钩吻素子调节AMPK/NLRP3信号通路对膝骨关节炎大鼠软骨损伤的影响

doi:10.11958/20252961

摘要/Abstract

摘要：

目的探究钩吻素子（KM）调节单磷酸腺苷活化蛋白激酶（AMPK）/Nod样受体蛋白3（NLRP3）通路对膝骨关节炎（KOA）大鼠软骨损伤的影响。方法构建KOA大鼠模型并分为KOA组，低、高剂量KM（L、H-KM）组，阳性药（塞来昔布）组，H-KM+AMPK抑制剂Compound C（H-KM+Compound C）组，每组12只，12只大鼠作为对照（CK）组。检测膝关节宽度、关节肿胀度及步态评分；酶联免疫吸附试验检测白细胞介素（IL）-18、IL-1β、肿瘤坏死因子（TNF）-α水平；HE染色、番红-固绿染色、透射电镜观察软骨组织病理变化、结构变化、软骨微观结构；Western blot检测AMPK/NLRP3通路相关蛋白表达。结果 KOA组有明显病理改变，组织表层粗糙、完整性破坏，细胞肥大紊乱，潮线断裂；番红-固绿染色红色变浅，提示蛋白多糖流失等；细胞出现颗粒脱落、内容物肿胀断裂；KOA组较CK组膝关节宽度、关节肿胀度、步态评分，IL-18、IL-1β、TNF-α水平，NLRP3蛋白表达升高，p-AMPK/AMPK比值降低（P<0.05）；L-KM组、H-KM组、塞来昔布组较KOA组膝关节宽度、关节肿胀度、步态评分，IL-18、IL-1β、TNF-α水平，NLRP3蛋白表达及病理损伤程度降低，p-AMPK/AMPK比值升高（P<0.05）；H-KM+Compound C组较H-KM组膝关节宽度、关节肿胀度、步态评分，IL-18、IL-1β、TNF-α水平，NLRP3蛋白表达及病理损伤程度升高，p-AMPK/AMPK比值降低（P<0.05）。结论 KM可能通过激活AMPK/NLRP3通路减轻KOA大鼠软骨损伤。

关键词: 钩吻碱, 骨关节炎，膝, AMP活化蛋白激酶类, NLR家族，热蛋白结构域包含蛋白3, 软骨

Abstract:

Objective To discuss the effect of koumine (KM) on cartilage injury in rats with knee osteoarthritis (KOA) by regulating the adenosine monophosphate activated protein kinase (AMPK)/Nod-like receptor protein 3 (NLRP3) pathway. Methods The KOA rat model was constructed and separated into the KOA group, the low and high dose KM (L, H-KM) groups, the positive drug (celecoxib) group and the H-KM+AMPK inhibitor Compound C group, with 12 rats per group. Another 12 rats were used as the control (CK) group. The knee joint width, joint swelling and gait score were measured. ELISA was used to detect the levels of IL-18, IL-1β and TNF-α. HE staining, Safranin O-Fast Green staining and transmission electron microscopy were used to observe the pathological changes, structural changes and microstructure of cartilage tissue. In addition, Western blot assay was used to detect the expression of AMPK/NLRP3 pathway related proteins. Results The KOA group showed obvious pathological changes: rough tissue surface, disordered cell hypertrophy and broken tidal lines. The lightening of the red color in Safran-solid green staining indicated the loss of proteoglycans. The cells experienced particle shedding, swelling and rupture of their contents. The KOA group showed increased knee joint width, joint swelling degree, gait score, IL-18, IL-1β, TNF-α levels and NLRP3 protein expression, while decreased p-AMPK/AMPK ratio than those of the CK group (P<0.05). The L-KM group and the H-KM group and the celecoxib group showed decreased knee joint width, joint swelling degree, gait score, IL-18, IL-1β, TNF-α levels, NLRP3 protein expression and pathological damage, while increased p-AMPK/AMPK ratio than those of the KOA group (P<0.05). The H-KM+Compound C group showed increased knee joint width, joint swelling degree, gait score, IL-18, IL-1β, TNF-α levels, NLRP3 protein expression and pathological damage, while decreased p-AMPK/AMPK ratio than those of the CK group (P<0.05). Conclusion KM may alleviate cartilage injury in KOA rats by activating the AMPK/NLRP3 pathway.

Key words: gelsemine, osteoarthritis, knee, AMP-activated protein kinases, NLR family, pyrin domain-containing 3 protein, cartilage

中图分类号:

R684.3

图/表 7

表1 各组大鼠膝关节宽度、关节肿胀度、步态评分比较（n=12，$\bar{x}±s$）

Tab.1 Comparison of knee joint width, joint swelling degree and gait score between six groups of rats

组别	膝关节宽度/mm	关节肿胀度/%	步态评分/分
CK组	4.69±0.56	0.00±0.00	0.00±0.00
KOA组	9.15±0.93^a	36.52±3.71^a	2.10±0.22^a
L-KM组	7.83±0.81^b	23.67±2.89^b	1.30±0.18^b
H-KM组	5.27±0.61^bc	11.05±1.53^bc	0.50±0.13^bc
塞来昔布组	5.24±0.59^bc	11.86±1.58^bc	0.40±0.09^bc
H-KM+Compound C组	8.22±0.85^d	28.37±3.02^d	1.70±0.20^d
F	78.210^＊＊	355.539^＊＊	335.802^＊＊

