黄芩素对IL-1β诱导的骨关节炎软骨细胞NF-κB信号通路的影响
发布时间：2019-03-18 11:18
【摘要】：临床上对于骨关节炎(Osteoarthritis,简称OA)患畜的治疗方法主要侧重于缓解症状。如临床上多向患畜的关节腔内注射透明质酸等药物;或使患畜服用解热镇痛药“扑热息痛”、非甾体类抗炎药“NSAIDs”或“COX-2抑制剂”等药物。这些方法虽然可以对OA的临床症状起到缓解作用,但它们对患畜的机体均具有不同程度的副作用。OA的治疗目的不应仅仅是改善症状,而还应达到消除和预防OA的目的。因此,有必要在治疗研究的基础上,应进一步阐明其发病机制及药物作用机理。近年来,许多实验验证的黄芩素(简称BAI)具有多种生物学活性,如抗炎、抗细胞凋亡等。本实验利用IL-1β刺激大鼠软骨细胞,诱导其产生OA模型,将BAI提取液==作用于该模型的软骨细胞中,以探究在IL-1β诱导的大鼠OA中,BAI的干预对NF-κB信号通路及其基因产物表达的影响,为今后OA的临床治疗中BAI可作为快速有效的药物而提供可靠的理论依据。实验对SD孕鼠产下的哺乳期幼鼠的软骨细胞进行分离培养,取2代细胞,首先用IL-1β(10ng/ml)进行预处理,24h后将IL-1β(10ng/ml)与(50μM)BAI共同作用于软骨细胞中,分别处理0h、4h、8h、12h和24h。而后利用CCK-8试剂盒对于这些时间点的软骨细胞进行检测,探究BAI是否对软骨细胞的活力产生影响;再利用蛋白免疫印迹法检测软骨细胞中基质金属蛋白酶MMP-3、MMP-9,和化学因子COX-2的表达水平;及BAI对细胞质和细胞核内NF-κB p65和phospho-NF-κB p65表达水平的影响。应用免疫荧光法检测BAI对软骨细胞NF-κB通路中p65核转位的影响。实验结果表明不同浓度的BAI干预并没有对软骨细胞造成损伤。BAI能够抑制由IL-1β诱导的OA软骨细胞中MMP-3、MMP-9的表达,并调控了软骨细胞中COX-2的表达下调;抑制细胞质中p65的磷酸化及核转位。综上所述,本实验通过对IL-1β诱导的OA软骨细胞的研究,证实了BAI可以抑制OA软骨细胞的凋亡,阻断软骨基质的降解。BAI通过遏制p65的磷酸化及核转位而阻断NF-κB信号通路的激活,从而实现其对软骨细胞的保护机制。为今后临床上应用BAI治疗OA提供了理论依据。
[Abstract]:Clinical treatment of osteoarthritis (OA) mainly focuses on relieving symptoms. For example, many drugs such as hyaluronic acid were injected into the articular cavity of animals, or antipyretic analgesic "paracetamol", non-steroidal anti-inflammatory drug "NSAIDs" or "COX-2 inhibitor" were given to the affected animals. Although these methods can relieve the clinical symptoms of OA, they all have different degree side effects on the infected animals. The treatment of OA should not only improve the symptoms, but also achieve the purpose of eliminating and preventing OA. Therefore, it is necessary to further clarify its pathogenesis and drug action mechanism on the basis of therapeutic research. In recent years, baicalin (BAI) has many biological activities, such as anti-inflammatory, anti-apoptosis and so on. In this study, IL-1 尾 was used to stimulate rat chondrocytes to induce the production of OA model. BAI extract = was applied to chondrocytes of the model to explore the effects of IL-1 尾 on the OA of rats. The effect of BAI intervention on the expression of NF- 魏 B signaling pathway and its gene products provides a reliable theoretical basis for BAI as a rapid and effective drug in the clinical treatment of OA in the future. Chondrocytes were isolated and cultured from neonatal lactation rats of SD pregnant rats. The second generation of chondrocytes were pretreated with IL-1 尾 (10ng/ml), and the chondrocytes were treated with IL-1 尾 (10ng/ml) and (50 渭 M) BAI 24 hours later, the chondrocytes were treated with IL-1 尾 (10ng/ml) and (50 渭 M) BAI. They were treated at 0 h, 4 h, 8 h, 12 h and 24 h, respectively. Then CCK-8 kit was used to detect chondrocytes at these time points to explore whether BAI had an effect on chondrocyte viability. The expression of matrix metalloproteinase MMP-3,MMP-9, and chemical factor COX-2 in chondrocytes and the effects of BAI on the expression of NF- kappa B p65 and phospho-NF- 魏 B p65 in cytoplasm and nucleus were detected by Western blot. The effect of BAI on p65 nuclear translocation in chondrocytes NF- 魏 B pathway was detected by immunofluorescence. The results showed that different concentrations of BAI did not cause damage to chondrocytes. Bai could inhibit the expression of MMP-3,MMP-9 in OA chondrocytes induced by IL-1 尾 and regulate the down-regulation of COX-2 expression in chondrocytes. Inhibition of p65 phosphorylation and nuclear translocation in cytoplasm. In conclusion, through the study of OA chondrocytes induced by IL-1 尾, we confirmed that BAI can inhibit the apoptosis of OA chondrocytes. Bai blocks the activation of NF- 魏 B signaling pathway by inhibiting the phosphorylation of p65 and nuclear translocation, thus realizing the protective mechanism of Bai on chondrocytes. It provides a theoretical basis for the clinical application of BAI in the treatment of OA.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S857.16
