齐墩果酸抑制骨关节炎大鼠肌肉氧化损伤及软骨下骨异常骨重建

doi:10.11843/j.issn.0366-6964.2022.11.033

摘要/Abstract

摘要： 旨在探讨齐墩果酸（oleanolic acid，OLA）对于骨关节炎（osteoarthritis，OA）大鼠肌肉氧化损伤和软骨下骨异常骨重建的作用。选取18只Sprague-Dawley大鼠随机分为3组：对照组（CON组）、模型组（OA组）和齐墩果酸给药组（OLA组）。OA组和OLA组利用前十字韧带切断联合半月板部分切除手术（ACLT+PMMx）建立大鼠膝OA模型，CON组进行假手术。OLA组大鼠每天灌胃50 mg·（kg·d）^-1 OLA，CON组和OA组灌服等体积含20 mL·L^-1 Tween 80的无菌生理盐水，连续给药4周后取膝关节、股四头肌和血清样本。利用伸膝发声试验和热敏感试验检测大鼠关节疼痛情况。酶联免疫吸附测定试验检测大鼠肌肉中柠檬酸合酶（citrate synthase，CS）、肌球蛋白重链（myosin heavy chain，MHC）、白细胞介素1（interleukin 1，IL-1）和IL-6的水平，以及大鼠血清中骨钙素（osteocalcin，OCN）和Ⅰ型胶原羧基末端肽（C-terminal typeⅠ collagen telopeptide，CTX-Ⅰ）的水平。免疫印迹法检测肌肉组织Nrf2/NQO1/HO-1通路蛋白表达情况。微型计算机断层扫描（Micro-CT）用于软骨下骨扫描和骨组织显微结构定量分析。结果显示，与OA组比较，OLA可以降低OA疼痛评分（P<0.05），激活肌肉Nrf2/NQO1/HO-1通路，促进肌肉CS和MHC表达，并下调IL-1和IL-6水平（P<0.05）。此外，补充OLA后大鼠软骨下骨的骨体积分数（BV/TV）、骨矿物质密度（BMD）和骨小梁厚度（Tb.Th）显著升高（P<0.05），骨代谢标志物OCN和CTX-Ⅰ表达显著降低（P<0.05）。结果提示，OLA能够抑制OA大鼠关节疼痛反应，通过调控Nrf2/NQO1/HO-1通路抑制OA大鼠肌肉功能障碍，改善OA早期软骨下骨生物力学性能和微结构改变，从而延缓OA大鼠软骨下骨异常骨重建的发生。

关键词: 骨关节炎, 齐墩果酸, 软骨下骨, 肌肉, 氧化应激

Abstract: We aimed to investigate the effects of oleanolic acid (OLA) on oxidative damage of muscle and abnormal subchondral bone reconstruction in rats with osteoarthritis (OA). Eighteen Sprague-Dawley rats were selected and randomly divided into three groups:the control group (CON group), the model group (OA group) and the oleanolic acid administration group (OLA group).The OA and OLA groups were established using anterior cruciate ligament dissection combined with partial meniscectomy (ACLT+PMMx) to establish a rat knee OA model, and the CON group underwent sham surgery. Rats in the OLA group were given 50 mg·(kg·d)^-1 OLA by gavage daily, and rats in the CON and OA groups were given equal volumes of sterile saline containing 20 mL·L^-1 Tween 80. After four consecutive weeks of dosing, knee, quadriceps and serum samples were taken. The rats were tested for joint pain using knee extension vocalization test and heat sensitivity test. Enzyme-linked immunosorbent assay to detect the levels of citrate synthase (CS), myosin heavy chain (MHC), interleukin 1 (IL-1) and interleukin 6 (IL-6) in rat muscle and the levels of osteocalcin (OCN) and C-terminal telopeptide of type I collagen (CTX-Ⅰ) in rat serum. Western blot was used to detect changes in Nrf2/NQO1/HO-1 pathway protein expression in muscle tissue. Micro-CT was used for subchondral bone scanning and quantitative analysis of bone tissue microstructure. The results showed that compared with the OA group, OLA could reduce the OA pain score (P<0.05), activate the muscle Nrf2/NQO1/HO-1 pathway, promote the expression of muscle CS and MHC, and down-regulate the levels of IL-1 and IL-6 (P<0.05). In addition, percent bone volume (BV/TV), bone mineral density (BMD) and trabecular thickness (Tb.Th) were significantly increased (P<0.05) and expression of bone metabolic markers OCN and CTX-Ⅰ were significantly decreased (P<0.05) in subchondral bone of rats after OLA supplementation. The results suggest that OLA can inhibit the joint pain response in OA rats, inhibit muscle dysfunction in OA rats by regulating the Nrf2/NQO1/HO-1 pathway, improve the biomechanical properties and microstructural changes of subchondral bone in early OA, thereby delaying the occurrence of abnormal bone remodeling of subchondral bone in OA rats.

