基于Th1、Th2细胞因子探讨清肠合剂改善术后腹腔粘连的机制

doi:10.11958/20241146

天津医药 ›› 2024, Vol. 52 ›› Issue (12): 1244-1250.doi: 10.11958/20241146

基于Th1、Th2细胞因子探讨清肠合剂改善术后腹腔粘连的机制

史镜铂¹(), 李昌年¹, 孟宇轩¹, 解广东², 徐杰³, 荣宝海²^,^△()

1 山东中医药大学第一临床医学院（邮编250355）
2 山东中医药大学附属医院胃肠与疝外科
3 血液病科

收稿日期:2024-08-19 修回日期:2024-10-10 出版日期:2024-12-15 发布日期:2024-12-17
通讯作者: △E-mail：rbh666666@126.com
作者简介:史镜铂（1999），男，硕士在读，主要从事中西医结合临床方面研究。E-mail:19862121910@163.com
基金资助:
山东省中医药科技项目(M-2022152)

Mechanism study of Qingchang mixture in the treatment of postoperative abdominal adhesions by regulating the expression of Th1 and Th2 cytokines

SHI Jingbo¹(), LI Changnian¹, MENG Yuxuan¹, XIE Guangdong², XU Jie³, RONG Baohai²^,^△()

1 First Clinical Medical College, Shandong University of Traditional Chinese Medicine, Ji’nan 250355, China
2 Department of Gastrointestinal and Hernia Surgery
3 Department of Hematology, the Affiliated Hospital of Shandong University of Traditional Chinese Medicine

Received:2024-08-19 Revised:2024-10-10 Published:2024-12-15 Online:2024-12-17
Contact: △E-mail：rbh666666@126.com

史镜铂, 李昌年, 孟宇轩, 解广东, 徐杰, 荣宝海. 基于Th1、Th2细胞因子探讨清肠合剂改善术后腹腔粘连的机制[J]. 天津医药, 2024, 52(12): 1244-1250.

SHI Jingbo, LI Changnian, MENG Yuxuan, XIE Guangdong, XU Jie, RONG Baohai. Mechanism study of Qingchang mixture in the treatment of postoperative abdominal adhesions by regulating the expression of Th1 and Th2 cytokines[J]. Tianjin Medical Journal, 2024, 52(12): 1244-1250.

摘要/Abstract

摘要：

目的探讨清肠合剂改善大鼠术后腹腔粘连的效果及其可能的作用机制。方法 75只SD大鼠随机分为5组：空白组、假手术组、模型组、清肠合剂组及透明质酸钠组。除空白组和假手术组外，其余3组采用盲肠刮擦法建立腹腔粘连大鼠模型；透明质酸钠组用2 mL透明质酸钠凝胶均匀涂抹受损的腹壁及盲肠后关腹。清肠合剂组造模成功后每日予2 mL清肠合剂（14.58 g/kg）灌胃1次，其余4组予等体积生理盐水灌胃。每组分别于术后第3、7、14天处死5只大鼠，采用Nair’s评分评估腹腔粘连情况，并收集术后7 d粘连组织或正常腹膜组织，应用荧光定量PCR法检测白细胞介素（IL）-4、信号转导和转录激活因子6（STAT-6）及干扰素-γ（IFN-γ）的mRNA表达水平；Western blot检测IL-4、IL-10、STAT-6及IFN-γ的蛋白表达情况；苏木精-伊红染色后光镜下观察各组粘连组织病理变化，评估炎症和纤维形成情况。结果与空白组和假手术组相比，模型组粘连组织中IL-4、STAT-6的mRNA和蛋白表达水平以及IL-10的蛋白表达水平下降，IFN-γ的mRNA和蛋白表达水平、炎症评分及纤维评分均升高（P<0.05）。与模型组相比，清肠合剂组和透明质酸钠组的IL-4和STAT-6的mRNA和蛋白表达水平以及IL-10蛋白表达水平均升高；Nair’s评分、炎症评分、纤维评分以及IFN-γ的mRNA和蛋白表达水平均降低（P<0.05）。结论清肠合剂可以抑制术后腹腔粘连的形成，其机制可能与调控辅助性T细胞1、辅助性T细胞2相关的细胞因子，降低炎症反应水平有关。

