舒筋活血胶囊通过JAK2/STAT3通路缓解大鼠膝骨关节炎的机制研究

doi:10.11958/20221924

摘要/Abstract

摘要：

目的探讨舒筋活血胶囊调控酪氨酸蛋白激酶2（JAK2）/信号转导及转录激活蛋白3（STAT3）信号通路对大鼠膝骨关节炎（KOA）的影响及其机制。方法采用Hulth法制备大鼠KOA模型，将60只大鼠分为假手术组（Sham组）、KOA组、舒筋活血胶囊组（SJHX组）（472.5 mg/kg 舒筋活血胶囊灌胃）、AG490组（5.0 mg/kg的JAK2抑制剂AG490腹腔注射），每组15只。HE染色与番红O-固绿染色观察滑膜组织与软骨组织病理变化，酶联免疫吸附试验（ELISA）检测血清白细胞介素（IL）-10、IL-1β、肿瘤坏死因子α（TNF-α）水平，免疫荧光法检测M1/M2型巨噬细胞极化，免疫组化染色检测IL-1β、诱导型一氧化氮合成酶（iNOS）、基质金属蛋白酶（MMP）-13蛋白表达，Western blot检测JAK2/STAT3通路相关蛋白。结果与Sham组比较，KOA组大鼠关节面不平整，滑膜组织与软骨组织结构破坏，病理评分增加，血清IL-1β、TNF-α水平明显升高，IL-10水平降低，滑膜组织IL-1β、iNOS、MMP-13、p-JAK2/JAK2、p-STAT3/STAT3表达水平及M1、M2型巨噬细胞、M1/M2比值升高（P<0.05）；与KOA组比较，SJHX组与AG490组大鼠滑膜组织与软骨组织病理损伤减轻，病理评分降低，血清IL-1β、TNF-α水平降低，IL-10水平升高，滑膜组织IL-1β、iNOS、MMP-13、p-JAK2/JAK2、p-STAT3/STAT3表达水平及M1巨噬细胞、M1/M2比值降低（P<0.05）。结论舒筋活血胶囊可通过抑制JAK2/STAT3信号通路激活，调节M1/M2巨噬细胞极化，改善KOA大鼠滑膜炎症状。

关键词: 骨关节炎, 膝, 活血舒筋, 巨噬细胞, Janus激酶2, STAT3转录因子, 舒筋活血胶囊

Abstract:

Objective To investigate the effect and mechanism of Shujin Huoxue Capsule on knee osteoarthritis (KOA) in rats by regulating tyrosine protein kinase 2 (JAK2)/signal transduction and transcription-activating protein 3 (STAT3) signaling pathway. Methods The Hulth method was used to prepare rat KOA model. Sixty rats were divided into the Sham group, the KOA group, the Shujin Huoxue Capsule (SJHX) group (472.5 mg/kg Shujin Huoxue Capsule gavage) and the AG490 group (5.0 mg/kg JAK2 inhibitor AG490 intraperitoneally) with 15 rats in each group. The pathological changes of synovial tissue and cartilage tissue were observed by HE staining and Safranin O-fast green staining. Serum levels of interleukin-10 (IL-10), IL-1β and tumor necrosis factor-α (TNF-α) were detected by enzyme-linked immunosorbent assay (ELISA). M1/M2 macrophage polarization was detected by immunofluorescence assay. The expression levels of IL-1β, inducible nitric oxide synthase (iNOS) and matrix metalloproteinase 13 (MMP-13) were detected by immunohistochemical staining. JAK2/STAT3 pathway related proteins were detected by Western blot assay. Results Compared with the Sham group, the articular surface was uneven in rats of the KOA group. The synovial tissue and cartilage tissue were destroyed, pathological scores were increased. Serum levels of IL-1β and TNF-α were obviously increased, and the level of IL-10 was obviously reduced. Expression levels of IL-1β, iNOS, MMP-13, p-JAK2/JAK2 and p-STAT3/STAT3 in synovial tissue, and the proportions of M1, M2 macrophages and M1/M2 were obviously increased (P<0.05). Compared with the KOA group, the pathological damage of synovial tissue and cartilage tissue was reduced in the SJHX group and the AG490 group. Pathological scores were reduced. Serum levels of IL-1β and TNF-α were obviously reduced, and the level of IL-10 was obviously increased. Expression levels of IL-1β, iNOS, MMP-13, p-JAK2/JAK2, p-STAT3/STAT3 in synovial tissue, and the proportions of M1, M2 macrophages and M1/M2 were obviously reduced (P<0.05). Conclusion Shujin Huoxue Capsule can improve the symptoms of synovitis in KOA rats by inhibiting the activation of JAK2/STAT3 signaling pathway and regulating the polarization of M1/M2 macrophages.

