补脾益肠丸重塑CD4+ T细胞亚群稳态缓解溃疡性结肠炎的机制研究

doi:10.11958/20230776

天津医药 ›› 2023, Vol. 51 ›› Issue (12): 1332-1338.doi: 10.11958/20230776

补脾益肠丸重塑CD4⁺T细胞亚群稳态缓解溃疡性结肠炎的机制研究

肖秋萍¹^,²(), 赵畅³, 刘端勇⁴, 李姗姗¹, 施旻⁵, 陈丽玲¹, 钟友宝¹^,⁴^,^△()

1.江西中医药大学实验动物科技中心（邮编330004）
2.江西中医药大学研究生院（邮编330004）
3.江西中医药大学针灸推拿学院（邮编330004）
4.江西中医药大学方证研究中心（邮编330004）
5.江西中医药大学中医学院（邮编330004）

收稿日期:2023-05-22 修回日期:2023-08-07 出版日期:2023-12-15 发布日期:2023-12-22
通讯作者: ^△ E-mail：767363512@qq.com
作者简介:肖秋萍（1991），女，博士在读，主要从事中药药效基础方面研究。E-mail：1933067805@qq.com
基金资助:
国家自然科学基金资助项目(82260863);江西省卫生和计划生育委员会科技计划项目(202110121);江西省教育厅科学技术研究项目(GJJ201239);江西省教育厅科学技术研究项目(GJJ2200961);江西省中医药管理局科技计划项目(2022A344)

Study on mechanism of Bupi Yichang pill in alleviating experimental ulcerative colitis by restoring the homeostasis of CD4⁺T cell subpopulations

XIAO Qiuping¹^,²(), ZHAO Chang³, LIU Duanyong⁴, LI Shanshan¹, SHI Min⁵, CHEN Liling¹, ZHONG Youbao¹^,⁴^,^△()

1. Laboratory Animal Research Center for Science and Technology, Jiangxi University of Chinese Medicine, Nanchang 330004, China
2. Department of Postgraduate, Jiangxi University of Chinese Medicine, Nanchang 330004, China
3. College of Acupuncture and Massage, Jiangxi University of Chinese Medicine, Nanchang 330004, China
4. the Research Center of Formula and Syndrome, Jiangxi University of Chinese Medicine, Nanchang 330004, China
5. College of Traditional Chinese Medicine, Jiangxi University of Chinese Medicine, Nanchang 330004, China

Received:2023-05-22 Revised:2023-08-07 Published:2023-12-15 Online:2023-12-22
Contact: ^△ E-mail：767363512@qq.com

肖秋萍, 赵畅, 刘端勇, 李姗姗, 施旻, 陈丽玲, 钟友宝. 补脾益肠丸重塑CD4⁺T细胞亚群稳态缓解溃疡性结肠炎的机制研究[J]. 天津医药, 2023, 51(12): 1332-1338.

XIAO Qiuping, ZHAO Chang, LIU Duanyong, LI Shanshan, SHI Min, CHEN Liling, ZHONG Youbao. Study on mechanism of Bupi Yichang pill in alleviating experimental ulcerative colitis by restoring the homeostasis of CD4⁺T cell subpopulations[J]. Tianjin Medical Journal, 2023, 51(12): 1332-1338.

