益气活络方调节PI3K/Akt/mTOR通路抑制胶原诱导型关节炎大鼠滑膜炎症的作用机制

doi:10.12092/j.issn.1009-2501.2026.02.009

中国临床药理学与治疗学 ›› 2026, Vol. 31 ›› Issue (2): 213-222.doi: 10.12092/j.issn.1009-2501.2026.02.009

益气活络方调节PI3K/Akt/mTOR通路抑制胶原诱导型关节炎大鼠滑膜炎症的作用机制

王鹏飞¹(), 王雨薇², 金芳梅¹, 陈平¹, 李伟青¹, 田雪梅¹, 王爱华¹, 王海东¹^,*()

1. 甘肃省中医院风湿骨病中心，兰州 730050，甘肃
2. 甘肃中医药大学，兰州 730000，甘肃

收稿日期:2025-03-25 修回日期:2025-06-01 出版日期:2026-02-26 发布日期:2026-03-17
通讯作者: 王海东 E-mail:wpengfei1011@163.com;whaidong5895@163.com
作者简介:王鹏飞，男，硕士研究生，主治医师，从事风湿病的中医药防治研究。E-mail：wpengfei1011@163.com
基金资助:
国家中医药管理局高水平重点学科建设项目（国中医药人教函[2023]85号）；甘肃省中医药项目（GZKP-2022-17）；兰州市青年科技人才创新专项（2023-QN-12）

The mechanism of action of Yiqi Huoluo formula in regulating the PI3K/Akt/mTOR pathway to suppress synovial inflammation in collagen-induced arthritis rats

Pengfei WANG¹(), Yuwei WANG², Fangmei JIN¹, Ping CHEN¹, Weiqing LI¹, Xuemei TIAN¹, Aihua WANG¹, Haidong WANG¹^,*()

1. Rheumatology and Bone Disease Center, Gansu Provincial Hospital of Traditional Chinese Medicine, Lanzhou 730050, Gansu, China
2. Gansu University of Traditional Chinese Medicine, Lanzhou 730000, Gansu, China

Received:2025-03-25 Revised:2025-06-01 Online:2026-02-26 Published:2026-03-17
Contact: Haidong WANG E-mail:wpengfei1011@163.com;whaidong5895@163.com

摘要/Abstract

摘要：

目的: 研究益气活络方对胶原诱导型关节炎（collagen-induced arthritis，CIA）大鼠的治疗作用及其对PI3K/Akt/mTOR信号通路的影响。方法: 将60只SD大鼠随机分为空白组、模型组、甲氨蝶呤组（1 mg/mL）、益气活络方低、中、高剂量组（9.45、18.9、37.8 g/kg），每组各10只。除空白组外，其余大鼠均建立CIA大鼠模型。加强免疫后连续干预28 d后取材。期间观察大鼠一般情况、踝关节肿胀度以及关节炎指数评分，检测血清炎症因子TNF-α、IL-1β、IL-6、IL-10水平；采用苏木精-伊红（HE）染色观察病理变化；实时荧光定量PCR（RT-qPCR）检测膝关节滑膜组织中PI3K、Akt、mTOR、CD86、CD206 mRNA表达；蛋白免疫印记法（Western blot）检测p-PI3K、p-Akt、p-mTOR蛋白水平；免疫组化测定CD86、CD206蛋白表达。结果: 与空白组相比，模型组大鼠关节肿胀度及关节炎指数评分升高（P<0.01），踝关节病理切片可见大量炎性细胞浸润，纤维组织及滑膜细胞增生，排列紊乱，滑膜组织周边可见少量毛细血管增生，血清中TNF-α、IL-1β、IL-6水平升高，IL-10水平降低（P<0.05，P<0.01），膝关节滑膜组织中PI3K、Akt、mTOR、CD86 mRNA表达水平增加，而CD206 mRNA的表达降低（P<0.05，P<0.01），滑膜组织中p-PI3K、p-Akt、p-mTOR蛋白的表达水平升高（P<0.05，P<0.01），踝关节滑膜中CD86蛋白表达升高，CD206蛋白表达降低（P<0.05，P<0.01）。与模型组相比，甲氨蝶呤组、益气活络方中、高剂量组大鼠踝关节肿胀程度减轻（P<0.05，P<0.01），关节炎指数评分降低（P<0.05，P<0.01），血清中TNF-α、IL-1β、IL-6水平降低，IL-10水平升高（P<0.05，P<0.01），膝关节滑膜组织中PI3K、Akt、mTOR、CD86、mRNA表达水平降低，而CD206 mRNA的表达升高（P<0.05，P<0.01），滑膜组织中p-PI3K、p-Akt、p-mTOR蛋白的表达水平降低（P<0.05），踝关节滑膜中CD86蛋白表达降低，CD206蛋白表达升高（P<0.05），并能改善滑膜炎症。结论: 益气活络方通过下调PI3K/Akt/mTOR通路，调控巨噬细胞极化，减少促炎因子，促进抑炎因子表达，从而缓解类风湿关节炎滑膜炎症，减轻关节病理损伤。

