10%比38.38%，P均<0.05），免疫组化结果显示上述4组的VEGF蛋白平均光密度值上升（0.24±0.04）、（0.29±0.02）、（0.32±0.06）、（0.33±0.10）比（0.22±0.07），P均<0.05]，WB结果显示上述4组的VEGF、VEGFR-2、p-ERK、ERK蛋白表达上升VEGF：（3.13±0.09）、（2.93±0.73）、（3.31±0.18）、（3.38±0.15）比（2.01±0.08）；VEGFR-2：（3.19±0.02）、（3.53±0.10）、（3.77±0.12）、（3.83±0.09）比（2.62±0.01）；p-ERK：（1.57±0.35）、（1.79±0.06）、（1.73±0.07）、（1.75±0.06）比（1.39±0.04）；ERK：（3.82±0.08）、（4.22±0.09）、（4.24±0.04）、（4.19±0.27）比（3.42±0.14），P均<0.05]。与LMWH组比较，滋肾活血方低剂量+LMWH组胚胎丢失率无明显差异（P＞0.05），滋肾活血方中剂量+LMWH组、滋肾活血方高剂量+LMWH组胚胎丢失率降低更显著（P均<0.05）；免疫组化结果显示VEGF蛋白平均光密度值在滋肾活血方低剂量+LMWH组、滋肾活血方中剂量+LMWH组、滋肾活血方高剂量+LMWH组上升（P均<0.05）；WB结果显示上述3组的VEGFR-2、p-ERK、ERK蛋白表达上升（P均<0.05），VEGF蛋白表达无明显差异（P均>0.05）。结论 滋肾活血法联合LMWH能够改善复发性流产小鼠妊娠结局，其机制可能与激活VEGF/VEGFR-2/p-ERK/ERK信号通路有关。

Kidney-nourishing and blood-activating therapy combined with low molecular weight heparin improves recurrent abortion in mice and its effects on VEGF/VEGFR-2/p-ERK/ERK signaling pathway

Objective To investigate the effects of kidney-nourishing and blood-activating combined with low molecular weight heparin on abortion rate and VEGF/VEGFR-2/p-ERK/ERK signaling pathway in decidua of mice with recurrent abortion. Methods 68 non-pregnant CBA/J female mice, 26 DBA/2 male mice and 8 BALB/c male mice, which according to female: male=2:1, were used to establish 50 mice of CBA/J×DBA/2 habitual abortion mouse model and 10 mice of normal pregnancy mouse model. 50 mice of habitual abortion model were randomly divided into model group (distilled water), LMWH group (1000U/kg), low-dose Zishenhuoxue Decoction (3.9g/kg) + LMWH (1000U/kg) group, medium-dose Zishenhuoxue Decoction (11.7g/kg) + LMWH (1000U/kg) group and high-dose Zishenhuoxue Decoction (35.1g/kg) + LMWH (1000U/kg) group, and 10 mice of normal pregnant model were used as normal control group (distilled water). The daily dose of gavage of Zishenhuoxue Decoction and LMWH injection were calculated according to conversion formula of human and mice surface area. The time of treatment was 2 weeks. Calculate the rate of mouse embryo loss in each group; immunohistochemical method was used to detect the expression of VEGF protein in mouse decidua; Western blot method was used to detect the protein expression of VEGF, VEGFR-2, p-ERK and ERK in mouse decidua. Results Compared with the normal control group, the embryo loss rate in model group was increased (38.38% vs 7.29%, P <0.05), immunohistochemistry showed that the mean optical density value of VEGF protein in model group decreased (0.22±0.07) vs (0.23±0.01), P < 0.01], and Western Blot method showed that the protein expression of VEGF and VEGFR-2 in model group decreased VEGF: (2.01±0.08) vs (2.90±0.06); VEGFR-2: (2.62±0.01) vs (3.15±0.08), P < 0.05]. Compared with model group, the embryo loss rate in LMWH group, Zishenhuoxue Decoction low-dose + LMWH group, Zishenhuoxue Decoction medium-dose + LMWH group and Zishenhuoxue Decoction high-dose + LMWH group decreased (23.76%, 16.83%, 12.75%, 10.10% vs 38.38%, P < 0.05), immunohistochemistry showed the mean optical density value of VEGF protein increased in LMWH group, Zishenhuoxue Decoction low-dose + LMWH group, Zishenhuoxue Decoction medium-dose + LMWH group and Zishenhuoxue Decoction high-dose + LMWH group (0.24±0.04), (0.29±0.02), (0.32±0.06), (0.33±0.10) vs (0.22±0.07), P < 0.05], and Western Blot method showed the protein expression of VEGF, VEGFR-2, p-ERK, ERK increased in LMWH group, Zishenhuoxue Decoction low-dose + LMWH group, Zishenhuoxue Decoction medium-dose + LMWH group and Zishenhuoxue Decoction high-dose + LMWH group VEGF: (3.13±0.09), (2.93±0.73), (3.31±0.18), (3.38±0.15) vs (2.01±0.08); VEGFR-2: (3.19±0.02), (3.53±0.10), (3.77±0.12), (3.83±0.09) vs (2.62±0.01); p-ERK: (1.57±0.35), (1.79±0.06), (1.73±0.07), (1.75±0.06) vs (1.39±0.04); ERK: (3.82±0.08), (4.22±0.09), (4.24±0.04), (4.19±0.27) vs (3.42±0.14), all P < 0.05]. Compared with LMWH group, there was no significant difference on embryo loss rate in Zishenhuoxue Decoction low-dose + LMWH group (P > 0.05), whereas the embryo loss rate of Zishenhuoxue Decoction medium-dose + LMWH group and Zishenhuoxue Decoction high-dose + LMWH group decreased significantly (P < 0.05), immunohistochemistry showed the mean optical density of VEGF protein increased in Zishenhuoxue Decoction low-dose + LMWH group, Zishenhuoxue Decoction medium-dose + LMWH group and Zishenhuoxue Decoction high-dose + LMWH group (P < 0.05), Western Blot method showed the protein expression of VEGFR-2, p-ERK, ERK in Zishenhuoxue Decoction low-dose + LMWH group, Zishenhuoxue Decoction medium-dose + LMWH group and Zishenhuoxue Decoction high-dose + LMWH group increased (P < 0.05) and there was no significant difference on the protein expression of VEGF in Zishenhuoxue Decoction low-dose + LMWH group, Zishenhuoxue Decoction medium-dose + LMWH group and Zishenhuoxue Decoction high-dose + LMWH group (P > 0.05). Conclusion Kidney-nourishing and blood-activating therapy combined with LMWH can improve the pregnancy outcome of recurrent abortion mice, and the mechanism may be related to the activation of VEGF/VEGFR-2/p-ERK/ERK signaling pathway.