摘要/Abstract

摘要：

目的探究补脾益肠丸（BPYCP）对实验性溃疡性结肠炎（UC）小鼠CD4⁺T细胞亚群的调控作用。方法 48只C57BL/6小鼠采用随机数字表法分为对照组（Control组，10只）、模型组（DSS组， 13只）、模型+补脾益肠丸组（DSS+BPYCP组， 13只）、模型+5-ASA组（DSS+5-ASA组， 12只）；采用自由饮用2.5%葡聚糖硫酸钠（DSS）溶液诱导小鼠UC模型，DSS+BPYCP组和DSS+5-ASA组分别采用BPYCP或美沙拉嗪（5-ASA）连续灌胃2周。观察小鼠粪便黏稠度及便血情况，测量结肠长度，结肠称质量并计算结肠质量指数及单位结肠质量指数；苏木素-伊红（HE）染色法观察结肠病理变化，并进行病理损伤评分；流式细胞术检测肠系膜淋巴结中的CD4⁺T细胞亚群水平；酶联免疫吸附试验检测干扰素（INF）-γ、白细胞介素（IL）-4、IL-17A、IL-10及IL-21表达水平；实时荧光定量PCR检测结肠组织T-框蛋白21（T-bet）、GATA结合蛋白3（GATA-3）、维甲酸相关核孤儿受体γt（RORγt）、B细胞淋巴瘤-6（Bcl-6）及大鼠叉头蛋白P3（Foxp3）mRNA表达水平。结果与DSS组比较，DSS+BPYCP组和DSS+5-ASA组UC小鼠腹泻、便血等症状得到改善，小鼠体质量及结肠长度均增加，且结肠质量、结肠质量指数及单位结肠质量指数均下降，黏膜上皮较完整，腺体排列更规整，炎性细胞浸润更少，病理组织损伤评分显著降低，肠系膜淋巴结中的Th2细胞比例降低，Th17细胞比例及IL-17A水平降低，结肠组织T-bet、GATA-3、RORγt、Bcl-6 mRNA水平降低（P<0.05）；DSS+BPYCP组的Th1细胞比例降低，CD4⁺CD25⁺Treg、CD4⁺CD25⁺Foxp3⁺Treg细胞比例及IL-10水平均升高，CD4⁺CXCR5⁺Tfh细胞比例和IL-21水平降低，Foxp3 mRNA水平升高（P<0.05）；DSS+5-ASA组的Th1细胞比例及IFN-γ水平降低（P<0.05）。结论 BPYCP可能通过重塑肠道组织的CD4⁺T细胞亚群稳态缓解实验性UC。

关键词: 结肠炎, 溃疡性, 补脾益肠丸, CD4⁺T细胞亚群, 细胞稳态

Abstract:

Objective To investigate the regulatory effect of Bupi Yichang pill (BPYCP) on CD4⁺T cell subsets of ulcerative colitis (UC) mice. Methods Forty-eight C57BL/6 mice were randomly divided into 4 groups: the control group (n=10), the model group (DSS group, n=13), the model +BPYCP group (DSS+BPYCP group, n=13) and the model+mesalazine (5-ASA) group (DSS+5-ASA group, n=12). The mouse UC model was induced by 2.5% dextrosan sulfate (DSS) solution. The DSS+BPYCP group and the DSS+5-ASA group were given BPYCP or 5-ASA for 2 weeks, respectively, and fecal viscosity and blood in stool were observed. The colon length was measured. Colonic mass index and unit colonic mass index were calculated. Hematoxylin-eosin (HE) staining was used to observe pathological changes of colon and to score the pathological tissue damage. The level of CD4⁺T cell subsets in mesenteric lymph nodes was detected by flow cytometry. The expression levels of cytokines interferon-γ (INF-γ), interleukin (IL-4), IL-17A, IL-10 and IL-21 secreted by CD4⁺T cell subsets in colon tissue were detected by ELISA. Real-time fluorescence quantitative PCR was used to detect colon tissue CD4⁺T cell subset nuclear transcription factors, mRNA expression levels of T-frame protein 21 (T-bet), GatA-binding protein 3 (GATA-3), retinoa-associated nuclear orphan receptor γt (RORγt), B cell lymphoma-6 (Bcl-6) and Foxp3 in rats. Results Compared with the DSS group, the diarrhea and hematostoecium symptoms of UC mice in the DSS+BPYCP group and the DSS+5-ASA group were significantly improved, body weight and colon length of mice were increased, and colon mass, colon mass index and unit colon mass index were decreased (P<0.05). The mucosal epithelium was more complete than that in the DSS group, and gland arrangement was more regular. The inflammatory cell infiltration was less, and the pathological tissue damage score was significantly decreased (P<0.01). The proportion of Th2 cells in mesenteric lymph nodes was decreased, the proportion of Th17 cells and the level of IL-17A were decreased, and the mRNA levels of T-bet, GATA-3, RORγt and Bcl-6 in colon tissue were decreased (P<0.05). In the DSS+BPYCP group, the proportion of Th1 cells decreased, the proportion of CD4⁺CD25⁺Treg cells, CD4⁺CD25⁺Foxp3+Treg cells and the level of IL-10 increased, and the proportion of CD4⁺CXCR5⁺Tfh cells and the level of IL-21 decreased. The level of Foxp3 mRNA increased (P<0.05). The proportion of Th1 cells and the level of IFN-γ were decreased in the DSS+5-ASA group (P<0.05). Conclusion BPYCP may alleviate UC by remodeling the homeostasis of CD4⁺T cell subpopulations.