关键词: 磷脂酰肌醇3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶点通路, 类风湿关节炎, 滑膜炎, 益气活络方

Abstract:

AIM: To study the therapeutic effect of Yiqi Huoluo formula on collagen-induced arthritis (CIA) rats and its effect on PI3K/Akt/mTOR signaling pathway. METHODS: A total of 60 SD rats were randomly divided into blank group, model group, methotrexate group (1 mg/mL), and Yiqi Huoluo Fang low, medium and high dose groups (9.45 g/kg, 18.9 g/kg, 37.8 g/kg), with 10 rats in each group. Except for the blank group, CIA rat models were established for the rest of the rats. After 28 days of continuous intervention after booster immunization, the samples were collected. During the period, the general condition, ankle swelling and arthritis index score of the rats were observed. Enzyme linked immunosorbent assay (ELISA) was used to detect the levels of TNF-α, IL-1β, IL-6 and IL-10 in the serum of rats. Hematoxylin-eosin staining (HE) was used to observe the histopathological changes of the synovial synovium of the ankle joint in rats. Quantitative Real-time polymerase chain reaction (RT-qPCR) was used to detect the mRNA expressions of PI3K, Akt, mTOR, CD86 and CD206 in the synovial tissue of rat knee. Western blot was used to detect the expression levels of p-PI3K, p-Akt and p-mTOR proteins in the synovial tissue of rat knee. Immunohistochemistry was used to determine the positive expression of CD86 and CD206 in rat ankle joints. RESULTS: Compared with the blank group, the joint swelling and AI index of the model group increased (P<0.01), a large number of inflammatory cell infiltration, fibrous tissue and synovial cell proliferation and disordered arrangement were seen in the pathological sections of the ankle joint, a small number of capillaries were found around the synovial tissue, the serum levels of TNF-α, IL-1β and IL-6 were increased, and the levels of IL-10 were decreased (P<0.05, P<0.01), the expression levels of PI3K, Akt, mTOR, CD86 and mRNA in the synovial tissue of knee joint were increased, while the expression of CD206 mRNA was decreased (P<0.05, P<0.01), the expression levels of p-PI3K, p-Akt and p-mTOR proteins in synovial tissue increased (P<0.05, P<0.01), and the expression of CD86 protein and CD206 protein decreased in synovial membrane of ankle joint (P<0.05, P<0.01). Compared with the model group, the degree of ankle swelling in the methotrexate group, the Yiqi Huoluo formula and the high-dose group decreased (P<0.05, P<0.01), the AI index decreased (P<0.05, P<0.01), the serum levels of TNF-α, IL-1β and IL-6, the levels of IL-10 increased (P<0.05, P<0.01), the expression levels of PI3K, Akt, mTOR, CD86 and mRNA in the synovial tissue of the knee joint decreased, and the expression of CD206 mRNA increased (P<0.05, P<0.01), the expression levels of p-PI3K, p-Akt and p-mTOR proteins in synovial tissue decreased (P<0.05), the expression of CD86 protein decreased in synovial membrane of ankle joint, and the expression of CD206 protein increased (P<0.05) and can improve synovial inflammation. CONCLUSION: The Yiqi Huoluo formula alleviates synovial inflammation in rheumatoid arthritis (RA) and reduces joint pathological damage by down-regulating the PI3K/Akt/mTOR pathway, modulating macrophage polarization, decreasing pro-inflammatory factors, and promoting the expression of anti-inflammatory factors.