Key words: osteoarthritis, oleanolic acid, subchondral bone, muscle, oxidative stress

中图分类号:

S857.165

马天文, 吕良钰, 于跃, 贾丽娜, 陈鸿, 汤继浪, 赵明超, 王新宇, 张建涛, 高利. 齐墩果酸抑制骨关节炎大鼠肌肉氧化损伤及软骨下骨异常骨重建[J]. 畜牧兽医学报, 2022, 53(11): 4081-4088.

MA Tianwen, LÜ Liangyu, YU Yue, JIA Lina, CHEN Hong, TANG Jilang, ZHAO Mingchao, WANG Xinyu, ZHANG Jiantao, GAO Li. Oleanolic Acid Inhibits Muscle Oxidative Damage and Abnormal Subchondral Bone Remodeling in Osteoarthritis Rats[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(11): 4081-4088.

参考文献 33

[1]	GLYN-JONES S, PALMER A J R, AGRICOLA R, et al.Osteoarthritis[J].Lancet, 2015, 386(9991):376-387.
[2]	HEINOLA T, DE GRAUW J C, VIRKKI L, et al.Bovine chronic osteoarthritis causes minimal change in synovial fluid[J].J Comp Pathol, 2013, 148(4):335-344.
[3]	HUNTER D J, BIERMA-ZEINSTRA S.Osteoarthritis[J].Lancet, 2019, 393(10182):1745-1759.
[4]	SÁNCHEZ-QUESADA C, LÓPEZ-BIEDMA A, GAFORIO J J.Oleanolic acid, a compound present in grapes and olives, protects against genotoxicity in human mammary epithelial cells[J].Molecules, 2015, 20(8):13670-13688.
[5]	LI Y L, NIE J L, JIANG P.Oleanolic acid mitigates interleukin-1β-induced chondrocyte dysfunction by regulating miR-148-3p-modulated FGF2 expression[J].J Gene Med, 2020, 22(5):e3169.
[6]	BAO J P, YAN W F, XU K, et al.Oleanolic acid decreases IL-1β-induced activation of fibroblast-like synoviocytes via the SIRT3-NF-κB axis in osteoarthritis[J].Oxid Med Cell Longev, 2020, 2020:7517219.
[7]	KANG D G, LEE H J, KIM K T, et al.Effect of oleanolic acid on the activity, secretion and gene expression of matrix metalloproteinase-3 in articular chondrocytes in vitro and the production of matrix metalloproteinase-3 in vivo[J].Korean J Physiol Pharmacol, 2017, 21(2):197-204.
[8]	陆锦炜, 陈曦, 叶陈毅, 等.骨性关节炎中的软骨下骨囊肿:软骨下骨异常重建[J].中国组织工程研究, 2019, 23(32):5209-5215.LU J W, CHEN X, YE C Y, et al.Subchondral bone cysts in osteoarthritis:abnormal reconstruction of subchondral bone[J].Chinese Journal of Tissue Engineering Research, 2019, 23(32):5209-5215.(in Chinese)
[9]	ZHU X B, CHAN Y T, YUNG P S H, et al.Subchondral bone remodeling:a therapeutic target for osteoarthritis[J].Front Cell Dev Biol, 2020, 8:607764.
[10]	BECKER R, BERTH A, NEHRING M, et al.Neuromuscular quadriceps dysfunction prior to osteoarthritis of the knee[J].J Orthop Res, 2004, 22(4):768-773.
[11]	CALLAHAN D M, MILLER M S, SWEENY A P, et al.Muscle disuse alters skeletal muscle contractile function at the molecular and cellular levels in older adult humans in a sex-specific manner[J].J Physiol, 2014, 592(20):4555-4573.
[12]	IKEDA S, TSUMURA H, TORISU T.Age-related quadriceps-dominant muscle atrophy and incident radiographic knee osteoarthritis[J].J Orthop Sci, 2005, 10(2):121-126.
[13]	LEI M, GUO C H, HUA L M, et al.Crocin attenuates joint pain and muscle dysfunction in osteoarthritis rat[J].Inflammation, 2017, 40(6):2086-2093.
[14]	HAO Y, MIAO J, LIU W J, et al.Formononetin protects against cisplatin-induced acute kidney injury through activation of the PPARα/Nrf2/HO-1/NQO1 pathway[J].Int J Mol Med, 2020, 47(2):511-522.
[15]	HUAI B, DING J Y.Atractylenolide III attenuates bleomycin-induced experimental pulmonary fibrosis and oxidative stress in rat model via Nrf2/NQO1/HO-1 pathway activation[J].Immunopharmacol Immunotoxicol, 2020, 42(5):436-444.
[16]	DAI M L, SUI B, XUE Y, et al.Cartilage repair in degenerative osteoarthritis mediated by squid type II collagen via immunomodulating activation of M2 macrophages, inhibiting apoptosis and hypertrophy of chondrocytes[J].Biomaterials, 2018, 180:91-103.