本文编号：2442814
[Abstract]:Clinical treatment of osteoarthritis (OA) mainly focuses on relieving symptoms. For example, many drugs such as hyaluronic acid were injected into the articular cavity of animals, or antipyretic analgesic "paracetamol", non-steroidal anti-inflammatory drug "NSAIDs" or "COX-2 inhibitor" were given to the affected animals. Although these methods can relieve the clinical symptoms of OA, they all have different degree side effects on the infected animals. The treatment of OA should not only improve the symptoms, but also achieve the purpose of eliminating and preventing OA. Therefore, it is necessary to further clarify its pathogenesis and drug action mechanism on the basis of therapeutic research. In recent years, baicalin (BAI) has many biological activities, such as anti-inflammatory, anti-apoptosis and so on. In this study, IL-1 尾 was used to stimulate rat chondrocytes to induce the production of OA model. BAI extract = was applied to chondrocytes of the model to explore the effects of IL-1 尾 on the OA of rats. The effect of BAI intervention on the expression of NF- 魏 B signaling pathway and its gene products provides a reliable theoretical basis for BAI as a rapid and effective drug in the clinical treatment of OA in the future. Chondrocytes were isolated and cultured from neonatal lactation rats of SD pregnant rats. The second generation of chondrocytes were pretreated with IL-1 尾 (10ng/ml), and the chondrocytes were treated with IL-1 尾 (10ng/ml) and (50 渭 M) BAI 24 hours later, the chondrocytes were treated with IL-1 尾 (10ng/ml) and (50 渭 M) BAI. They were treated at 0 h, 4 h, 8 h, 12 h and 24 h, respectively. Then CCK-8 kit was used to detect chondrocytes at these time points to explore whether BAI had an effect on chondrocyte viability. The expression of matrix metalloproteinase MMP-3,MMP-9, and chemical factor COX-2 in chondrocytes and the effects of BAI on the expression of NF- kappa B p65 and phospho-NF- 魏 B p65 in cytoplasm and nucleus were detected by Western blot. The effect of BAI on p65 nuclear translocation in chondrocytes NF- 魏 B pathway was detected by immunofluorescence. The results showed that different concentrations of BAI did not cause damage to chondrocytes. Bai could inhibit the expression of MMP-3,MMP-9 in OA chondrocytes induced by IL-1 尾 and regulate the down-regulation of COX-2 expression in chondrocytes. Inhibition of p65 phosphorylation and nuclear translocation in cytoplasm. In conclusion, through the study of OA chondrocytes induced by IL-1 尾, we confirmed that BAI can inhibit the apoptosis of OA chondrocytes. Bai blocks the activation of NF- 魏 B signaling pathway by inhibiting the phosphorylation of p65 and nuclear translocation, thus realizing the protective mechanism of Bai on chondrocytes. It provides a theoretical basis for the clinical application of BAI in the treatment of OA.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S857.16
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