Key words: phosphatidylinositol 3-kinase/protein kinase B/mammalian rapamycin target pathway, rheumatoid arthritis, synovitis, Yiqi Huoluo formula

中图分类号:

R965.2

王鹏飞, 王雨薇, 金芳梅, 陈平, 李伟青, 田雪梅, 王爱华, 王海东. 益气活络方调节PI3K/Akt/mTOR通路抑制胶原诱导型关节炎大鼠滑膜炎症的作用机制[J]. 中国临床药理学与治疗学, 2026, 31(2): 213-222.

Pengfei WANG, Yuwei WANG, Fangmei JIN, Ping CHEN, Weiqing LI, Xuemei TIAN, Aihua WANG, Haidong WANG. The mechanism of action of Yiqi Huoluo formula in regulating the PI3K/Akt/mTOR pathway to suppress synovial inflammation in collagen-induced arthritis rats[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2026, 31(2): 213-222.

图/表 9

表 1

Table 1 Primer sequences

Gene	Sequence	Base pair
PI3K	F:TTCTTGGGATAGGTGACAGGC	21
PI3K	R:CGGGGTTAGGACAAAAGGTAC	21
Akt	F:CGACACCAGGTATTTTGATGAGG	23
Akt	R:GCTGAGTAGGAGAACTGGGGAAA	23
mTOR	F:AAGTGAAGCCGAGAGCAATGAG	22
mTOR	R:CTGAGTGTATCCTGGAGGTTGTTG	24
CD86	F:TCAACTAATGAGTATGGCGACAAC	24
CD86	R:AAACTGGGGCTGCGAAAA	18
CD206	F:ATCACTCCTGTCCCTGACTCCC	22
CD206	R:CACCATCTCCCCTGCCATT	19

表 2

Table 2 Comparison of body weight among different groups of rats ($\bar x $±s, n=10)

Group	Dose	0 d	7 d	14 d	21 d	28 d
Control		221.880±5.225	256.060±5.931	264.160±4.601	272.700±4.008	283.760±3.472
Model		227.140±4.277	235.880±2.111^c	244.66±4.227^c	253.640±2.214^c	262.120±3.885^c
MTX	1 mg/mL	218.100±5.028	240.120±3.666	262.120±4.396^e	264.160±1.924^e	277.600±3.700^f
YQHLF L	9.45 g/kg	209.733±6.359	221.467±5.626	230.400±4.729	245.867±6.428	257.000±2.483
YQHLF M	18.9 g/kg	217.133±8.431	227.333±8.490	242.333±2.718	251.500±3.936	266.967±3.403
YQHLF H	37.8 g/kg	210.467±6.235	223.100±4.965	242.767±7.678	267.200±1.253^e	285.467±5.044^f

表 3

Table 3 Effects of Yiqi Huoluo formula on rat joint swelling (mm, $\bar x $±s, n=10)

