目的 探究丹参酮ⅡA(Tan ⅡA)通过抑制相关信号通路延缓膝骨关节炎(KOA)大鼠模型软骨退变和抑制局部炎症的作用机制。方法 将60只SD大鼠随机分为假手术组、KOA组、阳性药物塞来昔布20 mg·kg-1组、Tan ⅡA不同剂量处理组(Tan Ⅱ A10、20、50 mg·kg-1),每组各12只。采用前交叉韧带切断术(ACLT)建立KOA大鼠模型,造模成功次日,各实验组腹腔注射不同剂量Tan ⅡA,假手术组和KOA组给予等体积生理盐水,共给药28 d。通过步态行为分析、机械刺激缩足反应阈值(PWMT)和热刺激缩足反应潜伏期(PWTL)来评价大鼠疼痛行为;改良的Mankin's评分和OARSI评分评价软骨病理损伤程度;酶联免疫吸附测定法检测关节组织和血清中炎症因子、骨代谢生化指标水平;免疫组化法检测软骨组织中核因子κB p65(NF-κB p65)和Ⅱ型胶原(COL2A1)蛋白表达;Western blot法检测TLR4/Myd88/NF-κB信号通路相关蛋白Toll样受体4(TLR4)、髓样分化蛋白(Myd88)及NF-κB p65表达水平;分离胞核和胞浆蛋白,检测NF-κB p65核转位。结果 实验结束时,与假手术组相比,KOA组和各实验组大鼠步态行为评分、PWTL、软骨组织COL2A1蛋白表达MOD值降低,同时PWMT、Mankin's评分和OARSI评分、关节组织和外周血清白细胞介素(IL)-1β、IL-6、肿瘤坏死因子α(TNF-α)、Ⅰ型胶原交联C-末端肽(CTX-Ⅰ)、Ⅱ型胶原交联C-末端肽(CTX-Ⅱ)水平升高,软骨组织中TLR4、Myd88和NF-κB p65蛋白表达上调,同时NF-κB p65核转位明显(均P<0.05)。此外,与KOA组相比,塞来昔布组和Tan ⅡA不同剂量处理组大鼠步态行为评分、PWTL、软骨组织COL2A1蛋白表达MOD值升高,同时PWMT、Mankin's评分和OARSI评分、关节组织和外周血清IL-1β、IL-6、TNF-α、CTX-Ⅰ、CTX-Ⅱ水平降低,软骨组织中TLR4、Myd88和NF-κB p65蛋白表达下调,同时NF-κB p65核转位被抑制(均P<0.05)。尤其是Tan ⅡA 50 mg·kg-1组上述指标改善更显著。结论 Tan ⅡA通过抑制TLR4/Myd88/NF-κB信号通路相关蛋白表达,减少细胞炎症因子释放,进而抑制关节软骨退变,达到改善KOA大鼠膝关节功能的目的。
Abstract
OBJECTIVE To study the mechanism of tanshinone ⅡA (Tan ⅡA) on ameliorating the cartilage degeneration and inhibiting local inflammation in knee osteoarthritis (KOA) rats by inhibiting the related pathway. METHODS Sixty rats were divided into sham-operation group, KOA model group, positive drug celecoxib 20 mg·kg-1 group and experimental groups (Tan ⅡA10, 20,50 mg·kg-1), with 12 rats in each. Except the sham-operation group, other rats were operated by anterior cruciate ligament transection (ACLT) to construct the KOA models. On the second day after successful modeling, the experimental groups were intraperitoneally injected with different doses of Tan ⅡA, the sham-operated group and the KOA model group were given the same volume of normal saline for 28 days. Pain behavior was evaluated by Gait behavior analysis, paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL), the degree of cartilage injury was evaluated by modified Mankin's score and OARSI score, and the levels of inflammatory factors and biochemical markers of bone metabolism in joint tissue and serum were detected by enzyme-linked immunosorbent assay. The expression of nuclear factor kappa B p65 (NF-κB p65) and type Ⅱ collagen (COL2A1) in cartilage was detected by immunohistochemistry. The expression of Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88) and NF-κB p65 proteins were detected by Western blot. Nuclear and cytoplasmic proteins were extracted and detected for the nuclear translocation of NF-κB p65. RESULTS At the end of the experiment, compared with the sham-operation group, the gait behavior scores, PWTL, COL2A1 expression MOD values of the cartilage in KOA model group and the experimental groups were decreased, while the levels of interleukin (IL)-1β, IL-6, tumor necrosis factor alpha (TNF-α), C-terminal crosslinking telopeptide of type Ⅰ collagen (CTX-Ⅰ) and C-terminal crosslinking telopeptide of type Ⅱ collagen (CTX-Ⅱ) in joint tissues or serum samples increased, and