良方温经汤调控CHCHD4表达干预寒凝血瘀型子宫内膜异位症乏氧的机制研究

Mechanism of Liangfang Wenjing Decoction in treatment of hypoxia on endometriosis with cold coagulation and blood stasis by regulating CHCHD4 expression

探究良方温经汤调控卷曲螺旋卷曲螺旋结构域4(CHCHD4)表达干预寒凝血瘀型子宫内膜异位症(EMs)乏氧环境的机制。冰水浴法制备大鼠寒凝血瘀证模型,在此基础上采用自体移植法建立EMs模型,随机分为模型组,良方温经汤低、中、高(4.7、9.4、18.8 g·kg^(-1))剂量组及少腹逐瘀汤组,另设假手术组,每组10只,灌胃给药4周。造模期间进行大鼠一般情况观察及阴道涂片,并通过激光散斑衬比成像(LSCI)检测耳部及子宫血流,以判断寒凝血瘀证候情况;给药结束后观察大鼠一般情况;卡尺测量异位病灶面积;免疫组织化学法检测CHCHD4、乏氧诱导因子^(-1)α(HIF^(-1)α)的定位与表达;实时定量聚合酶链反应(RT-qPCR)及蛋白免疫印迹检测CHCHD4、HIF^(-1)αmRNA和蛋白的表达。进行EMs患者异位子宫内膜间质细胞(ESCs)原代培养,构建CHCHD4过表达质粒,转染建立CHCHD4过表达ESCs模型。将细胞分为空白对照组、CHCHD4过表达组、CHCHD4过表达+空白血清组、CHCHD4过表达+良方温经汤血清组,蛋白免疫印迹检测CHCHD4、HIF^(-1)α蛋白的表达;并检测葡萄糖消耗量及乳酸水平;MTT法检测细胞增殖情况。结果显示,与正常大鼠相比,造模大鼠出现精神萎靡、饮食饮水减少、动情周期紊乱,耳部及子宫微血管血液循环受阻等寒凝血瘀证候表征。与假手术组相比,模型组大鼠异位病灶隆起,CHCHD4、HIF^(-1)αmRNA及蛋白表达显著升高(P<0.05);与模型组相比,各给药组大鼠寒凝血瘀表征均有改善,异位病灶面积显著缩小(P<0.05或P<0.01),CHCHD4、HIF^(-1)αmRNA与蛋白表达显著降低(P<0.05或P<0.01)。在细胞模型中,与空白对照组相比,CHCHD4过表达组CHCHD4、HIF^(-1)α蛋白表达、葡萄糖消耗量、乳酸水平、细胞增殖活力均显著升高(P<0.01);与CHCHD4过表达组相比,空白血清组各指标均无明显变化,良方温经汤血清组CHCHD4、HIF^(-1)α蛋白表达显著下降(P<0.05或P<0.01),葡萄糖消耗量、乳酸水平显著降低(P<0.01),细胞增殖活力显著下降(P<0.01)。由上可知,良方温经汤对寒凝血瘀型EMs的治疗作用与降低CHCHD4表达,进而改善异位病灶及异位ESCs乏氧有关。

To explore the mechanism of Liangfang Wenjing Decoction regulating coiled-coil-helix coiled-coil-helix domain containing 4(CHCHD4)in the treatment of hypoxia on endometriosis(EMs)with cold coagulation and blood stasis.The rat model of cold coagulation and blood stasis syndrome was prepared by the ice-water bath method,and then the EMs model was established by autologous intimal transplantation.The rats were randomly divided into model group,low,medium,and high(4.7,9.4,and 18.8 g·kg^(-1))dose groups of Liangfang Wenjing Decoction,Shaofu Zhuyu Decoction group,and sham group,with 10 rats in each group.The rats were given intragastric administration for four weeks.During the modeling,the general condition and vaginal smear of rats were observed,and the blood flow of ears and uterus were detected by laser speckle contrast imaging(LSCI)to judge the syndrome of cold coagulation and blood stasis.After the administration,the general condition of the rats was observed,and the area of ectopic lesions was measured by caliper.The localization and expression of CHCHD4 and hypoxia inducible factors-1α(HIF-1α)were detected by immunohistochemistry,and the mRNA and protein expressions of CHCHD4 and HIF-1αwere detected by real-time quantitative polymerase chain reaction(RT-qPCR)and Western blot.The primary culture of ectopic endometrial stromal cells(ESCs)from EMs patients was performed,and the CHCHD4 overexpression plasmid was constructed and transfected to establish the ESCs model of CHCHD4 overexpression.The cells were divided into the control group,CHCHD4 overexpression group,CHCHD4 overexpression+control serum group,and CHCHD4 overexpression+Liangfang Wenjing Decoction serum group.The protein expression of CHCHD4 and HIF-1αwas detected by Western blot,and the glucose consumption and lactic acid level were detected.The cell proliferation was detected by MTT assay.The experiment found that compared with normal rats,the modeling rats showed symptoms of cold coagulation and blood stasis,such as mental malaise,reduced diet and drinking water,disordered estrous cycle,and blocked blood circulation in ears and uterine microvessels.Compared with the sham group,the ectopic lesions in the model group were uplifted,and the mRNA and protein expressions of CHCHD4 and HIF-1αwere significantly increased(P<0.05).Compared with the model group,the symptoms of cold coagulation and blood stasis in each treatment group were improved,and the area of ectopic lesions was significantly reduced(P<0.05 or P<0.01).The mRNA and protein expression levels of CHCHD4 and HIF-1αwere significantly decreased(P<0.05 or P<0.01).In the cell model,compared with the control group,the expression of CHCHD4,HIF-1αprotein,glucose consumption,lactic acid level,and cell proliferation activity in the CHCHD4 overexpression group were significantly increased(P<0.01).Compared with the CHCHD4 overexpression group,there was no significant change in each index in the control serum group,while the protein expression of CHCHD4 and HIF-1αin the Liangfang Wenjing Decoction serum group was decreased significantly(P<0.05 or P<0.01).The glucose consumption,lactic acid level,and cell proliferation activity decreased significantly(P<0.01).It can be seen from the above that the therapeutic effect of Liangfang Wenjing Decoction on EMs with cold coagulation and blood stasis might be related to reducing the expression of CHCHD4 and then improving the hypoxia of ectopic lesions and ectopic ESCs.