Group	Dose	0 d	7 d	14 d	21 d	28 d
Control		2.575±0.075	5.875±0.515	7.500±0.289	8.625±0.239	8.750±0.479
Model		66.375±4.270^c	114.700±3.590^c	112.200±4.991^c	107.525±4.526^c	101.975±4.101^c
MTX	1 mg/mL	57.175±1.768	101.450±1.445	86.175±3.242^f	73.600±3.408^f	60.900±3.484^f
YQHLF L	9.45 g/kg	59.075±2.099	114.850±4.101	101.650±6.521	88.525±4.786^f	70.275±3.100^f
YQHLF M	18.9 g/kg	62.350±3.395	115.025±6.678	98.875±6.089^e	72.925±2.407^f	62.250±1.149^f
YQHLF H	37.8 g/kg	56.800±2.171	111.675±4.906^e	92.225±4.250^f	67.550±3.992^f	55.525±2.351^f

表 4

Table 4 Effects of Yiqi Huoluo formula on the joint inflammation index of CIA rats in each group ($\bar x $±s, n=10)

Group	Dose	0 d	7 d	14 d	21 d	28 d
Control		0.000±0.000	0.000±0.000	0.000±0.000	0.000±0.000	0.000±0.000
Model		6.800±1.398	11.500±0.850^c	10.90±0.876^c	9.800±0.919^c	9.400±0.699^c
MTX	1 mg/mL	6.700±1.251	11.100±0.994	6.700±0.949^f	6.000±0.816^f	4.800±0.789^f
YQHLF L	9.45 g/kg	6.400±1.075	11.300±0.949	9.100±1.595^f	8.000±1.155^f	7.100±0.994^f
YQHLF M	18.9 g/kg	6.100±0.738	11.100±1.197	8.500±1.080^f	6.900±0.738^f	5.700±1.160^f
YQHLF H	37.8 g/kg	6.500±0.850	11.400±0.966	7.700±1.160^f	5.300±0.823^f	4.300±0.675^f

图 1

Fig.1 Expression levels of TNF-α, IL-1β, IL-6 and IL-10 in serum of rats treated with Yiqi Huoluo formula ($\bar x $±s, n=10) cP<0.01, compared with the control group; eP<0.05, fP<0.01, compared with the model group.

图 2

Fig.2 Effects of Yiqi Huoluo formula on histopathology of ankle joint cartilage and synovial tissue in each group of rats (×200) and Comparison of pathological scores of ankle joints in each group of rats ($\bar x $±s, n=10) A: Control; B: Model; C: MTX; D: YQHLF L; E: YQHLF M; F: YQHLF H. The red arrows indicate inflammatory cell infiltration, the green arrows indicate synovial tissue hyperplasia, the blue arrows indicate neovascularization, and the yellow arrows indicate cartilage erosion. cP<0.01, compared with the control group; fP<0.01, compared with the model group.

图 3 bP<0.05, cP<0.01, compared with the control group; eP<0.05, fP<0.01, compared with the model group.

Fig.3 Effects of Yiqi Huoluo formula on the mRNA expression of PI3K, Akt, mTOR, CD86 and CD206 in the synovial tissue of rat knee joints ($\bar x $±s, n=10) bP<0.05, cP<0.01, compared with the control group; eP<0.05, fP<0.01, compared with the model group.

图 4

Fig.4 Effects of Yiqi Huoluo formula on the protein expression of p-PI3K, p-Akt, and p-mTOR in rat synovial tissues ($\bar x $±s, n=10) A: Control; B: Model; C: MTX; D:YQHLF L; E: YQHLF M; F: YQHLF H. cP<0.01, compared with the control group; eP<0.05, fP<0.01, compared with the model group.

图 5

Fig.5 Effect of Yiqi Huoluo formula on the protein expression of CD86 and CD206 in rat ankle joint synovium ($\bar x $±s, n=10) cP<0.01, compared with the control group; eP<0.05, fP<0.01, compared with the model group.

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益气活络方调节PI3K/Akt/mTOR通路抑制胶原诱导型关节炎大鼠滑膜炎症的作用机制

The mechanism of action of Yiqi Huoluo formula in regulating the PI3K/Akt/mTOR pathway to suppress synovial inflammation in collagen-induced arthritis rats

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