健脾利湿解毒颗粒预防脾虚湿毒型大肠腺瘤患者内镜术后复发及转录组学研究

Preventive effect and transcriptomic analysis of Jianpi Iishi Jiedu Granule in patients with spleen deficiency and dampness toxicity type colorectal adenoma treated by endoscopy

关键词:  健脾利湿解毒颗粒  大肠腺瘤  复发  转录组学  信号通路

Key words:  Jianpi Lishi Jiedu Granule  colorectal adenomas  recurrence  transcriptomics  signaling pathway

摘要:

[目的] 观察健脾利湿解毒颗粒治疗脾虚湿毒型大肠腺瘤（CRAs）内镜术后患者的复发率，并通过转录组学方法初步探究其可能的作用机制。[方法] 将符合纳入标准的80例脾虚湿毒型CRAs内镜治疗术后患者随机分为试验组、对照组，每组各40例。试验组患者服用健脾利湿解毒颗粒，对照组患者服用安慰剂对照颗粒，疗程3个月。所有患者于 12个月后复查肠镜，记录CRAs复发情况。收集所有患者治疗前的CRAs组织和瘤旁组织（腺瘤旁5 cm正常结肠组织）、治疗后复发的CRAs组织。提取CRAs组织和瘤旁组织的总RNA，进行转录组学测序并用实时定量逆转录聚合酶链反应（qRT-PCR）和蛋白免疫印迹法（Western Blot）法验证关键信号通路中相关基因和蛋白的表达。[结果] 试验组患者1年后CRAs复发率低于对照组患者（P<0.05）；转录组学测序结果显示，试验组患者治疗前后对比，CRAs组织中共筛选出差异基因471个，其中上调315个，下调156个；治疗后试验组与对照组患者对比，CRAs组织中共筛选出差异基因1 518个，其中上调952个，下调566个。基因本体（GO）和京都基因与基因组百科全书（KEGG）分析显示差异基因主要富集在Wnt/β-catenin信号通路；qRT-PCR验证结果显示试验组患者复发的CRAs组织中FZD10、DVL1、GSK3B、WNT5、CCND1、MYC、MMP7基因表达量较对照组降低（P<0.05），APC2较对照组升高（P<0.05）；Western Blot验证结果显示试验组患者复发的CRAs组织中Wnt5a、GSK-3β、β-catenin、Cyclin D1蛋白表达量较对照组降低（P<0.05）。[结论] 健脾利湿解毒颗粒在预防患者CRAs复发方面具有良好的效果，且安全性良好，抑制Wnt/β-catenin信号通路的过度激活、调节下游相关基因转录和蛋白的表达可能是其预防CRAs复发的作用机制之一。

Abstract:

[Objective] To observe the recurrence rate of Jianpi Lishi Jiedu Granules in treating patients with colorectal adenomas induced by spleen deficiency and dampness toxin after endoscopic treatment and preliminarily explore the potential mechanism through transcriptomics. [Methods] The 80 criteria-fulfilling patients with colorectal adenoma induced by spleen deficiency and dampness toxin were randomly divided into the experimental group（40 cases） and the control group（40 cases）. Afterward，patients in the experimental and control groups were respectively prescribed Jianpi Lishi Jiedu Granules and placebo control granules for 3 months. All patients were also scheduled for colonoscopy follow-ups after 12 months for inspections of colorectal adenoma recurrences. Pre-treatment samples of colorectal adenoma tissues and peritumoral tissues（normal colon tissues located within a 5 cm radius from the adenomas），as well as post-treatment samples of recurrent colorectal adenoma tissues were collected from all patients. Moreover，the total RNA was extracted from the colorectal adenoma tissues and adjacent tissues，after which transcriptomic sequencing was performed to verify the expression of related genes and proteins in key signaling pathways through qRT-PCR and Western blot. [Results] The recurrence rate of colorectal adenoma in the experimental group was lower than that in the control group after 1 year（P<0.05）. The results of transcriptomic sequencing suggested that，compared with the pre-treatment status，a total of 471 differential genes were selected from colorectal adenoma tissues in the experimental group post-treatment，of which 315 were up-regulated and 156 were down-regulated；after treatment，a total of 1 518 differential genes were selected from colorectal adenoma tissues in the experimental group compared with the control group，of which 952 were up-regulated and 566 were down-regulated. Meanwhile，GO and KEGG analysis showed an enrichment of differential genes primarily in the Wnt/β-catenin signaling pathway. Moreover，qRT-PCR validation indicated a decreased expression of FZD10，DVL1，GSK3B，WNT5，CCND1，MYC，and MMP7 gene（P<0.05） and an elevated expression of APC2（P<0.05） in recurrent colorectal adenoma tissues of patients in the experimental group compared with the control group. Furthermore，Western blot validation revealed a lower expression of Wnt5a，GSK-3β，β-catenin，and cyclin D1 protein（P<0.05） in recurrent colorectal adenoma tissues of patients in the experimental group than in the control group. [Conclusion] The Jianpi Lishi Jiedu Granules have a good effect in preventing the recurrence of colorectal adenomas（CRAs） in patients，and possess good safety. Inhibiting the excessive activation of the Wnt/β-catenin signaling pathway and regulating the transcription of downstream related genes and the expression of proteins may be one of the mechanisms by which it prevents the recurrence of CRAs.