the expressions of TLR4, MyD88 and NF-κB p65 proteins in cartilage tissues were up-regulated, while the nuclear translocation NF-κB p65 was significantly increased (P<0.05). In addition, compared with KOA model group, the gait behavior score, PWTL, COL2A1 protein expression MOD value in cartilages were increased in experimental groups, while PWMT, Mankin's score and OARSI score, the levels of IL-1β, IL-6, TNF-α, CTX-Ⅰ, CTX-Ⅱ in joint tissues or serum samples were decreased, and the expressions of TLR4, MyD88 and NF-κB p65 proteins in cartilage were down-regulated, and the nuclear translocation NF-κB p65 was inhibited (P<0.05). In particular, the improvement of the above-mentioned indicators in Tan ⅡA mg·kg-1 group were more significant. CONCULUSION Tan ⅡA can inhibit the expression of TLR4/Myd88/nfb signaling pathway-related proteins, decrease the release of inflammatory cytokines, and then inhibit the degeneration of articular cartilage, so as to improve the knee joint function of KOA rats.
关键词
丹参酮ⅡA /
膝骨关节炎 /
TLR4/Myd88/NF-κB通路 /
软骨退变 /
炎症反应
{{custom_keyword}} /
Key words
tanshinone ⅡA;knee osteoarthritis /
TLR4/MyD88/NF-κB pathway /
cartilage degeneration /
inflammatory response
{{custom_keyword}} /
中图分类号：
R965
{{custom_clc.code}}
({{custom_clc.text}})
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
[1] WANG B, XING D, DONG S J, et al. Prevalence and disease burden of knee osteoarthritis in China: a systematic review. Chin J Evid-Based Med (中国循证医学杂志), 2018, 18(2):134-142.
[2] LI Z, ZOU Y, FAN D, et al. The mechanism of medial collateral ligament repair in knee?osteoarthritis?based on the?TLR4/MyD88/NF-kappa B inflammatory signaling pathway. J Musculoskelet Neuronal Interact, 2020, 20(3):398-403.
[3] WAN Y Y, ZHANG H, ZHANG C Y, et al. Study on the medication rule of the method of promoting blood circulation and removing blood stasis in treating knee osteoarthritis based on apriori and cluste-ring algorithm. World J Integ Tradit Western Med (世界中西医结合杂志), 2021, 16(1):29-33.
[4] KE Q, GUO X M. Clinical observation of efficacy and quality of life improvement with danshen injection treating osteoarthritis of the knee. Clin J Chin Med(中医临床研究), 2011, 3(24):5-7.
[5] DING H S. Effects of tanshinone ⅡA on the expression of HMGB1, IL-23/IL-17 axis and myeloperoxidase in rats with myocardial ischemia-reperfusion injury. Bachu Med J (巴楚医学), 2020, 3(3):19-23.
[6] YOU M C, SHEN C W, XU Y Y, et al. Effects of tanshinone ⅡA on expression of Bcl-2 and Bax and in spinal of rats with neuropathic pain. Chin J Basic Med Tradit Chin Med (中国中医基础医学杂志), 2020, 26(4):479-482.
[7] JIA P T, ZHANG X L, ZUO H N, et al. Articular cartilage degradation is prevented by tanshinone ⅡA through inhibiting apoptosis and the expression of inflammatory cytokines. Mol Med Rep, 2017, 16(5):6285-6289.
[8] KOTHARI P, SINHA S, SARDAR A, et al. Inhibition of cartilage degeneration and subchondral bone deterioration by Spinacia oleracea in human mimic of ACLT-induced osteoarthritis. Food Funct, 2020, 11(9):8273-8285.
