Mechanism of Shujin Huoxue Capsule in relieving knee osteoarthritis in rats through JAK2/STAT3 pathway

doi:10.11958/20221924

Abstract

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

CLC Number:

R684.3

Figures/Tables 11

References 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.

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^＊＊

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^＊＊

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^＊＊

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^＊＊

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^＊＊