中药理疗结合仿生物电刺激对宫腔粘连的疗效及机制

Effect and mechanism of traditional Chinese medicine physiotherapy combined with bionic electrical stimulation on intrauterine adhesions

摘要:

目的 探索中药理疗结合仿生物电刺激对宫腔粘连(IUA)的疗效及机制。方法 选取2021年6月至2022年9月在海口市中医医院妇产科就诊的84例IUA患者作为研究对象，采用随机数字表法将其分成宫腔粘连分离术(TCRA)中药组(TCRA+中药)、西药组[TCRA+戊酸雌二醇(3 mg, 1次/d)+黄体酮胶囊(100 mg, 2次/d)]和TCRA中药理疗组(TCRA+中药+仿生物电刺激),每组28例。采用免疫组织化学检测3组TGF-β1、SMAD2的表达；采用定量反转录聚合酶链式反应(qRT-PCR)检测3组MIR-29A的表达。Starbase数据库分析MIR-29A与SMAD2的靶向结合。使用HEC-1-A细胞进行转染实验，将HEC-1-A细胞随机分为Smad2-WT+miR-NC组、Smad2-WT+miR-29a组、Smad2-MUT+miR-NC组和Smad2-MUT+miR-29a组，采用双荧光素酶报告基因实验检测4组荧光素酶活性；将HEC-1-A细胞随机分为miR-29a mimic组、miR-29a inhibitor组和NC组，采用qRT-PCR检测SMAD2和MIR-29A基因的表达，采用蛋白质印迹法检测SMAD2和MIR-29A蛋白的表达。结果 治疗后3个月，3组子宫内膜厚度比较，差异无统计学意义(P>0.05)。与治疗前比较，治疗后TCRA中药理疗组粘连分级评分和中医证候评分明显低于TCRA中药组和西药组(P<0.05)。与治疗前比较，治疗后3组TGF-β1和SMAD2表达明显下降(P<0.001),MIR-29A表达明显上升(P<0.001)。Starbase数据库分析显示，MIR-29A和SMAD2存在靶向结合位点。双荧光素酶报告基因实验显示，与Smad2-WT+miR-NC组比较，Smad2-WT+miR-29a组荧光素酶活性明显降低(P<0.001)。此外，与对照组比较，miR-29a mimc组SMAD2 mRNA及蛋白表达明显降低，而在miR-29a inhibitor组中明显增加(P<0.001)。结论 中药理疗通过MIR-29A可抑制TGF-β1/Smad信号通路，改善IUA患者术后粘连程度。

Abstract:

Objective Toexplorethetherapeuticeffectand mechanism oftraditionalChinese medicine (TCM) physiotherapycombinedwithbiomimeticelectricalstimulationinthetreatmentofintrauterineadhesions(IUA).Methods A totalof84patientswithIUAintheDepartmentofObstetricsandGynecologyofHaikou HospitalofTraditionalChinese MedicinefromJune2021toSeptember2022wereselectedastheresearchobjects.Aftertranscervicaladhesiolysis(TCRA), theyweredividedintotheTCMgroup(n=28,oraladministrationofChineseherbaldecoction),theWesternmedicinegroup (n=28,estradiolvalerate3mgonceaday+ progesterone100mgtwiceaday),andtheTCMphysiotherapygroup(n=28, oraladministrationofChineseherbaldecoction + biomimeticelectricalstimulation)accordingtoacomputer-generated randomnumbertable.Immunohistochemicalanalysiswasutilizedtodetecttheexpressionlevelsoftransforminggrowth factor(TGF)-β1andSMAD2.Quantitativereversetranscriptionpolymerasechainreaction(qRT-PCR)wasusedtomeasure theexpressionofmicroRNA-29A (MIR-29A).TheStarbasedatabasewasutilizedtoanalyzethetargetbindingbetween MIR-29A andSMAD2.HumanendometrialcancerHEC-1-Acellswereusedfortransfectionexperiments.Thecultured HEC-1-Acellswererandomlydividedintofourgroups:Smad2-WT + miR-NCgroup,Smad2-WT + miR-29agroup, Smad2-MUT + miR-NCgroup,andSmad2-MUT + miR-29agroup.Adual-luciferasereportergeneassaywasconductedto measuretheluciferaseactivityinthesefourgroups.The HEC-1-Acellswererandomlydividedintothe miR-29a mimic group,miR-29ainhibitorgroup,andNCgroup.qRT-PCR wasperformedtoassessthegeneexpressionofSMAD2and MIR-29A,while Western blotanalysis wasusedto detectthe protein expressionlevelsofSMAD2 and MIR-29A. Results Therewasnostatisticallysignificantdifferenceinendometrialthicknessamongthethreegroupsat3 monthsafter treatment(P >0.05).Comparedwithpre-treatment,theadhesiongradingscoreandTCM syndromescoreintheTCM physiotherapygroupweresignificantlylowerthanthoseintheTCM groupandthe Western medicinegroup (P <0.05). Compared withpre-treatment,theexpressionofTGF-β1andSMAD2significantlydecreased (P <0.001),whilethe expressionofMIR-29A significantlyincreased (P <0.001)inallthreegroupspost-treatment.TheStarbasedatabase analysisshowedthatMIR-29A targetsSMAD2 withabindingsite.Dual-luciferasereporterassaysdemonstratedthat, comparedtotheSmad2-WT + miR-NCgroup,theSmad2-WT + miR-29agroupexhibitedasignificantdecreaseinluciferase activity(P<0.001).Additionally,comparedtothecontrolgroup,themiR-29amimicgroupshowedasignificantdecreasein SMAD2mRNAandproteinexpression,whilethe miR-29ainhibitorgroupshowedasignificantincrease (P <0.001). Conclusion TCM physiotherapyinhibitsthe TGF-β1/Smadsignalingpathwaythrough MIR-29A,whichcanimprove post-operativeadhesionseverityinIUApatients.