水火种玉汤治疗宫腔粘连肾虚血瘀证患者的临床疗效及转录组学研究

Clinical Efficacy and Transcriptomics Study of Shuihuo Zhongyu Decoction in the Treatment of Patients with Intrauterine Adhesion of Kidney Deficiency and Blood Stasis Syndrome

目的 观察水火种玉汤联合雌孕激素序贯治疗宫腔粘连(IUA)肾虚血瘀证患者的临床疗效,并通过转录组学方法探讨其可能的作用机制。方法 回顾性选取符合纳入标准的IUA肾虚血瘀证住院患者70例,观察组和对照组各35例,对照组行宫腔镜下宫腔粘连分离术(TCRA)后予雌孕激素序贯治疗,观察组在对照组基础上加服水火种玉汤治疗,2组疗程均为3个月。评估2组患者治疗前后中医证候积分、疗效、美国生育学会(AFS)评分、子宫内膜厚度(ED)、子宫内膜容受性[子宫内膜体积(EV)、子宫内膜血流指数(FI)、血管化指数(VI)、血管化血流指数(VFI)]、妊娠结局及宫腔再粘连情况。选取2组患者子宫内膜组织各10例进行转录组测序,筛选出差异基因,并对2组差异基因进行基因本体(GO)功能和京都基因与基因组百科全书(KEGG)信号通路富集分析。结果 治疗后,2组中医证候积分、AFS评分、CD138阳性率均明显下降(P<0.05),观察组优于对照组(P<0.05);观察组治疗后ED、EV、FI、VI、VFI均显著增加(P<0.01),优于对照组(P<0.05,P<0.01);观察组疗效、妊娠率、宫腔再粘连发生率优于对照组(P<0.05,P<0.01)。转录组学结果显示,观察组相比对照组共发现365个差异表达基因(DEGs),其中132个上调,包括磺基转移酶家族2B成员1(SULT2B1)、载脂蛋白A1(APOA1)等;233个基因下调,包括乳铁蛋白(LTF)、C-X-C趋化因子配体1(CXCL1)等;GO功能和KEGG通路富集分析表明,水火种玉汤可能于膜内组分、质膜、细胞外部分、细胞外基质等与金属离子、糖胺聚糖、蛋白质丝氨酸/苏氨酸激酶、内切肽酶等相互作用,通过调节细胞黏附、细胞外组织结构、抗原受体调节等生物进程而发挥作用,涉及的信号通路包括趋化因子、细胞因子受体相互作用、白介素17(IL-17)、核因子激活的B细胞的κ-轻链增强(NF-κB)等。结论 水火种玉汤能有效改善IUA肾虚血瘀证患者的临床症状,降低AFS评分,提高子宫内膜厚度,增加子宫内膜容受性,改善妊娠结局,降低宫腔再粘连发生率,其机制可能与上调SULT2B1、APOA1,下调LTF、CXCL1等基因表达,调节趋化因子信号通路、IL-17通路、NF-κB通路等相关。

OBJECTIVE To observe the clinical efficacy of Shuihuo Zhongyu Decoction combined with estrogen and progesterone sequential treatment for patients with intrauterine adhesion(IUA)of kidney deficiency and blood stasis syndrome,and to explore its possible mechanism of action by transcriptomics.METHODS A total of 70 hospitalized patients with IUA of kidney deficiency and blood stasis syndrome who met the inclusion criteria were retrospectively selected,with 35 cases in the observation group and 35 cases in the control group.The control group underwent hysteroscopic transcervical resection of adhesion(TCRA)and then received estrogen and progesterone sequential treatment.The observation group was treated with Shuihuo Zhongyu Decoction on the basis of the treatment of the control group.The treatment course of both groups was 3 months.The TCM syndrome scores,therapeutic efficacy,AFS scores,endometrial thickness(ED),endometrial receptivity[endometrial volume(EV),endometrial flow index(FI),vascularization index(VI),vascularization flow index(VFI)],pregnancy outcomes and intrauterine re-adhesion were evaluated in the 2 groups before and after treatment.10 cases of endometrial tissues in each group were selected for transcriptomic sequencing,and differentially expressed genes were verified by qPCR.RESULTS After treatment,the TCM syndrome scores,AFS scores and CD138 positive rate in the 2 groups were significantly decreased(P<0.05),and the observation group was better than the control group(P<0.05);after treatment,ED,EV,FI,VI and VFI in the observation group were significantly increased(P<0.01),which were better than those in the control group(P<0.05,P<0.01);the therapeutic efficacy,pregnancy rate and incidence of intrauterine re-adhesion in the observation group were better than those in the control group(P<0.05,P<0.01).Transcriptomics results showed that a total of 365 differentially expressed genes(DEGs)were found in the observation group compared with the control group,of which 132 were upregulated,including sulfotransferase family 2B member 1(SULT2B1),apolipoprotein A1(APOA1),etc.;233 were downregulated,including lactoferrin(LTF),C-X-C chemokine ligand 1(CXCL1),etc.;GO function and KEGG pathway enrichment analysis showed that SHT may interact with metal ions,glycosaminoglycans,protein serine/threonine kinases,endopeptidases,etc.in the membrane components,plasma membrane,extracellular part,extracellular matrix,etc.,and play a role by regulating biological processes such as cell adhesion,extracellular tissue structure,and antigen receptor regulation.The signaling pathways involved include chemokine signaling pathway,chemokine and cytokine receptor interactions,IL-17 signaling pathway,nuclear factor-κ-light chain enhanced of activated B cells(NF-κB),etc.CONCLUSION Shuihuo Zhongyu Decoction can effectively improve the clinical symptoms of IUA patients with kidney deficiency and blood stasis syndrome,reduce AFS scores,increase endometrial thickness,improve endometrial receptivity,improve pregnancy outcomes,and decrease the incidence of intrauterine re-adhesion.Its mechanism may be related to upregulating SULT2B1 and APOA1,downregulating the expression of genes such as LTF and CXCL1,and regulating signal pathways such as chemokines,IL-17,and NF-κB.