表2 各组大鼠IL-18、IL-1β、TNF-α水平比较（n=12，ng/L，$\bar{x}±s$）

Tab.2 Comparison of IL-18, IL-1β and TNF-α levels between six groups of rats

组别	IL-18	IL-1β	TNF-α
CK组	96.58±12.55	49.21±6.15	102.54±13.62
KOA组	143.78±14.62^a	78.94±7.95^a	150.73±15.53^a
L-KM组	124.69±13.89^b	64.85±7.11^b	123.56±14.26^b
H-KM组	104.25±13.21^bc	53.18±6.83^bc	107.58±14.05^bc
塞来昔布组	103.16±13.10^bc	52.37±6.79^bc	105.16±13.87^bc
H-KM+Compound C组	133.91±14.25^d	71.36±7.63^d	139.18±15.04^d
F	23.671^＊＊	34.083^＊＊	23.015^＊＊

图1 各组大鼠软骨组织病理变化（HE染色，×200）

Fig.1 Pathological changes of cartilage tissue in each group of rats (HE staining, ×200)

图2 各组大鼠软骨组织结构变化（番红-固绿染色，×400）

Fig.2 Structural changes of cartilage tissue in each group of rats (Safranin-O/Fast Green staining, ×400)

图3 各组大鼠软骨微观结构（透射电镜，×30 000）

Fig.3 Ultrastructure of cartilage in each group of rats (transmission electron microscopy, ×30 000)

图4 各组大鼠软骨组织p-AMPK、AMPK、NLRP3蛋白表达 A：CK组；B：KOA组；C：L-KM组；D：H-KM组；E：塞来昔布组；F：H-KM+Compound C组。

Fig.4 The protein expressions of p-AMPK, AMPK and NLRP3 in cartilage tissue of each group of rats

表3 各组大鼠AMPK/NLRP3通路相关蛋白表达比较（n=6，$\bar{x}±s$）

Tab.3 Comparison of AMPK/NLRP3 pathway-related protein expression levels between six groups of rats

组别	p-AMPK/AMPK	NLRP3/β-actin
CK组	0.87±0.09	0.46±0.05
KOA组	0.49±0.05^a	0.82±0.09^a
L-KM组	0.61±0.06^b	0.71±0.08^b
H-KM组	0.79±0.08^bc	0.63±0.07^bc
塞来昔布组	0.82±0.08^bc	0.51±0.06^bc
H-KM+Compound C组	0.53±0.06^d	0.76±0.08^d
F	30.859^＊＊	22.725^＊＊

