活血消异方改善子宫内膜异位症大鼠卵泡发育的机制探讨

Effects of Huoxue Xiaoyi Decoction on follicular development in endometriosis rats

目的 从卵巢颗粒细胞凋亡与自噬相互作用的角度,探讨活血消异方对子宫内膜异位症大鼠卵泡发育的影响及作用机制。方法 将动情周期正常的48只SD大鼠分为空白组、假手术组各10只,剩余的分为供体组(n=5)和受体组(n=23)用于造模,造模成功后的20只大鼠又分为模型组、活血消异方组各10只。活血消异方组予活血消异方汤剂2 ml灌胃,空白组、假手术组、模型组均予蒸馏水2 ml灌胃,各组连续灌胃15 d后,检测大鼠血清活性氧(ROS)、总超氧化物歧化酶(T-SOD)、过氧化氢酶(CAT)水平。取各组大鼠的卵巢组织行HE染色,光镜下观察各级卵泡形态并计数;免疫组化染色定位颗粒细胞,并比较凋亡相关因子Bax、Bcl-2、Caspase-3的表达;TUNEL染色观察颗粒细胞凋亡情况并计算凋亡率;蛋白免疫印迹法(Western blot)检测卵巢组织中凋亡及自噬相关蛋白p-JNK、Bax、Bcl-2、Caspase-3、Beclin-1、LC3Ⅱ的表达;电镜观察各组大鼠卵巢颗粒细胞自噬体形态,并计算自噬体胞质面积比。结果 (1)与空白组、假手术组比较,模型组血清ROS含量显著升高,T-SOD、CAT水平显著降低(P<0.05);与模型组比较,活血消异方组血清ROS含量显著降低,T-SOD、CAT水平显著升高(P<0.05)。(2)与空白组、假手术组比较,模型组次级卵泡数目显著下降(P<0.05),卵巢颗粒细胞凋亡率显著升高(P<0.001);与模型组比较,活血消异方组次级卵泡数目显著升高(P<0.05),凋亡率显著降低(P<0.001)。(3)与空白组、假手术组比较,模型组卵巢颗粒细胞中Bax、Caspase-3蛋白表达显著升高(P<0.01),Bcl-2蛋白表达显著降低(P<0.001);与模型组比较,活血消异方组卵巢颗粒细胞中Bax、Caspase-3蛋白表达显著降低(P<0.01),Bcl-2蛋白表达显著升高(P<0.001)。(4)与空白组、假手术组比较,模型组卵巢组织中p-JNK、Bax、Caspase-3蛋白表达水平显著升高(P<0.05),Bcl-2、Beclin-1、LC3Ⅱ蛋白表达水平显著降低(P<0.05);与模型组比较,活血消异方组卵巢组织中p-JNK、Bax、Caspase-3蛋白表达水平显著降低(P<0.05),Bcl-2、Beclin-1、LC3Ⅱ蛋白表达水平显著升高(P<0.05)。(5)电镜下观察卵巢颗粒细胞中自噬体结果显示,4组间自噬体胞质面积比的差异均无统计学意义(P>0.05)。结论 活血消异方可能通过ROS-JNK信号通路,改善子宫内膜异位症大鼠氧化应激状态,减少卵巢颗粒细胞凋亡,启动颗粒细胞保护性自噬,改善子宫内膜异位症大鼠卵泡发育。

Objective:To explore the effect and the possible mechanism of Huoxue Xiaoyi Decoction on follicular development in endometriosis model rats based on the interaction between ovarian granulosa cell apoptosis and autophagy.Methods:Forty-eight SD rats with normal estrous cycles were divided into the blank group and the sham operation group,with 10rats in each group.The remaining rats were divided into the donor group(n=5)and the recipient group (n=23)for modeling.After successful modeling,20rats were furtherdivided into the model group and the Huoxue Xiaoyi Decoction group,with 10rats in each group.The ratsin Huoxue Xiaoyi Decoction group were given 2ml of Huoxue Xiaoyi Decoction by gavage,while the blankgroup,sham operation group and model group were all given 2ml of distilled water by gavage.Aftercontinuous gastric lavage for 15days,the levels of serum reactive oxygen species(ROS),total superoxidedismutase(T-SOD),and catalase(CAT)were measured.The ovarian tissues were taken from each groupof rats for HE staining to observe the morphology of follicles at all stage under light microscopy,and countthem.Immunohistochemical staining was used to locate granulosa cells and compare the expression ofapoptosis related factors Bax,Bcl-2,and Caspase-3.TUNEL staining was used to observe the apoptosis ofgranulosa cells and calculate the apoptosis rate.The apoptosis and expression of autophagy related proteinsincluding p-JNK,Bax,Bcl-2,Caspase-3,Beclin-1and LC3Ⅱin ovarian tissue were detected by Westernblot.The morphology of autophagosomes in ovarian granulosa cells of rats in each group was observedunder electron microscopy,and the cytoplasmic area ratio of autophagosomes was calculated.Results:(1)Compared with the blank group and the sham operation group,the serum ROS content inthe model group was significantly increased,while T-SOD and CAT levels were significantly decreased(P<0.05).Compared with the model group ,the serum ROS content in the Huoxue Xiaoyi Decoctiongroup was significantly decreased,while T-SOD and CAT significantly increased(P<0.05).(2)Comparedwith the blank group and the sham operation group,the number of secondary follicles in the model groupwas significantly decreased(P<0.05),while the apoptosis rate of ovarian granulosa cells in the modelgroup was significantly increased(P<0.001).Compared with the model group,the apoptosis rate in theHuoxue Xiaoyi Decoction group was significantly decreased(P<0.001),while the number of secondaryfollicles was significantly increased (P <0.05).(3)Compared with the blank group and the shamoperation group,protein expressions of Bax and Caspase-3in ovarian granulosa cells of the model groupwere significantly increased(P<0.01),while protein expression of Bcl-2was significantly decreased(P<0.001).Compared with the model group,protein expressions of Bax and Caspase-3in ovarian granulosacells of the Huoxue Xiaoyi Decoction group were significantly decreased,while protein expression of Bcl-2were significantly increased(P<0.001).(4)Compared with the blank group and the sham operationgroup,the protein expressions of p-JNK,Bax,Caspase-3protein in ovarian tissue of the model group weresignificantly increased,while the protein expression of Bcl-2,Beclin-1,LC3Ⅱ protein were significantlydecreased(P<0.05).Compared with the model group,the expression of p-JNK,Bax,Caspase-3protein inovarian tissue of the Huoxue Xiaoyi Decoction group were significantly decreased,while the expression ofBcl-2,Beclin-1,LC3Ⅱprotein were significantly increased(P<0.05).(5)Observation of autophagosomesin ovarian granulosa cells under electron microscopy showed that there was no significant difference in thecytoplasmic area ratio of autophagosomes among the four groups(P>0.05).Conclusions:Huoxue Xiaoyi Decoction may improve the oxidative stress state,decrease apoptosis ofovarian granulosa cells,initiate granulosa cell protective autophagy and improve follicles development inendometriosis model rats through ROS-JNK signaling pathway.