[9] WANG X, FAN J, DING X, et al. Tanshinone I inhibits IL-1beta-induced apoptosis, inflammation and extracellular matrix degradation in chondrocytes CHON-001 cells and attenuates murine osteoarthritis.Drug Des Devel Ther, 2019, 13(10):3559-3568.
[10] DENG B, WANG X Y, XU D Q, et al. Research of mechanism on affect of alendronate sodium on Wnt signaling pathway and collagenⅡ in knee osteoarthritis rats. Chin Med Innovat (中国医学创新), 2019, 16(21):25-30.
[11] XU Q, CHEN B, WANG Y, et al. The effectiveness of manual therapy for relieving pain, stiffness, and dysfunction in knee osteoarthritis: a systematic review and meta-analysis.Pain Physician, 2017, 20(4):229-243.
[12] XING Z L, QI Z R, QIU Q Z. Effects of knee triple-organ decoction extracts obtained by various processes on serum inflammatory cytokines and cartilaginous telomerase activity in KOA rats. J Guangzhou Univ Tradit Chin Med (广州中医药大学学报), 2019, 36(7):1054-1059.
[13] CHAPLAN S R, BACH F W, POGREL J W, et al. Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods, 1994, 53(1):55-63.
[14] PRITZKER K P, GAY S, JIMENEZ S A, et al. Osteoarthritis cartilage histopathology: grading and staging. Osteoarthritis Cartilage, 2006, 14(1):13-29.
[15] ZHANG Y, ZENG Y. Curcumin reduces inflammation in knee osteoarthritis rats through blocking TLR4 /MyD88/NF-κB signal pathway. Drug Dev Res, 2019, 80(3):353-359.
[16] LIU X, CAI H X, CAO P Y, et al. TLR4 contributes to the damage of cartilage and subchondral bone in discectomy-induced TMJOA mice.J Cell Mol Med, 2020, 24(19):11489-11499.
[17] GU H, JIAO Y, YU X, et al. Resveratrol inhibits the IL-1beta-induced expression of MMP-13 and IL-6 in human articular chondrocytes via TLR4/MyD88-dependent and -independent signaling cascades.Int J Mol Med, 2017, 39(3):734-740.
[18] KIM S H, BANG J, SON C N, et al. Grape seed proanthocyanidin extract ameliorates murine autoimmune arthritis through regulation of TLR4/MyD88/NF-κB signaling pathway. Korean J Intern Med, 2018, 33(3):612-621.
[19] LIU M, XIE J, SUN Y.TLR4/MyD88/NF-kappaB-mediated inflammation contributes to cardiac dysfunction in rats of PTSD. Cell Mol Neurobiol, 2020, 40(6):1029-1035.
[20] YE J M, HUANG G X, HUANG A H. Inflammation through TLR4/MyD88 signaling pathway in low-Mg2+-induced epileptic hippocampal slice models. J Apoplexy Nervous Dis (中风与神经疾病杂志), 2019, 36(4):300-302.
[21] WANG Y, LI H, SHI Y, et al. miR-143-3p impacts on pulmonary inflammatory factors and cell apoptosis in mice with mycoplasmal pneumonia by regulating TLR4/MyD88/NF-kappaB pathway. Biosci Rep, 2020, 40(7):BSR20193419.
[22] MANZOOR S, KHALIL S, MALIK M A, et al. Induction of profibrotic microenvironment via TLR4 MyD88-dependent and -independent inflammatory signaling in chronic hepatitis C virus infection. Viral Immunol, 2020, 33(9):585-593.
[23] WANG D X, MIN J K, LI H, et al. Significance of serum COMP, COL10A1 and P Ⅱ ANP in diagnosis and evaluation of osteoarthritis. Chin Mod Doctor (中国现代医生), 2020, 14(12):18-21.
[24] ATTUR M, ZHOU H, SAMUELS J, et al. Interleukin 1 receptor antagonist (IL1RN) gene variants predict radiographic severity of knee osteoarthritis and risk of incident disease. Ann Rheum Dis, 2020, 79(3):400-407.
{{custom_fnGroup.title_cn}}
脚注
{{custom_fn.content}}
基金
青岛市2018年度医药科研指导计划项目资助(2018-WJZD029)
{{custom_fund}}
PDF(4612 KB)