Key words: osteoarthritis, knee, activating blood and relaxing muscle tendon, macrophages, Janus kinase 2, STAT3 transcription factor, Shujin Huoxue Capsule

中图分类号:

R684.3

图/表 11

图1 HE染色检测各组大鼠滑膜组织病理变化（×200）

Fig.1 Pathological changes of synovium detected by HE staining in each group (×200)

表1 各组大鼠Krenn评分与Mankin评分比较（n=5，分，$\bar{x}\pm s$）

Tab.1 Comparison of Krenn score and Mankin score between the four groups of rats

组别	Krenn评分	Mankin评分
Sham组	0.00±0.00	0.00±0.00
KOA组	4.60±1.20^a	5.80±1.65^a
SJHX组	2.40±0.60^ab	3.60±1.00^ab
AG490组	2.60±0.80^ab	3.40±1.15^ab
F	29.071^＊＊	22.729^＊＊

图2 番红O-固绿染色检测各组大鼠软骨组织病理变化（×200）

Fig.2 Pathological changes of cartilage tissue of rats detected by safranine O-solid green staining (×200)

表2 各组大鼠血清IL-10、IL-1β、TNF-α水平比较（n=15， $\bar{x}\pm s$）

Tab.2 Comparison of serum levels of IL-10, IL-1β and TNF-α between the four groups

组别	IL-10/（μg/L）	IL-1β/（ng/L）	TNF-α/（μg/L）
Sham组	23.56±4.51	12.26±5.36	16.15±4.31
KOA组	11.34±2.33^a	85.42±7.41^a	39.54±5.75^a
SJHX组	18.35±4.74^ab	47.46±6.15^ab	20.36±4.82^b
AG490组	20.12±4.63^b	48.43±5.63^ab	22.47±5.23^ab
F	22.797^＊＊	349.636^＊＊	62.044^＊＊

图3 免疫组化检测大鼠滑膜组织IL-1β、iNOS、MMP-13蛋白表达（×200）

Fig.3 Expression of IL-1β, iNOS and MMP-13 in synovial tissue of rats detected by immunohistochemistry (×200)

表3 各组大鼠滑膜组织IL-1β、iNOS、MMP-13蛋白表达（n=5，$\bar{x}\pm s$）

Tab.3 Expression levels of IL-1β, iNOS and MMP-13 in synovial tissue of rats in each group

组别	IL-1β	iNOS	MMP-13
Sham组	0.21±0.05	0.33±0.06	0.15±0.04
KOA组	0.86±0.11^a	0.76±0.12^a	0.93±0.15^a
SJHX组	0.45±0.08^ab	0.52±0.10^b	0.37±0.07^ab
AG490组	0.47±0.09^ab	0.55±0.11^b	0.35±0.05^b
F	49.731^＊＊	15.461^＊＊	71.280^＊＊

图4 各组大鼠滑膜组织F4/80+/CD68+免疫荧光染色（×400）

Fig.4 Immunofluorescence staining of synovial tissue F4/80+/CD68+ in each group (×400)

图5 各组大鼠滑膜组织F4/80+/CD206+免疫荧光染色（×400）

Fig.5 Immunofluorescence staining of synovial tissue F4/80+/CD206+ in each group (×400)

表4 各组大鼠滑膜M1、M2型巨噬细胞比例比较（n=5， $\bar{x}\pm s$）

Tab.4 Comparison of the proportion of M1 and M2 in synovial membrane of rats between the four groups

组别	M1/%	M2/%	M1/M2
Sham组	5.46±1.15	6.12±1.32	1.01±0.32
KOA组	20.55±3.34^a	9.24±1.85^a	2.46±0.65^a
SJHX组	12.45±2.78^ab	7.52±1.59	1.57±0.43^b
AG490组	13.26±2.85^ab	8.75±1.62	1.45±0.42^b
F	26.876^＊＊	3.768^＊	8.343^＊

图6 Western blot检测各组p-JAK2、JAK2、p-STAT3、STAT3蛋白表达 A：Sham组；B：KOA组；C：SJHX组；D：AG490组。

Fig.6 Western blot detection of p-JAK2, JAK2, p-STAT3 and STAT3 protein expression in each group

表5 各组大鼠滑膜组织p-JAK2、JAK2、p-STAT3、STAT3蛋白表达比较（n=5，$\bar{x}\pm s$）

Tab.5 Comparison of protein expression levels of p-JAK2, JAK2, p-STAT3 and STAT3 in synovium of rats between the four groups