Key words: colitis, ulcerative, Bupi Yichang pill, CD4⁺T cell subpopulations, cellular homeostasis

中图分类号:

R967

图/表 14

参考文献 18

[1]	RUBIN D T, ANANTHAKRISHNAN A N, SIEGEL C A, et al. ACG Clinical Guideline:Ulcerative Colitis in Adults[J]. Am J Gastroenterol, 2019, 114(3):384-413. doi:10.14309/ajg.0000000000000152.
[2]	NG S C, SHI H Y, HAMIDI N, et al. Worldwide incidence and prevalence of inflammatory bowel disease in the 21st century:a systematic review of population-based studies[J]. Lancet, 2017, 390(10114):2769-2778. doi:10.1016/S0140-6736(17)32448-0.
[3]	邢潇潇, 郭建波, 李华山. 补脾益肠丸治疗溃疡性结肠炎的系统评价与Meta分析[J]. 海南医学院学报, 2022, 28(1):41-47,57.
	XING X X, GUO J B, LI H S. Systematic review and meta-analysis of Bupi Yichang pills for ulcerative colitis[J]. Journal of Hainan Medical University, 2022, 28(1):41-47,57. doi:10.13210/j.cnki.jhmu.20210107.002.
[4]	CHANG J T. Pathophysiology of inflammatory bowel diseases[J]. N Engl J Med, 2020, 383(27):2652-2664. doi:10.1056/NEJMra2002697.
[5]	SCHMIDT N, GONZALEZ E, VISEKRUNA A, et al. Targeting the proteasome:partial inhibition of the proteasome by bortezomib or deletion of the immunosubunit LMP7 attenuates experimental colitis[J]. Gut, 2010, 59(7):896-906. doi:10.1136/gut.2009.203554.
[6]	AKKOL E K, KARPUZ B, SOBARZO-SÁNCHEZ E, et al. A phytopharmacological overview of medicinal plants used for prophylactic and treatment of colitis[J]. Food Chem Toxicol, 2020, 144:111628. doi:10.1016/j.fct.2020.111628.
[7]	艾永建, 何梦崎, 王一涛, 等. 经典方药治疗溃疡性结肠炎的研究进展[J]. 中国中药杂志, 2022, 47(21):5797-5805.
	AI Y J, HE M Q, WANG Y T, et al. Review of classical prescriptions in treatment of ulcerative colitis[J]. China Journal of Chinese Materia Medica, 2022, 47(21):5797-5805. doi:10.19540/j.cnki.cjcmm.20220713.501.
[8]	RUTERBUSCH M, PRUNER K B, SHEHATA L, et al. In vivo CD4(+)T cell differentiation and function:Revisiting the Th1/Th2 paradigm[J]. Annu Rev Immunol, 2020, 38:705-725. doi:10.1146/annurev-immunol-103019-085803.
[9]	SMIDS C, HORJUS TALABUR HORJE C S, DRYLEWICZ J, et al. Intestinal T cell profiling in inflammatory bowel disease:Linking T cell subsets to disease activity and disease course[J]. J Crohns Colitis, 2018, 12(4):465-475. doi:10.1093/ecco-jcc/jjx160.
[10]	RAHIMI K, AHMADI A, HASSANZADEH K, et al. Targeting the balance of T helper cell responses by curcumin in inflammatory and autoimmune states[J]. Autoimmun Rev, 2019, 18(7):738-748. doi:10.1016/j.autrev.2019.05.012.
[11]	HAGIHARA Y, YOSHIMATSU Y, MIKAMI Y, et al. Epigenetic regulation of T helper cells and intestinal pathogenicity[J]. Semin Immunopathol, 2019, 41(3):379-399. doi:10.1007/s00281-019-00732-9.
[12]	YANG Y X, YUAN Y, XIA B. Cinnamtannin D1 ameliorates DSS-induced colitis by preventing Th17/Treg imbalance through activation of the AMPK/mTOR pathway[J]. Allergol Immunopathol(Madr), 2022, 50(5):153-161. doi:10.15586/aei.v50i5.654.
[13]	ZHONG Y, LIU W, XIONG Y, et al. Astragaloside Ⅳ alleviates ulcerative colitis by regulating the balance of Th17/Treg cells[J]. Phytomedicine, 2022, 104:154287. doi:10.1016/j.phymed.2022.154287.
[14]	WU X, CHEN H, GAO X, et al. Natural herbal remedy Wumei Decoction ameliorates intestinal mucosal inflammation by inhibiting Th1/Th17 cell differentiation and maintaining microbial homeostasis[J]. Inflamm Bowel Dis, 2022, 28(7):1061-1071. doi:10.1093/ibd/izab348.
[15]	DOU D, JI Y, ZHENG J, et al. A new role for conivaptan in ulcerative colitis in mice:Inhibiting differentiation of CD4(+)T cells into Th1 cells[J]. Dig Dis Sci, 2022, 67(8):3683-3692. doi:10.1007/s10620-021-07300-y.
[16]	WANG H Y, GE W, LIU S Q, et al. Curcumin inhibits T follicular helper cell differentiation in mice with dextran sulfate sodium(DSS)-induced colitis[J]. Am J Chin Med, 2022, 50(1):275-293. doi:10.1142/s0192415x22500100.
[17]	HEIDARI N, ABBASI-KENARSARI H, NAMAKI S, et al. Adipose-derived mesenchymal stem cell-secreted exosome alleviates dextran sulfate sodium-induced acute colitis by Treg cell induction and inflammatory cytokine reduction[J]. J Cell Physiol, 2021, 236(8):5906-5920. doi:10.1002/jcp.30275.
[18]	GU D, NAN Q, MIAO Y, et al. KT2 alleviates ulcerative colitis by reducing Th17 cell differentiation through the miR-302c-5p/STAT3 axis[J]. Eur J Cell Biol, 2022, 101(2):151223. doi:10.1016/j.ejcb.2022.151223.