参考文献 21

[1]	DISTEFANO G, HARRISON S, LYNCH J, et al. Skeletal muscle composition,power,and mitochondrial energetics in older men and women with knee osteoarthritis[J]. Arthritis Rheumatol, 2024, 76(12):1764-1774. doi:10.1002/art.42953.
[2]	罗锟, 王智, 王柯. 山姜素调节VEGF/SphK1/S1P信号通路对膝骨关节炎大鼠血管生成的影响[J]. 天津医药, 2024, 52(5):480-485.
	LUO K, WANG Z, WANG K. Impacts of alpinetin on angiogenesis in knee osteoarthritis rats by regulating the VEGF/SphK1/S1P signaling pathway[J]. Tianjin Med J, 2024, 52(5):480-485. doi:10.11958/20230892.
[3]	ZHAO G, ZHU S, ZHANG F, et al. Global burden of osteoarthritis associated with high body mass index in 204 countries and territories,1990-2019:findings from the Global Burden of Disease Study 2019[J]. Endocrine, 2023, 79(1):60-71. doi:10.1007/s12020-022-03201-w.
[4]	FAN Y, BIAN X, MENG X, et al. Unveiling inflammatory and prehypertrophic cell populations as key contributors to knee cartilage degeneration in osteoarthritis using multi-omics data integration[J]. Ann Rheum Dis, 2024, 83(7):926-944. doi:10.1136/ard-2023-224420.
[5]	YANG W T, KE C Y, YEH K T, et al. Stromal-vascular fraction and adipose-derived stem cell therapies improve cartilage regeneration in osteoarthritis-induced rats[J]. Sci Rep, 2022, 12(1):2828. doi:10.1038/s41598-022-06892-3.
[6]	WANG Z, ZHAO C, LI M, et al. Efficacy and safety of external therapies of Traditional Chinese Medicine in patients with knee osteoarthritis:a systematic review and network meta-analysis[J]. Rejuvenation Res, 2025, 28(5):248-262. doi:10.1089/rej.2025.0039.
[7]	JIN G L, SU Y P, LIU M, et al. Medicinal plants of the genus Gelsemium (Gelsemiaceae,Gentianales)--a review of their phytochemistry,pharmacology,toxicology and traditional use[J]. J Ethnopharmacol, 2014, 152(1):33-52. doi:10.1016/j.jep.2014.01.003.
[8]	LIN Y R, ZHENG F T, XIONG B J, et al. Koumine alleviates rheumatoid arthritis by regulating macrophage polarization[J]. J Ethnopharmacol, 2023, 311:116474. doi:10.1016/j.jep.2023.116474.
[9]	LU J S, YANG L, CHEN J, et al. Basolateral amygdala astrocytes modulate diabetic neuropathic pain and may be a potential therapeutic target for koumine[J]. Br J Pharmacol, 2023, 180(10):1408-1428. doi:10.1111/bph.16011.
[10]	魏超, 于江, 盛关云, 等. 棕矢车菊素调节AMPK/NLRP3信号通路对骨关节炎大鼠炎性损伤的影响[J]. 中国药房, 2025, 36(4):421-426.
	WEI C, YU J, SHENG G Y, et al. Effects of jaceosidin on inflammatory injury in osteoarthritis rats by regulating AMPK/NLRP3 signaling pathway[J]. China Pharmacy, 2025, 36(4):421-426. doi:10.6039/j.issn.1001-0408.2025.04.06.
[11]	彭博文, 冯立, 覃剑. 贝母素乙调节RhoA/ROCK信号通路对膝骨关节炎大鼠软骨损伤的影响[J]. 中国骨质疏松杂志, 2024, 30(7):980-984.
	PENG B W, FENG L, QIN J. Effect of Peiminine on cartilage injury in knee osteoarthritis rats by regulating RhoA/ROCK signal pathway[J]. Chin J Osteoporos, 2024, 30(7):980-984. doi:10.3969/j.issn.1006-7108.2024.07.007.
[12]	杨婷婷. 钩吻素子、钩吻素甲对大鼠骨关节炎的疗效研究[D]. 湖南: 湖南农业大学, 2022.
	YANG T T. Effect of koumine and gelsemine on osteoarthritis in rats[D]. Hunan: Hunan Agricultural University, 2022.
[13]	郭文帆, 杨学钰, 郑雪君, 等. 茶黄素调节AMPK/SIRT1/NF-κB信号通路对膝骨关节炎大鼠的治疗作用[J]. 河北医学, 2023, 29(1):42-49.
	GUO W F, YANG X Y, ZHENG X J, et al. Therapeutic effect of theaflavins modulation of AMPK/SIRT1/NF-κB signaling pathway in rats with knee osteoarthritis[J]. Hebei Med, 2023, 29(1):42-49. doi:10.3969/j.issn.1006-6233.2023.01.008.
[14]	李志娟, 王鑫, 孙敬青, 等. 火针对膝骨关节炎大鼠关节功能及炎性反应的影响[J]. 针刺研究, 2020, 45(3):220-226.
	LI Z J, WANG X, SUN J Q, et al. Effect of fire-needle intervention on joint function,cartilage impairment and inflammatory response in knee osteoarthritis rats[J]. Acupuncture Research, 2020, 45(3):220-226. doi:10.13702/j.1000-0607.190963.
[15]	BENNELL K L, SCHWARTZ S, TEO P L, et al. Effectiveness of an unsupervised online yoga program on pain and function in people with knee osteoarthritis:a randomized clinical trial[J]. Ann Intern Med, 2022, 175(10):1345-1355. doi:10.7326/M22-1761.
[16]	郑凤婷. 钩吻素子抗类风湿关节炎的巨噬细胞极化调节作用机制[D]. 福建: 福建医科大学, 2023.
	ZHENG F T. Regulation mechanism of koumine on macrophage polarization regulation in rheumatoid arthritis[D]. Fujian: Fujian MEDICAL University, 2023.
[17]	YANG J, LIN Y R, XIONG B J, et al. Regulation effect of koumine on T-helper cell polarization in rheumatoid arthritis[J]. Eur J Pharmacol, 2022, 937:175387. doi:10.1016/j.ejphar.2022.175387.
[18]	SUN Y, LIU W, ZHANG H, et al. Curcumin prevents osteoarthritis by inhibiting the activation of inflammasome NLRP3[J]. J Interferon Cytokine Res, 2017, 37(10):449-455. doi:10.1089/jir.2017.0069.
[19]	DENG Z, NI J, WU X, et al. GPA peptide inhibits NLRP3 inflammasome activation to ameliorate colitis through AMPK pathway[J]. Aging (Albany NY), 2020, 12(18):18522-18544. doi:10.18632/aging.103825.
[20]	STROHM L, MIHALIKOVA D, CZARNOWSKI A, et al. Sex-specific antioxidant and anti-inflammatory protective effects of AMPK in cardiovascular diseases[J]. Antioxidants (Basel), 2025, 14(5):615-622. doi:10.3390/antiox14050615.
[21]	GUO R, GAO S, FENG Y, et al. Ulinastatin attenuates spinal cord injury by targeting AMPK/NLRP3 signaling pathway[J]. J Chem Neuroanat, 2022, 125:102145. doi:10.1016/j.jchemneu.2022.102145.