加味三棱丸对人子宫内膜异位症内膜雌激素分泌及血管生成的影响

Effects of Jiawei Sanlengwan on estrogen secretion and angiogenesis of human endometrium of endometriosis

目的研究加味三棱丸(SLW)对子宫内膜异位症(内异症)内膜细胞雌二醇分泌及血管内皮生长因子(VEGF)表达、分泌水平的影响,并进一步探讨其抑制血管生成能力的机制。方法培养子宫肌瘤和内异症病人在位子宫内膜细胞。以子宫肌瘤为对照,内异症在位内膜细胞分别经SLW高、中、低剂量(5.0、2.5、1.25g·kg-1·d-1,ig,bid×7d)给药大鼠血清处理后,在不同的时间点采用电化学发光免疫法检测内膜细胞雌二醇的分泌水平;采用免疫印迹(Western blot)法和酶联免疫吸附测定(ELISA)法,考察SLW中剂量(2.5g·kg-1·d-1)对VEGF表达和分泌的时效关系,明确SLW2.5g·kg-1·d-1抑制VEGF表达和分泌的最佳时间。随后设子宫肌瘤对照组,并将内异症在位内膜细胞分为:内异症对照组、SLW(2.5g·kg-1·d-1)组、阿那曲唑组、SLW(2.5g·kg-1·d-1)+雌二醇组和雌二醇组,并按上述方法在最佳时间点检测VEGF蛋白的表达及分泌水平。结果SLW高、中剂量可显著降低内异症在位内膜细胞分泌的雌二醇水平,并呈时间及剂量依赖性(P<0.01)。Western blot和ELISA法检测结果显示,内异症对照组在各检测时间内,VEGF蛋白的表达和分泌水平均明显高于子宫肌瘤对照组(P<0.01),经SLW2.5g·kg-1·d-1给药小鼠的血清处理24h后,VEGF蛋白的表达和分泌水平较内异症对照组显著降低(P<0.01);SLW2.5g·kg-1·d-1给药小鼠的血清与1.34ng·L-1雌二醇联合处理24h后,被SLW抑制的VEGF蛋白表达和分泌水平可被反向添加的雌二醇部分或完全恢复;雌二醇单独处理24h,VEGF蛋白的表达和分泌水平较内异症对照组明显增高(P<0.01),而阿那曲唑(0.1mg·kg-1·d-1)的作用恰好相反。结论SLW可能通过降低内异症在位内膜细胞雌二醇的分泌水平,从而降低VEGF的表达和分泌,发挥其抑制血管生成的作用。

AIM To study the effect of Jiawei Sanlengwan （SLW） on the secretion level of estradiol （E2） and the expression and secretion level of vascular endothelial growth factor （VEGF） in endometrial cells of endometriosis, and then to approach the mechanism of inhibiting angiogenic potential of endometriosis. METHODS The eutopic endometrial ceils of hysteromyoma and endometriosis patients were cultured, and the secretion level of E2 in endometrial cells in the culture media supematant at different time point with the treatment of serum of rats ig SLW （5.0, 2.5, 1.25 g·kg^-1·d^-1, ig, bid × 7 d） was detected by electrochemilu- minescence immunoassay, then the expression and the secretion level of VEGF with the treatment of serum of rats ig SLW 2.5 g·kg^-1·d^-1 was analyzed by Western blot and ELISA, respectively. After the optimal time for SLW 2.5g·kg^-1·d^-1 to inhibit the expression and secretion level of VEGF was identified based on the time-effect relationship, another endometrial cells were divided into six groups： hysteromyoma control group, endometriosis control group, endometriosis ＋ SLW （2.5 g·kg^-1·d^-1） group, endometriosis ＋ anastrozole group, endometriosis ＋ SLW （2.5 g·kg^-1·d^-1） ＋ E2 group, and endometriosis ＋ E2 group. The expression and secretion level of VEGF in endometrial cells of each group was detected according to the optimal time point respectively. RESULTS The secretion level of E2 of eutopic endometria with endometriosis could be decreased by high dose and middle dose of SLW （5.0, 1.25 g·kg^-1·d^-1）, which showed the dependence of time and dose. The result of Western blot and ELISA indicated that the expression and the secretion level of VEGF protein of the endometriosis control group were significantly higher than that of the hysteromyoma control group at each time point （P 〈 0.01）. After the 24-hour treatment with serum of rats admistrated by SLW 2.5g·kg^-1·d^-1, the expression and secretion level of VEGF protein was significantly lower than that of the endometriosis control group （P 〈 0.01 ）. After the 24-hour combined treatment of 1.34 ng·L^-1 E2 and serum of rats admistrated by SLW 2.5 g·kg^-1·d^-1, the angiogenesis promoting effect of eutopic endometrial cells reversed by SLW could be partly or completely recovered by E2, and there was no statistical difference （P 〉 0.05） in the secretion level of VEGF protein compared with the endometriosis control group. With the treatment of 1.34 ng·L^-1 E2 for 24 h, the expression and secretion level of VEGF protein in eutopic endometrial cells was significantly higher than that of the endometriosis control group （P 〈 0.01） , but anastrozole 0.1 mg·kg^-1·d^-1 had the contrary effect to E2. CONCLUSION SLW could decrease the secretion of E2 of the eutopic endometrial cells so as to inhibit the expression and secretion of VEGF to play a role in inhibition of angiogenesis in the eutopic endometrial cells of endometriosis.