[17]	LONG C M, YANG J R, YANG H, et al.Attenuation of renal ischemia/reperfusion injury by oleanolic acid preconditioning via its antioxidant, antiinflammatory, and antiapoptotic activities[J].Mol Med Rep, 2016, 13(6):4697-4704.
[18]	IM H J, KIM J S, LI X, et al.Alteration of sensory neurons and spinal response to an experimental osteoarthritis pain model[J].Arthritis Rheum, 2010, 62(10):2995-3005.
[19]	TALIYAN R, SHARMA P L.Possible mechanism of protective effect of thalidomide in STZ-induced-neuropathic pain behavior in rats[J].Inflammopharmacology, 2012, 20(2):89-97.
[20]	WANG X, YE X L, LIU R, et al.Antioxidant activities of oleanolic acid in vitro:possible role of Nrf2 and MAP kinases[J].Chem Biol Interact, 2010, 184(3):328-337.
[21]	张鹏, 李俞甫, 吴尚泽, 等.丹皮酚对L-精氨酸诱导的小鼠急性胰腺炎氧化损伤的影响[J].畜牧兽医学报, 2021, 52(7):1983-1990.ZHANG P, LI Y F, WU S Z, et al.Effects of paeonol on the oxidative damage of acute pancreatitis in mice induced by L-arginine[J].Acta Veterinaria et Zootechnica Sinica, 2021, 52(7):1983-1990.(in Chinese)
[22]	LOBODA A, DAMULEWICZ M, PYZA E, et al.Role of Nrf2/HO-1 system in development, oxidative stress response and diseases:an evolutionarily conserved mechanism[J].Cell Mol Life Sci, 2016, 73(17):3221-3247.
[23]	HSU D Z, CHU P Y, JOU I M.Daily sesame oil supplement attenuates joint pain by inhibiting muscular oxidative stress in osteoarthritis rat model[J].J Nutr Biochem, 2016, 29:36-40.
[24]	TONGE D P, BARDSLEY R G, PARR T, et al.Evidence of changes to skeletal muscle contractile properties during the initiation of disease in the ageing guinea pig model of osteoarthritis[J].Longev Healthspan, 2013, 2(1):15.
[25]	CHEN D S, CAO J G, ZHU B, et al.Baicalin attenuates joint pain and muscle dysfunction by inhibiting muscular oxidative stress in an experimental osteoarthritis rat model[J].Arch Immunol Ther Exp, 2018, 66(6):453-461.
[26]	LOESER R F, GOLDRING S R, SCANZELLO C R, et al.Osteoarthritis:a disease of the joint as an organ[J].Arthritis Rheum, 2012, 64(6):1697-1707.
[27]	KOVÁCS B, VAJDA E, NAGY E E.Regulatory effects and interactions of the wnt and OPG-RANKL-RANK signaling at the bone-cartilage interface in osteoarthritis[J].Int J Mol Sci, 2019, 20(18):4653.
[28]	HU W H, CHEN Y Q, DOU C, et al.Microenvironment in subchondral bone:predominant regulator for the treatment of osteoarthritis[J].Ann Rheum Dis, 2021, 80(4):413-422.
[29]	AIZAH N, CHONG P P, KAMARUL T.Early alterations of subchondral bone in the rat anterior cruciate ligament transection model of osteoarthritis[J].Cartilage, 2021, 13(2_suppl):1322S-1333S.
[30]	MOHAN G, PERILLI E, KULIWABA J S, et al.Application of in vivo micro-computed tomography in the temporal characterisation of subchondral bone architecture in a rat model of low-dose monosodium iodoacetate-induced osteoarthritis[J].Arthritis Res Ther, 2011, 13(6):R210.
[31]	SHARMA A R, JAGGA S, LEE S S, et al.Interplay between cartilage and subchondral bone contributing to pathogenesis of osteoarthritis[J].Int J Mol Sci, 2013, 14(10):19805-19830.
[32]	HAYAMI T, PICKARSKI M, ZHUO Y, et al.Characterization of articular cartilage and subchondral bone changes in the rat anterior cruciate ligament transection and meniscectomized models of osteoarthritis[J].Bone, 2006, 38(2):234-243.
[33]	MADSEN J O B, HERSKIN C W, ZERAHN B, et al.Decreased markers of bone turnover in children and adolescents with type 1 diabetes[J].Pediatr Diabetes, 2020, 21(3):505-514.