基因名称	引物序列（5'→3'）	产物大小/bp
β-actin	上游：GATTACTGCTCTGGCTCCTAG	147
β-actin	下游：GACTCATCGTACTCCTGCTTG	147
T-bet	上游：GCCATTACAGGATGTTTGTGGAT	101
T-bet	下游：CATGCTGCCTTCTGCCTTTC	101
GATA3	上游：GGAAAGCTGGTTCGGAGGCA	241
GATA3	下游：GCCGATTCATTCGGGCTCAG	241
RORγt	上游：CCGCTGAGAGGGCTTCAC	230
RORγt	下游：TGCAGGAGTAGGCCACATTACA	230
Foxp3	上游：TTTCACCTATGCCACCCTTATC	103
Foxp3	下游：GTAGGCGAACATGCGAGTAA	103
Bcl-6	上游：CCGGCTCAATAATCTCGTGAA	83
Bcl-6	下游：GGTGCATGTAGAGTGGTGAGTGA	83

基因名称	引物序列（5'→3'）	产物大小/bp
β-actin	上游：GATTACTGCTCTGGCTCCTAG	147
β-actin	下游：GACTCATCGTACTCCTGCTTG	147
T-bet	上游：GCCATTACAGGATGTTTGTGGAT	101
T-bet	下游：CATGCTGCCTTCTGCCTTTC	101
GATA3	上游：GGAAAGCTGGTTCGGAGGCA	241
GATA3	下游：GCCGATTCATTCGGGCTCAG	241
RORγt	上游：CCGCTGAGAGGGCTTCAC	230
RORγt	下游：TGCAGGAGTAGGCCACATTACA	230
Foxp3	上游：TTTCACCTATGCCACCCTTATC	103
Foxp3	下游：GTAGGCGAACATGCGAGTAA	103
Bcl-6	上游：CCGGCTCAATAATCTCGTGAA	83
Bcl-6	下游：GGTGCATGTAGAGTGGTGAGTGA	83

组别	体质量/g	结肠质量/g	结肠质量指数/%	结肠长度/cm	单位结肠质量指数/%
Control组	25.00±1.10	0.22±0.02	0.90±0.09	8.70±0.70	2.58±0.18
DSS组	20.90±1.14^a	0.34±0.05^a	1.64±0.20^a	7.16±0.65^a	4.82±0.70^a
DSS+BPYCP组	24.00±1.34^b	0.24±0.03^b	0.98±0.13^b	8.33±0.66^b	2.85±0.49^b
DSS+5-ASA组	23.19±1.35^ab	0.28±0.05^ab	1.19±0.23^ab	8.01±0.70^ab	3.45±0.60^ab
F	19.893^＊＊	20.206^＊＊	36.325^＊＊	9.216^＊＊	34.355^＊＊