组别	p-JAK2/JAK2	p-STAT3/STAT3
Sham组	0.32±0.04	0.19±0.04
KOA组	0.81±0.10^a	0.65±0.08^a
SJHX组	0.51±0.06^ab	0.37±0.05^ab
AG490组	0.53±0.05^ab	0.35±0.04^ab
F	46.055^＊＊	60.386^＊＊

参考文献 21

[1]	CURRY Z A, BELING A, BORG-STEIN J. Knee osteoarthritis in midlife women:unique considerations and comprehensive management[J]. Menopause, 2022, 29(6):748-755. doi:10.1097/GME.0000000000001966.
[2]	XIE J, HUANG Z, YU X, et al. Clinical implications of macrophage dysfunction in the development of osteoarthritis of the knee[J]. Cytokine Growth Factor Rev, 2019, 46(1):36-44. doi:10.1016/j.cytogfr.2019.03.004.
[3]	ZHONG Y, GU L, YE Y, et al. JAK2/STAT3 axis intermediates microglia/macrophage polarization during cerebral ischemia/reperfusion injury[J]. Neuroscience, 2022, 496(1):119-128. doi:10.1016/j.neuroscience.2022.05.016.
[4]	雷黎, 姜辉, 刘健, 等. 五味温通除痹胶囊对佐剂性关节炎大鼠Jak2/Stat3信号通路的调控作用[J]. 中药药理与临床, 2019, 35(4):174-178.
	LEI L, JIANG H, LIU J, et al. Effects of Wuwei Wentong Jiebi Capsule on Jak2/Stat3 signaling pathway in adjuvant arthritis rats[J]. Pharmacology and Clinics of Chinese Materia Medica, 2019, 35(4):174-178. doi:10.13412/j.cnki.zyyl.2019.04.035.
[5]	靳志海, 葛满意, 李高强, 等. 舒筋活血胶囊联合艾瑞昔布治疗膝骨关节炎的临床研究[J]. 现代药物与临床, 2019, 34(2):481-484.
	JIN Z H, GE M Y, LI G Q, et al. Clinical study of Shujinhuoxue capsule combined with eroxib in the treatment of knee osteoarthritis[J]. Drugs & Clinic, 2019, 34(2):481-484. doi:10.7501/j.issn.1674-5515.2019.02.046.
[6]	张亚. 舒筋活血胶囊治疗类风湿性关节炎的药效物质基础及药代动力学研究[D]. 杭州: 浙江工业大学, 2021.
	ZHANG Y. Study on pharmacodynamic substance basis and pharmacokinetics of Shujinhuoxue Capsule in the treatment of rheumatoid arthritis[D]. Hangzhou: Zhejiang University of Technology, 2021.
[7]	危一飞, 程桯, 肖潇, 等. 防己黄芪消肿方调控滑膜巨噬细胞极化治疗膝骨关节炎滑膜炎[J]. 中国实验方剂学杂志, 2022, 28(13):112-122.
	WEI Y F, CHENG W, XIAO X, et al. Fangji Huangqi Xiaozhong Fang regulates the polarization of synovial macrophages in treatment of synovitis of knee osteoarthritis[J]. Chinese Journal of Experimental Traditional Medical Formulae, 2022, 28 (13):112-122. doi:10.13422/j.carol carroll nki syfjx.20220703.
[8]	李金鹏, 刘涛, 何志军, 等. 消肿止痛合剂对大鼠膝骨性关节炎软骨中AMPK/mTOR信号通路影响的实验研究[J]. 中国骨质疏松杂志, 2020, 26(2):231-235.
	LI J P, LIU T, HE Z J, et al. Effect of Xiaozhongzhitong Mixture on AMPK/mTOR signaling pathway in cartilage of knee osteoarthritis in rats[J]. Chin J Osteopor, 2020, 26(2):231-235. doi:10.3969/j.issn.1006-7108.2020.02.015.
[9]	SIDDIQ M A B, CLEGG D, JANSEN T L, et al. Emerging and new treatment options for knee osteoarthritis[J]. Curr Rheumatol Rev, 2022, 18(1):20-32. doi:10.2174/1573397117666211116111738.
[10]	刘洋, 孙权, 杨砥, 等. 右归丸对骨性关节炎模型大鼠TGF-β1/Smads信号通路的影响[J]. 中国骨质疏松杂志, 2021, 27(10):1419-1424.
	LIU Y, SUN Q, YANG D, et al. Effect of Yougui Pill on TGF-β1/Smads signaling pathway in osteoarthritis model rats[J]. Chin J Osteopor, 2021, 27(10):1419-1424. doi:10.3969/j.iSSN.1006-7108.2021.10.003.
