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

doi:10.11958/20230776

Abstract

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

CLC Number:

R967

Figures/Tables 14

References 18

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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^＊