基于Th17/Treg免疫平衡探讨松节方对类风湿性关节炎风寒湿痹证模型大鼠的干预作用

doi:10.12122/j.issn.1673-4254.2026.08.17

摘要/Abstract

摘要：

目的基于Th17/Treg免疫平衡，探讨松节方（SJF）对类风湿性关节炎（RA）及风寒湿痹证（WCDA）的治疗机制。方法建立RA及WCDA大鼠模型，造模成功大鼠分别分为模型组、松节方组（n=10），另设正常对照组10只。连续给药2周。观察大鼠体质量、一般状态及足跖肿胀度；以酶联免疫吸附法检测各组大鼠血清白介素-10（IL-10）、白介素-17（IL-17）、白介素-21（IL-21）、肿瘤坏死因子α（TNF-α）水平；流式细胞仪检测外周血、脾细胞T细胞亚群Th17、Treg细胞的比例；以实时荧光定量聚合酶链反应法检测大鼠踝关节滑膜中维甲酸相关孤儿受体γt（RORγt）、叉头转录因子3（Foxp3）mRNA的表达情况； Western blotting法检测踝关节滑膜组织RORγt和Foxp3蛋白表达水平。结果在类风湿性关节炎（RA）及风寒湿痹证（WCDA）模型中，松节方可明显改善模型大鼠的一般状况，显著抑制足肿胀，下调促炎因子IL-17、IL-21、TNF-α水平（P<0.05），上调抗炎因子IL-10（P<0.01），同时显著升高Treg细胞百分比（P<0.01），降低Th17细胞百分比（P<0.01），并能抑制RORγt mRNA及蛋白表达（P<0.01），促进Foxp3 mRNA及蛋白表达，显著改善滑膜炎症浸润。结论松节方干预后模型大鼠的炎症因子水平、Th17/Treg 比例及 RORγt/Foxp3 表达均发生显著变化，提示松节方改善模型大鼠关节炎症及病理损伤的作用可能与调节Th17/Treg平衡有关。

关键词: 松节方, 类风湿性关节炎, 风寒湿痹证, Th17/Treg平衡, RORγt, 叉头转录因子3

Abstract:

Objective To investigate the therapeutic mechanism of Songjie Formula (SJF) for alleviating rheumatoid arthritis (RA) with wind-cold-dampness arthralgia syndrome (WCDA) from the perspective of Th17/Treg immune balance. Methods Twenty male SD rat models of RA with WCDA were randomized into two equal groups for daily gavage with saline (model group) or SJF for 14 consecutive days, with 10 normal rats serving as the normal control group. The changes in body weight, general condition, and plantar swelling of the rats were monitored. Serum levels of interleukin-10 (IL-10), IL-17, IL-21, and tumor necrosis factor alpha (TNF-α) were measured using ELISA, and Th17 and Treg cell percentages in peripheral blood and splenic T cell subsets were analyzed by flow cytometry. RT-qPCR and Western blotting were used to detect the mRNA and protein expression levels of retinoic acid-related orphan receptor γt (RORγt) and forkhead box P3 (Foxp3) in the ankle joint synovial tissue. Results In the rats models of RA with WCDA, SJF gavage significantly improved the general condition, reduced foot swelling, downregulated serum levels of IL-17, IL-21, and TNF-α, upregulated IL-10 level, and obviously increased Treg cell percentage while decreasing Th17 cell percentage. SJF treatment also significantly suppressed the expressions of RORγt mRNA and protein, promoted Foxp3 mRNA and protein expressions, and alleviated synovial inflammatory cell infiltration in the rat models. Conclusion SJF regulates inflammatory cytokine levels, Th17/Treg ratio, and RORγt/Foxp3 expressions in RA rat model with WCDA, suggesting that its therapeutic efficacy for RA is mediated primarily by ameliorating joint inflammation and pathologies via modulating the Th17/Treg balance.

Key words: Songjie Formula, rheumatoid arthritis, wind-cold-dampness arthralgia, Th17/Treg balance, retinoic acid-related orphan receptor γt, forkhead box P3

梅小利, 吴思澜, 罗金萍, 黄文涛, 孙建彬, 黄崇刚. 基于Th17/Treg免疫平衡探讨松节方对类风湿性关节炎风寒湿痹证模型大鼠的干预作用[J]. 南方医科大学学报, 2026, 46(8): 1896-1904.

Xiaoli MEI, Silan WU, Jinping LUO, Wentao HUANG, Jianbin SUN, Chonggang HUANG. Songjie Formula alleviates rheumatoid arthritis in rats with wind-cold-dampness syndrome by regulating Th17/Treg immune balance[J]. Journal of Southern Medical University, 2026, 46(8): 1896-1904.