关键词: Th1细胞, Th2细胞, 术后腹腔粘连, 清肠合剂, 炎症反应

Abstract:

Objective To elucidate the efficacy and possible mechanism of Qingchang mixture in ameliorating postoperative abdominal adhesions in rats. Methods Seventy-five Sprague-Dawley rats were randomly allocated into five experimental groups: the control group, the sham-operated group, the model group, the Qingchang mixture treatment group and the sodium hyaluronate treatment group. Except the control group and the sham-operated group, the other three groups were treated with cecal abrasion method to establish the rat model of abdominal adhesion. In the sodium hyaluronate group, 2 mL sodium hyaluronate gel was meticulously applied to the injured abdominal wall and cecum prior to abdominal closure. Following successful establishment of adhesion model, the Qingchang mixture group received a daily oral gavage of 2 mL Qingchang mixture (14.58 g/kg), while the other four groups were given equal volume normal saline administration. In each group, five rats were euthanized on postoperative days 3, 7 and 14 to assess abdominal adhesions using Nair’s scoring system. Adhesive tissue or normal peritoneal tissue were harvested on postoperative day 7, and mRNA expression levels of interleukin-4 (IL-4), signal transducer and activator of transcription 6 (STAT-6) and interferon-γ (IFN-γ) were quantified via fluorescence-based real-time PCR. Concurrently, Western blot analysis was employed to evaluate protein expression levels of IL-4, interleukin-10 (IL-10), STAT-6 and IFN-γ. Pathological alterations in adhesive tissue were visualized using hematoxylin-eosin (HE) staining under light microscopy, and inflammation and fibrotic changes were assessed accordingly. Results Compared with the blank and sham-operated groups, mRNA and protein expression levels of IL-4 and STAT-6 were significantly downregulated in the model group, protein expression level of IL-10 was also reduced. Conversely, the mRNA and protein expression levels of IFN-γ, as well as the inflammation and fibrosis scores were significantly elevated (P<0.05). In comparison to the model group, IL-4 and STAT-6 mRNA and protein expression levels were increased in the Qingchang mixture group and the hyaluronic acid group, along with an increase in IL-10 protein expression. Conversely, these groups exhibited a significant reduction in Nair’s scores, inflammation scores, fibrosis scores, and IFN-γ mRNA and protein expression levels were decreased (P<0.05). Conclusion Qingchang mixture appears to suppress the development of postoperative peritoneal adhesions, likely through mechanism that involves modulating the expression of T-helper 1 and T-helper 2 cytokines, thereby attenuating inflammatory response.

Key words: Th1 cells, Th2 cells, postoperative intra-abdominal adhesions, Qingchang mixture, inflammatory response

中图分类号:

R285.5，R656

图/表 8

表1 引物序列

Tab.1 Primer sequence

基因名称	引物序列（5′→3′）	产物大小/bp
IFN-γ	上游：GAGGAACTGGCAAAAGGACG	132
IFN-γ	下游：AGGTGCGATTCGATGACACT	132
IL-4	上游：TGTAGAGGTGTCAGCGGTCT	70
IL-4	下游：TCAGTGTTGTGAGCGTGGAC	70
STAT-6	上游：TCAGCACCGAGTAAGGGAGA	130
STAT-6	下游：GGCCAGATCCTCACTTGGAC	130
β-actin	上游：CTCTGIGTGGATTGGTGGCT	139
β-actin	下游：CGCAGCTCAGTAACAGTCCG	139

