木犀草素对子痫前期大鼠滋养层细胞凋亡的影响及其机制

The effects of luteolin on apoptosis of trophoblast cells in preeclampsia rats and the mechanisms

目的探讨木犀草素(LUT)对子痫前期(PE)大鼠滋养层细胞凋亡的影响及其机制。方法取妊娠10 d SD大鼠,按随机数字表法随机分为对照组、模型组、20、40、60 mmol/L LUT(LUT-L、LUT-M、LUT-H)组,每组各12只,模型组和给药组大鼠皮下注射100 mg/(kg·d)亚硝基左旋精氨酸甲酯建立PE大鼠模型,对照组大鼠皮下注射等量生理盐水,每天1次,注射6 d。妊娠16 d的大鼠分别予以20、40、60 mmol/L LUT腹腔注射,对照组、模型组大鼠腹腔注射等量生理盐水,每天1次,注射5 d。测量各组大鼠妊娠10、16、21 d尾动脉血压及24 h尿蛋白水平;妊娠21 d,原位末端标记法(TUNEL)检测滋养层组织细胞凋亡情况,Western blot法检测滋养层组织B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋(Bax)、磷脂酰肌醇3-激酶(PI3K)、磷酸化PI3K(p-PI3K)、蛋白激酶B(Akt)、磷酸化AKT(p-Akt)、内皮型一氧化氮合酶(eNOS)和磷酸化eNOS(p-eNOS)蛋白表达量。多组间比较采用单因素方差分析,组间两两比较采用SNK-q检验。结果妊娠10 d,各组大鼠尾动脉收缩压、舒张压、24 h尿蛋白含量差异无统计学意义;妊娠16 d,与对照组比较,模型组、LUT-L组、LUT-M组、LUT-H组大鼠尾动脉收缩压、舒张压、24 h尿蛋白含量升高(P均<0.05);妊娠21 d,与对照组比较,模型组、LUT-L组、LUT-M组、LUT-H组收缩压[(110.33±3.67)比(147.28±4.16),(131.29±4.31),(124.46±4.27),(118.54±4.18)mmHg]、24 h蛋白尿、细胞凋亡率[(1.38±0.34)%,(43.45±3.72)%,(39.21±3.53)%,(27.86±3.41)%,(23.21±3.28)%]和Bax蛋白表达量均升高;Bcl-2、p-PI3K/PI3K(1.06±0.09比0.25±0.02,0.37±0.03,0.57±0.06,0.73±0.08)、p-Akt/Akt(0.87±0.08比0.11±0.01,0.23±0.03,0.56±0.07,0.78±0.06)和p-eNOS/eNOS蛋白表达水平(0.85±0.07比0.09±0.01,0.16±0.02,0.38±0.04,0.69±0.07)均降低(P均<0.05)。与模型组比较,LUT-L组、LUT-M组、LUT-H组大鼠尾动脉收缩压、舒张压、滋养层组织细胞凋亡率和Bax蛋白表达量降低,Bcl-2、p-PI3K/PI3K、p-Akt/Akt和p-eNOS/eNOS蛋白表达量升高(P均<0.05)。结论LUT可抑制PE大鼠滋养层组织细胞凋亡,其机制可能与PI3K/Akt/eNOS信号通路激活,调控凋亡相关蛋白表达有关。

Objective To investigate the effects of luteolin(LUT)on trophoblast cells in preeclampsia rats and the mechanisms.Methods Ten day gestational SD rats were randomly divided into normal control(NC),model(M)and LUT groups with different concentrations(20,40 and 60μmol/L)(LUT-L,LUT-M,LUT-H),and there were 12 rats in each group.The rats in the model and the administration groups were subcutaneously injected with 100 mg/(kg·d)nitrosyl L-arginine methyl ester once a day for 6 days.And the rats in the control group were subcutaneously injected with the same amount of normal saline.Then,16-day gestational rats in LUT-L,LUT-M and LUT-H groups were intraperitoneally injected with LUT 20,40 and 60μmol/L respectively,and the 16-day gestational rats of in NC group and M group were intraperitoneally injected with the same amount of normal saline,once a day for 5 days.The tail artery blood pressure and 24 h urine protein were measured on the 10th,16th and 21st day of pregnancy;on the 21st day of gestation,apoptosis of trophoblast was detected by TUNEL,and the expressions of B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),phosphatidylinositol-3 kinase(PI3K),phospho-PI3K(p-PI3K),protein kinase B(Akt),phospho-AKT(p-Akt),endothelial nitric oxide synthase(eNOS)and phospho-eNOS(p-eNOS)proteins in trophoblast were detected by Western blot.One-way ANOVA was used for comparison between groups,SNK-q test was used for further comparison between the two groups.Results On the 10th day of gestation,there was no significant difference in systolic blood pressure and diastolic blood pressure of tail artery and 24-hour urinary protein content between each group(P>0.05);on the 16th day of gestation,compared with NC group,the systolic blood pressure and diastolic blood pressure of tail artery and 24-hour urinary protein content were significantly increased in M,LUT-L,LUT-M and LUT-H groups(P<0.05);on the 21st day of gestation,compared with NC group,the systolic blood pressure(110.33±3.67 vs 147.28±4.16,131.29±4.31,124.46±4.27,118.54±4.18)24-hour urinary protein content,the apoptosis rates(1.38±0.34 vs 43.45±3.72,39.21±3.53,27.86±3.41,23.21±3.28),and the protein expression of Bax were significantly increased(all P<0.05);and protein expression of Bcl2,p-PI3K/PI3K(1.06±0.09 vs 0.25±0.02,0.37±0.03,0.57±0.06,0.73±0.08),p-Akt/Akt(0.87±0.08 vs 0.11±0.01,0.23±0.03,0.56±0.07,0.78±0.06),p-eNOS/eNOS(0.85±0.07 vs 0.09±0.01,0.16±0.02,0.38±0.04,0.69±0.07)were significantly decreased(P<0.05);compared with M group,the systolic blood pressure,diastolic blood pressure of tail artery,24-h urinary protein apoptotic rate of trophoblast and Bax protein expression were significantly decreased in LUT-L,LUT-M,and LUT-H groups(P<0.05),where as the expressions of Bcl-2,p-PI3K/PI3K,p-Akt/Akt,p-eNOS/eNOS proteins were significantly increased(all P<0.05).Conclusions LUT can inhibit the apoptosis of trophoblast in PE rats,and PI3K/Akt/eNOS signaling pathway maybe involve in regulating these apoptotic related proteins.