葛根素干预软骨氧化应激和Nrf2/HO-1通路改善PTOA大鼠软骨退变的机制

doi:10.11843/j.issn.0366-6964.2023.09.033

摘要/Abstract

摘要： 旨在研究葛根素干预软骨氧化应激和Nrf2/HO-1通路改善创伤后骨关节炎(post-traumatic osteoarthritis,PTOA)大鼠软骨退变的作用机制,探究葛根素对骨关节炎的治疗作用。将40只雄性Sprague-Dawley大鼠随机分为4组:对照组(n=8)、模型组(n=8)、塞来昔布组(n=8)和葛根素组(n=16)。葛根素组随机分为低剂量组(n=8)和高剂量组(n=8)。除对照组外,其余各组通过前十字韧带横断(anterior cruciate ligament transection,ACLT)建立大鼠PTOA模型。低剂量组(50 mg·kg^-1)、高剂量组(100 mg·kg^-1)和塞来昔布组(2.86 mg·kg^-1)进行灌胃给药,对照组给予等量生理盐水,连续干预5周。每周进行关节肿胀程度、冷敏感反应和膝关节伸膝发声检测,评估大鼠关节肿胀和疼痛程度。给药结束后,收集各组大鼠膝关节和血清样本。对胫骨和股骨组织进行HE染色和改良的Mankin评分,以评估病理组织学变化。检测大鼠软骨中基质金属蛋白酶3(matrix metalloproteinase 3,MMP3)、基质金属蛋白酶13(MMP13)和血小板反应蛋白解整合素金属肽酶4(ADAMTS4)的水平以及血清中Ⅱ型胶原羧基末端肽(CTX-Ⅱ)和软骨寡聚基质蛋白(COMP)的含量,以评估葛根素对大鼠骨关节炎软骨退变的改善作用。通过检测大鼠血清中抗氧化酶(SOD、GSH-Px和CAT)活力以及大鼠软骨中丙二醛(MDA)含量和Nrf2/HO-1通路水平,以评估葛根素对大鼠机体氧化损伤的保护作用。检测大鼠血清中白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)和肿瘤坏死因子(TNF-α)水平,以评估葛根素对机体的抗炎作用。结果显示:与模型组比较,使用高剂量葛根素干预可以显著减轻大鼠关节肿胀程度和疼痛反应,改善关节软骨损伤程度,降低Mankin评分(P<0.01),激活软骨中Nrf2/HO-1通路,抑制MDA、MMP3、MMP13和ADAMTS4表达(P<0.05),上调血清中抗氧化酶活力(P<0.01),抑制炎症因子IL-1β、IL-6和TNF-α以及软骨代谢标志物CTX-Ⅱ和COMP水平(P<0.05)。综上,葛根素通过激活Nrf2/HO-1通路抑制氧化应激与炎症损伤改善PTOA大鼠软骨退变。

关键词: 葛根素, 创伤后骨关节炎, 软骨退变, 氧化应激, Nrf2/HO-1通路

Abstract: The aim of this experiment was to investigate the mechanism of puerarin in the intervention of oxidative stress of cartilage and the improvement of Nrf2/HO-1 pathway on cartilage degeneration in rats with post-traumatic osteoarthritis (PTOA), and to explore the therapeutic effect of puerarin on osteoarthritis. Forty male Sprague-Dawley rats were randomly divided into four groups:control group (n=8), model group (n=8), celecoxib group (n=8) and puerarin group (n=16). Puerarin group was divided into low dose group (n=8) and high dose group (n=8). The PTOA model was established by anterior cruciate ligament transection (ACLT) in all groups except the control group. Low-dose group (50 mg·kg^-1), high-dose group (100 mg·kg^-1) and celecoxib group (2.86 mg·kg^-1) were administered by gavage, and the control group was given equal amount of saline for 5 weeks. The degree of joint swelling, cold sensitivity response and knee extension vocalization tests were performed weekly to assess the degree of joint swelling and pain in rats. After the administration, knee joint and serum samples of rats in each group were collected. The tibial and femoral tissues were subjected to HE staining and a modified Mankin score to assess histopathological changes. The histopathological changes were evaluated using the Mankin method. The levels of MMP3, MMP13 and ADAMTS4 in rat cartilage, as well as the levels of CTX-Ⅱ and COMP in serum were measured to evaluate the improvement of puerarin on cartilage degeneration in rats with osteoarthritis. To evaluate the protective effect of puerarin on oxidative damage in rats, the activity of antioxidant enzymes (SOD, GSH-Px, and CAT) in rat serum, the content of MDA and the level of Nrf2/HO-1 pathway in rat cartilage were measured. Detection of IL-1β, IL-6 and TNF-α in rat serum to evaluate the anti-inflammatory effect of puerarin on the body. The results showed that, compared with the model group, the high dose group intervention could significantly reduce the degree of joint swelling and pain response in rats, improve the degree of articular cartilage injury, reduce the Mankin score (P<0.01), activate the Nrf2/HO-1 pathway in cartilage tissue, inhibit the expression of MDA, MMP3, MMP13, and ADAMTS4 (P<0.05), upregulate the activity of antioxidant enzymes in serum (P<0.01), and inhibit the inflammatory factor IL-1β, IL-6 and TNF-α and the levels of cartilage metabolic markers CTX-Ⅱ and COMP in serum (P<0.05). In summary, puerarin improves cartilage degeneration in PTOA rats by activating the Nrf2/HO-1 pathway to inhibit oxidative stress and inflammatory damage.

Key words: puerarin, post-traumatic osteoarthritis, cartilage degeneration, oxidative stress, Nrf2/HO-1 pathway

中图分类号:

S857.16

陈鸿, 阮红日, 马天文, 李亚楠, 苗雪, 杨雯越, 高利, 魏成威. 葛根素干预软骨氧化应激和Nrf2/HO-1通路改善PTOA大鼠软骨退变的机制[J]. 畜牧兽医学报, 2023, 54(9): 3951-3963.

CHEN Hong, RUAN Hongri, MA Tianwen, LI Yanan, MIAO Xue, YANG Wenyue, GAO Li, WEI Chengwei. The Mechanism of Puerarin Improving Cartilage Degeneration in PTOA Rats by Interfering with Oxidative Stress and Nrf2/HO-1 Pathway of Cartilage[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(9): 3951-3963.

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