组别	体质量/g	结肠质量/g	结肠质量指数/%	结肠长度/cm	单位结肠质量指数/%
Control组	25.00±1.10	0.22±0.02	0.90±0.09	8.70±0.70	2.58±0.18
DSS组	20.90±1.14^a	0.34±0.05^a	1.64±0.20^a	7.16±0.65^a	4.82±0.70^a
DSS+BPYCP组	24.00±1.34^b	0.24±0.03^b	0.98±0.13^b	8.33±0.66^b	2.85±0.49^b
DSS+5-ASA组	23.19±1.35^ab	0.28±0.05^ab	1.19±0.23^ab	8.01±0.70^ab	3.45±0.60^ab
F	19.893^＊＊	20.206^＊＊	36.325^＊＊	9.216^＊＊	34.355^＊＊

组别	CD4⁺CXCR3⁺Th1/%	IFN-γ/（ng/L）
Control组	1.08±0.55	472.00±81.82
DSS组	2.37±0.62^a	714.67±181.52^a
DSS+BPYCP组	1.22±0.42^b	517.83±107.22
DSS+5-ASA组	1.12±0.37^b	467.33±103.84^b
F	9.056^＊＊	5.280^＊

补脾益肠丸重塑CD4⁺T细胞亚群稳态缓解溃疡性结肠炎的机制研究

Study on mechanism of Bupi Yichang pill in alleviating experimental ulcerative colitis by restoring the homeostasis of CD4⁺T cell subpopulations

引用本文

RichHTML

PDF (PC)

可视化

摘要/Abstract

使用本文

图/表 14

参考文献 18

相关文章 13

编辑推荐

Metrics

本文评价

组别	CD4⁺IL-33R⁺Th2/%	IL-4/（ng/L）
Control组	0.44±0.17	360.17±97.07
DSS组	2.02±0.52^a	530.00±110.16^a
DSS+BPYCP组	0.52±0.13^b	394.83±88.43
DSS+5-ASA组	1.14±0.54^ab	463.83±84.08
F	21.051^＊＊	3.742^＊

组别	CD4⁺CCR6⁺Th17/%	IL-17A/（ng/L）
Control组	0.99±0.26	50.55±16.53
DSS组	3.59±0.91^a	161.50±44.18^a
DSS+BPYCP组	1.15±0.27^b	65.75±21.19^b
DSS+5-ASA组	1.42±0.33^b	80.23±19.13^b
F	33.020^＊＊	19.347^＊＊

组别	CD4⁺CD25⁺ Treg/%	CD4⁺CD25⁺Foxp3⁺ Treg/%	IL-10/（ng/L）
Control组	13.19±3.52	18.73±4.58	1 138.50±230.64
DSS组	4.16±1.88^a	8.60±2.87^a	651.33±139.71^a
DSS+BPYCP组	15.37±3.76^b	19.10±5.16^b	1 455.67±288.92^b
DSS+5-ASA组	7.94±2.89^a	12.42±2.90	828.33±137.10
F	16.072^＊＊	9.715^＊＊	17.212^＊＊

组别	CD4⁺CXCR5⁺ Tfh/%	IL-21/（ng/L）
Control组	4.81±1.46	294.67±98.06
DSS组	21.65±6.22^a	602.67±122.60^a
DSS+BPYCP组	6.19±2.08^b	391.83±92.46^b
DSS+5-ASA组	5.95±2.45^b	448.83±86.15
F	30.210^＊＊	9.820^＊＊

组别	T-bet	GATA-3	RORγt	Bcl-6	Foxp3
Control组	0.91±0.44	1.09±0.42	1.22±0.53	0.83±0.27	1.01±0.34
DSS组	3.15±1.05^a	5.10±1.67^a	12.16±3.61^a	20.27±6.30^a	0.20±0.11^a
DSS+BPYCP组	1.80±0.57^b	2.30±0.69^b	5.38±1.92^ab	8.69±2.80^ab	0.55±0.18^ab
DSS+5-ASA组	1.59±0.54^b	3.19±0.62^ab	7.71±2.20^ab	13.49±3.72^ab	0.35±0.15^a
F	11.174^＊＊	17.970^＊＊	22.917^＊＊	26.148^＊＊	16.252^＊＊