表2 各组大鼠的术后Nair’s评分（n=15，$\bar{x}\pm s$）

Tab. 2 Comprehensive Nair’s scores of rats in each group

组别	0	Ⅰ	Ⅱ	Ⅲ	Ⅳ	Nair’s评分/分
假手术组	9	4	2	0	0	1.28±0.25
模型组	0	3	6	5	1	2.27±0.42^b
清肠合剂组	2	8	5	0	0	1.33±0.31^c
透明质酸钠组	1	9	5	0	0	1.37±0.32^c
F						6.143^＊

图1 各组大鼠腹腔粘连情况

Fig.1 Adhesion status in abdominal cavity of rats in each group

表3 术后7 d各组大鼠腹腔粘连组织中IL-4、STAT-6、IFN-γ mRNA表达水平（n=5，$\bar{x}\pm s$）

Tab. 3 mRNA expression levels of IL-4，STAT-6 and IFN-γ in abdominal adhesion tissue of rats in each group on postoperative day 7

组别	IL-4	STAT-6	IFN-γ
空白组	1.01±0.16	1.01±0.15	1.00±0.10
假手术组	0.95±0.11	0.84±0.15	1.14±0.20
模型组	0.25±0.06^ab	0.31±0.03^ab	2.06±0.11^ab
清肠合剂组	0.66±0.16^c	0.70±0.09^c	1.35±0.14^c
透明质酸钠组	0.60±0.10^c	0.67±0.04^c	1.48±0.24^c
F	18.486^＊＊	18.388^＊＊	18.522^＊＊

表4 术后7 d各组大鼠粘连组织中IL-4、STAT-6、IL-10和IFN-γ蛋白表达情况（n=5，$\bar{x}\pm s$）

Tab.4 The protein expression levels of IL-4，STAT-6，IL-10 and IFN-γ in adhesion tissue of rats in each group on postoperative day 7

组别	STAT-6	IL-4	IL-10	IFN-γ
空白组	0.93±0.13	1.37±0.10	1.28±0.11	0.41±0.10
假手术组	0.86±0.03	1.17±0.08	1.12±0.12	0.42±0.08
模型组	0.35±0.10^ab	0.61±0.05^ab	0.53±0.02^ab	1.16±0.14^ab
清肠合剂组	0.67±0.04^c	0.97±0.11^c	0.85±0.13^c	0.64±0.13^c
透明质酸钠组	0.59±0.06^c	0.92±0.02^c	0.79±0.02^c	0.83±0.14^c
F	24.014^＊＊	40.128^＊＊	28.678^＊＊	21.100^＊＊

图2 Western blot检测术后7 d各组粘连组织中STAT-6、IL-4、IL-10、IFN-γ蛋白表达水平 A：空白组；B：假手术组；C：清肠合剂组；D：透明质酸钠组；E：模型组。

Fig.2 Western blot analysis of STAT-6, IL-4, IL-10 and IFN-γ protein expression levels in adhesive tissue of each group at 7 days after surgery

图3 各组大鼠腹腔粘连组织HE染色结果 A：空白组；B：假手术组；C：清肠合剂组；D：透明质酸钠组；E：模型组；红色箭头示淋巴细胞，黄色箭头示粒细胞，黑色箭头示纤维细胞形成。

Fig.3 HE staining results of abdominal adhesion tissue of rats in each group

表5 各组大鼠炎症评分和纤维评分（n=15，分，$\bar{x}\pm s$）

Tab. 5 The inflammation score and fibrosis score of rats in each group

组别	炎症评分	纤维评分
空白组	1.00±0.00	0.00±0.00
假手术组	1.11±0.19	0.60±0.20
模型组	2.56±0.51^ab	2.07±0.42^ab
清肠合剂组	1.78±0.19^c	1.13±0.50^c
透明质酸钠组	2.11±0.19	1.40±0.20
F	17.650^＊＊	18.263^＊＊

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基于Th1、Th2细胞因子探讨清肠合剂改善术后腹腔粘连的机制

Mechanism study of Qingchang mixture in the treatment of postoperative abdominal adhesions by regulating the expression of Th1 and Th2 cytokines

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