图/表 10

表1 引物序列

Tab.1 Primer sequences for RT-qPCR

Primer name	Primer sequence	Expansion length (bp)
GAPDH-F	TGTGAACGGATTTGGCCGTA	183
GAPDH-R	ACCAGCTTCCCATTCTCAGC	183
ROR-γt-F	AGCAGGAGCAATGGAAGTCG	216
ROR-γt-R	ATGAGAACCAAGGCCGTGTAG	216
Foxp3 -F	CGCCTACTTCAGAAACCACC	90
Foxp3 -R	TCACTCTCCACTCGCACAAA	90

表2 SJF对大鼠体质量的影响

Tab.2 Effect of Songjie Formula (SJF) on body weight in rat models (Mean±SD, n=10)

Group	Dose (g/kg)	Pre-modeling	Pre-dose	Day 7 post-dose	Day 14 post-dose
Control	-	243±14	344±12	364±10	382±11
RA model	-	237±11	319±16**	340±23**	366±25
RA+SJF	6.16	251±11	325±24*	347±30	371±32
WCDA model	-	244±11	287±18**	322±25**	346±25**
WCDA+SJF	6.16	247±11	292±22**	327±24**	355±26*

表3 SJF对大鼠足肿胀的影响

Tab.3 Effect of SJF on paw edema in the rat models (Mean±SD, n=10)

Group	Dose(g/kg)	Pre-modeling	Post-modeling	Day 14 post-dose
Control	-	1.66±0.07	1.77±0.07	1.89±0.09
RA model	-	1.75±0.11	2.99±0.24**	2.97±0.31**
RA+SJF	6.16	1.79±0.10	3.02±0.31**	2.54±0.27**^##
WCDA model	-	1.74±0.08	3.04±0.27**	3.05±0.29^**
WCDA+SJF	6.16	1.70±0.09	2.97±0.19**	2.58±0.29**^▲▲

表4 SJF对大鼠血清炎症因子水平的影响

Tab.4 Effect of SJF on serum levels of inflammatory factors in the rats (Mean±SD, n=10)

Group	Dose (g/kg)	IL-10 (pg/mL)	IL-17 (pg/mL)	IL-21 (pg/mL)	TNF-α (pg/mL)
Control	-	38.1±7.5	12.0±3.6	586±56	73.6±19.6
RA model	-	29.2±4.2**	18.4±3.8**	761±111**	103.2±19.6**
RA+SJF	6.16	36.2±7.9^##	13.4±4.3^##	620±79^##	81.6±11.7^##
WCDA model	-	28.6±4.2**	19.2±4.7**	722±71**	110.9±29.7**
WCDA+SJF	6.16	35.5±7.2^▲	14.9±4.1^▲	634±71^▲▲	82.4±16.7^▲▲

图1 各组大鼠外周血及脾脏Th17、Treg细胞流式检测代表图

Fig.1 Flow cytometry for analyzing Th17 and Treg cells in peripheral blood and spleen in each group.

图2 SJF对大鼠外周血及脾脏Th17细胞及Treg细胞比例的影响

Fig.2 Effect of SJF on Th17 and Treg cell percentages in peripheral blood and spleen of the rats (Mean±SD, n=3). A: Changes of Th17 cells in the blood. B: Changes of Th17 cells in the spleen. C: Changes of Treg cells in the blood. D: Changes of Treg cells in the spleen. **P<0.01 vs Control; ##P<0.01 vs RA model; ▲▲P<0.01 vs WCDA model.

图3 SJF对大鼠滑膜组织RORγt、Foxp3 mRNA相对表达量的影响

Fig.3 Effect of SJF on relative expression levels of RORγt and Foxp3 mRNA in synovial tissue of rats (Mean±SD, n=3). **P<0.01 vs Control; ##P<0.01 vs RA model;△△P<0.01 vs RA+SJF; ▲▲P<0.01 vs WCDA model.

图4 SJF对大鼠滑膜组织RORγt、Foxp3 蛋白表达水平的影响

Fig.4 Effect of SJF on protein expression levels of RORγt and Foxp3 in synovial tissue of the rats (Mean±SD, n=3). **P<0.01 vs Control; ##P<0.01 vs RA model; △△P<0.01 vs RA+SJF;▲▲P<0.01 vs WCDA model.

表5 SJF对大鼠关节组织半定量评分的影响

Tab.5 Effect of SJF on the semi-quantitative score of rat joint tissue (Mean±SD, n=7)

Group	Dose (g/kg)	Semi-quantitative scoring (Point)
Control	-	0
RA model	-	2.43±0.79**
RA+SJF	6.16	1.57±0.79**^#
WCDA model	-	2.71±0.49**
WCDA+SJF	6.16	1.71±0.76**^▲▲

图5 SJF对大鼠关节病理的影响

Fig.5 Effect of SJF on joint pathology in the rat models (HE staining, original magnification: ×40).

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