[11]	谷右天, 杨占华, 安龙, 等. 舒筋活血汤治疗老年膝骨关节炎的效果分析[J]. 吉林中医药, 2020, 40(9):1209-1212.
	GU Y T, YANG Z H, AN L, et al. Relaxing tendons decoction for the treatment of senile knee osteoarthritis analysis[J]. Jilin J Tradit Chin Med, 2020, 40(9):1209-1212. doi:10.13463/j.cnki.jlzyy.2020.09.025.
[12]	SUN Y, ZUO Z, KUANG Y. An emerging target in the battle against osteoarthritis:macrophage polarization[J]. Int J Mol Sci, 2020, 21(22):8513-8532. doi:10.3390/ijms21228513.
[13]	TIAN Z, ZENG F, ZHAO C, et al. Angelicin alleviates post-trauma osteoarthritis progression by regulating macrophage polarization via STAT3 signaling pathway[J]. Front Pharmacol, 2021, 12(1):1-11. doi:10.3389/fphar.2021.669213.
[14]	杨利斌, 杨林, 赵恩典, 等. miR-106a-5p过表达对ACLT诱导的骨关节炎大鼠软骨退变和细胞外基质降解的修复及血清炎症因子的影响[J]. 中国免疫学杂志, 2021, 37(13):1553-1557.
	YANG L B, YANG L, ZHAO Y H, et al. Effects of miR-106a-5p overexpression on the repair of cartilage degeneration and extracellular matrix degradation and serum inflammatory cytokines in ACLT-induced osteoarthritis rats[J]. Chin J Immunology, 2021, 37(13):1553-1557. doi:10.3969/j.issn.1000-484X.2021.13.004.
[15]	FATTORI V, STAURENGO-FERRARI L, ZANINELLI T H, et al. IL-33 enhances macrophage release of IL-1β and promotes pain and inflammation in gouty arthritis[J]. Inflamm Res, 2020, 69(12):1271-1282. doi:10.1007/s00011-020-01399-x.
[16]	GRIFFIN T M, SCANZELLO C R. Innate inflammation and synovial macrophages in osteoarthritis pathophysiology[J]. Clin Exp Rheumatol, 2019, 120(5):57-63.
[17]	周琦, 孙慧娟, 刘树民. 穿山龙总皂苷调控巨噬细胞M1/M2极化治疗痛风性关节炎的作用机制[J]. 中国实验方剂学杂志, 2021, 27(24):92-99.
	ZHOU Q, SUN H J, LIU S M. Effect of total saponins of Pangolin on macrophage M1/M2 polarization in the treatment of gout arthritis[J]. Chinese Journal of Experimental Traditional Medical Formulae, 2021, 27(24):92-99. doi:10.13422/j.cnki.syfjx.20212340.
[18]	ZHU J, TANG Y, WU Q, et al. HIF-1α facilitates osteocyte-mediated osteoclastogenesis by activating JAK2/STAT3 pathway in vitro[J]. J Cell Physiol, 2019, 234(11):21182-21192. doi:10.1002/jcp.28721.
[19]	TONG J, FANG J, ZHU T, et al. Pentagalloylglucose reduces AGE-induced inflammation by activating Nrf2/HO-1 and inhibiting the JAK2/STAT3 pathway in mesangial cells[J]. J Pharmacol Sci, 2021, 147(4):305-314. doi:10.1016/j.jphs.2021.08.006.
[20]	TENGY, NI G, ZHANG W, et al. TRIM59 attenuates IL-1β-driven cartilage matrix degradation in osteoarthritis via direct suppression of NF-κB and JAK2/STAT3 signaling pathway[J]. Biochem Biophys Res Commun, 2020, 529(1):28-34. doi:10.1016/j.bbrc.2020.05.130.
[21]	张艳玲, 刘君伟, 李春, 等. 基于JAK2/STAT3信号通路探讨温针灸改善膝关节骨关节炎兔软骨损伤的机制[J]. 针刺研究, 2022, 47(12):1088-1094.
	ZHANG Y L, LIU J W, LI C, et al. Based on JAK2/STAT3 signal pathway,to explore the mechanism of warming acupuncture and moxibustion to improve cartilage injury in rabbits with knee osteoarthritis[J]. Acupunct Res, 2022, 47(12):1088-1094. doi:10.13702/j.1000-0607.20211331.