基于JAK 2/STAT 3信号通路探讨三棱莪术及配伍干预大鼠子宫内膜异位症的配伍研究

Compatibility Analysis of SPARGANII RHIZOMA and CURCUMAE RHIZOMA on Endometriosis in Rats Based on JAK 2/STAT 3 Signaling Pathway

目的:基于Janus激酶2/信号转导与转录激活因子3(JAK 2/STAT 3)信号通路,探讨三棱莪术配伍抗炎、促进内膜细胞凋亡,干预大鼠实验性子宫内膜异位症(EMS)的配伍机制。方法:通过自体子宫移植建立大鼠EMS模型,B超检测异位病灶体积。将造模成功的大鼠,按照病灶体积均衡分为模型对照组、三棱20 g/kg组、莪术20 g/kg组、配伍组(三棱莪术等比配伍,20 g生药/kg)和阳性组(AG4904 mg/kg),每组10只。另设10只大鼠为假手术对照组。给药4 w后,分别以B超和卡尺测量病灶体积,吸取腹腔液后,取异位内膜组织,苏木素-伊红(HE)染色观察组织形态;酶联免疫吸附(Elisa)法测定腹腔液中肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1β和IL-6含量,实时荧光定量聚合酶链式反应(Real Time-PCR)技术检测内膜组织中含半胱氨酸的天冬氨酸蛋白水解酶3(Caspase 3)、Bax和Bcl 2的mRNA表达,蛋白免疫印迹(Western blot)法测定内膜组织中JAK 2和STAT 3蛋白的磷酸化表达。结果:造模成功后,B超检测腹壁下方可见子宫内膜异位病灶,HE染色显示异位内膜形态与正常子宫内膜相似。与假手术对照组大鼠相比,模型对照组大鼠腹腔液中TNF-α、IL-1β和IL-6含量显著升高(P<0.01),内膜组织中JAK 2和STAT 3蛋白的磷酸化表达显著上调(P<0.01)。与模型对照组大鼠相比,AG4904 mg/kg、三棱、莪术以及配伍组异位病灶体积均显著减小(P<0.01),异位内膜组织萎缩,腹腔液中TNF-α、IL-1β和IL-6含量明显降低(P<0.05或P<0.01),内膜组织中Caspase 3和Bax的mRNA表达显著上调(P<0.01),Bcl 2的mRNA表达显著下调(P<0.01),莪术组和配伍组的JAK 2和STAT 3蛋白的磷酸化表达明显下调(P<0.01)。结论:三棱莪术配伍能够通过抗炎、促进细胞凋亡,使EMS大鼠病灶缩小,其作用与抑制JAK 2/STAT 3信号通路有关。

Objective:To explore the compatibility mechanism of SPARGANII RHIZOMA and CURCUMAE RHIZOMA in resisting inflammation,promoting endometrial cell apoptosis,and intervening in experimental endometriosis(EMS)in rats based on the Janus kinase 2-signal transducer and activator of transcription protein 3(JAK 2/STAT 3)signaling pathway.Methods:The EMS model was induced by uterus transplant in rats,and then the volume of ectopic endometrium was measured by ultrasound B.The model rats were divided into a model group,a SPARGANII RHIZOMA group(20 g/kg),a CURCUMAE RHIZOMA group(20 g/kg),a compatibility group(1:1,20 g/kg),and a positive group(AG4904 mg/kg),with 10 rats in each group.Another 10 rats were assigned to a sham operation group.After four weeks of drug treatment,the volume of ectopic endometrium was measured by ultrasound and caliper respectively.After the peritoneal fluid was drawn,the ectopic endometrium tissues were collected and observed by hematoxylin-eosin(HE)staining.The content of tumor necrosis factor(TNF)-α,interleukin(IL)-1β,and IL-6 in the peritoneal fluid was determined by enzyme-linked immunosorbent assay(ELISA),and real-time quantitative polymerase chain reaction(real-time PCR)was used to detect the mRNA expression of Caspase 3,Bax,and Bcl 2.The phosphorylation levels of JAK 2 and STAT 3 were determined by Western blot.Results:After modeling,ultrasound B results showed EMS lesions below the abdominal wall,and HE staining showed that the ectopic endometrium was similar to normal endometrium in morphology.Compared with the sham operation group,the model group showed increased content of TNF-α,IL-1β,and IL-6 in the peritoneal fluid(P<0.01),and up regulated phosphorylation levels of JAK 2 and STAT 3(P<0.01).Compared with the model group,the groups with drug intervention showed reduced volume of ectopic lesions(P<0.01),atrophy of ectopic endometrium,decreased content of TNF-α,IL-1β,and IL-6 in the peritoneal fluid(P<0.05 or P<0.01),up-regulated mRNA expression of Caspase 3 and Bax(P<0.01),and down-regulated mRNA expression of Bcl-2(P<0.01).The phosphorylation levels of JAK 2 and STAT 3 proteins were reduced in the CURCUMAE RHIZOMA group and the compatibility group(P<0.01).Conclusion:CURCUMAE RHIZOMA can shrink the lesions in EMS rats by resisting inflammation and promoting apoptosis,and the mechanism may be related to the inhibition of the JAK2/STAT3 signaling pathway.