[1]	陈静, 魏运姣, 罗超, 黄利华, 陈橙, 段莎莎. 基于Nrf2/ARE抗氧化应激途径探究乌梅丸对溃疡性结肠炎小鼠的作用机制[J]. 天津医药, 2024, 52(3): 278-254.
[2]	卢年华, 靳展洪叶, 张茜, 张萌, 李俊珂, 赵慧巧, 张永鹏. 湿生扁蕾呫吨酮联合益生菌调控TGF-β1/Smads通路及炎性因子干预大鼠结肠炎癌转化[J]. 天津医药, 2024, 52(2): 136-141.
[3]	张婕, 苗耐英, 李欢, 刘新建. 奥曲肽对新生儿坏死性小肠结肠炎患儿粪便性状及体液免疫的影响[J]. 天津医药, 2024, 52(12): 1331-1334.
[4]	张萌, 崔瑞洁, 李彩芳, 高琦, 李斐. 盐酸小檗碱片联合复方谷氨酰胺对溃疡性结肠炎患者肠黏膜屏障功能的影响[J]. 天津医药, 2024, 52(1): 102-106.
[5]	史刚刚, 郑冰, 韩梅. 溃疡性结肠炎患者发生阑尾口周围炎的危险因素及预后分析[J]. 天津医药, 2023, 51(4): 404-408.
[6]	邹大军, 胡福德, 周启立, 徐晓青. miR-301a对小鼠坏死性小肠结肠炎的影响及作用机制[J]. 天津医药, 2022, 50(9): 938-942.
[7]	杨天骄, 柯创宏, 曾词正, 林丽曼, 胡海玲, 莫笑欢 . 腹部超声对新生儿食物蛋白诱导的小肠结肠炎综合征与早期新生儿坏死小肠结肠炎的鉴别价值[J]. 天津医药, 2022, 50(2): 210-213.
[8]	李娟, 袁向飞 , 赵煜 . 小肠结肠炎综合征小鼠肠黏膜中NLRP3表达与炎症分子关系的研究[J]. 天津医药, 2021, 49(8): 829-832.
[9]	孟祥鹿, 孙际伟, 王欢, 代立梅, 赵少莉, 赵雨蒙, 冯玲玲, 王文红△. MRC结合FC对溃疡性结肠炎活动程度的诊断效能[J]. 天津医药, 2020, 48(7): 635-641.
[10]	刘晓兰, 张永忠, 张俊玲, 聂晓博, 贾琳, 刘会丽△. 苍术挥发油对溃疡性结肠炎大鼠的改善作用[J]. 天津医药, 2020, 48(10): 956-960.
[11]	杜小燕, 李玲. 坏疽性脓皮病伴溃疡性结肠炎继发肛瘘一例[J]. 天津医药, 2019, 47(1): 90-93.
[12]	胡丽丽，李辛子，何长江，孙焱，王春祥. 婴儿结肠狭窄的临床及影像学特点[J]. 天津医药, 2017, 45(12): 1286-1288.
[13]	王倩1 ,吕永慧2 ,周丽梅1 ,陈文剑2 ,江桂英1 ,江沂1 . Bcl-2与Caspase-3在溃疡性结肠炎中的表达与意义*[J]. , 2013, (9): 849-851 .

补脾益肠丸重塑CD4+ T细胞亚群稳态缓解溃疡性结肠炎的机制研究

Study on mechanism of Bupi Yichang pill in alleviating experimental ulcerative colitis by restoring the homeostasis of CD4+T cell subpopulations

引用本文

RichHTML

PDF (PC)

可视化

摘要/Abstract

使用本文

图/表 14

参考文献 18

相关文章 13

编辑推荐

Metrics

本文评价

补脾益肠丸重塑CD4⁺T细胞亚群稳态缓解溃疡性结肠炎的机制研究

Study on mechanism of Bupi Yichang pill in alleviating experimental ulcerative colitis by restoring the homeostasis of CD4⁺T cell subpopulations