黃芩素對IL-1β誘導的骨關節(jié)炎軟骨細胞NF-κB信號通路的影響
發(fā)布時間：2019-03-18 11:18
【摘要】：臨床上對于骨關節(jié)炎(Osteoarthritis,簡稱OA)患畜的治療方法主要側(cè)重于緩解癥狀。如臨床上多向患畜的關節(jié)腔內(nèi)注射透明質(zhì)酸等藥物;或使患畜服用解熱鎮(zhèn)痛藥“撲熱息痛”、非甾體類抗炎藥“NSAIDs”或“COX-2抑制劑”等藥物。這些方法雖然可以對OA的臨床癥狀起到緩解作用,但它們對患畜的機體均具有不同程度的副作用。OA的治療目的不應僅僅是改善癥狀,而還應達到消除和預防OA的目的。因此,有必要在治療研究的基礎上,應進一步闡明其發(fā)病機制及藥物作用機理。近年來,許多實驗驗證的黃芩素(簡稱BAI)具有多種生物學活性,如抗炎、抗細胞凋亡等。本實驗利用IL-1β刺激大鼠軟骨細胞,誘導其產(chǎn)生OA模型,將BAI提取液==作用于該模型的軟骨細胞中,以探究在IL-1β誘導的大鼠OA中,BAI的干預對NF-κB信號通路及其基因產(chǎn)物表達的影響,為今后OA的臨床治療中BAI可作為快速有效的藥物而提供可靠的理論依據(jù)。實驗對SD孕鼠產(chǎn)下的哺乳期幼鼠的軟骨細胞進行分離培養(yǎng),取2代細胞,首先用IL-1β(10ng/ml)進行預處理,24h后將IL-1β(10ng/ml)與(50μM)BAI共同作用于軟骨細胞中,分別處理0h、4h、8h、12h和24h。而后利用CCK-8試劑盒對于這些時間點的軟骨細胞進行檢測,探究BAI是否對軟骨細胞的活力產(chǎn)生影響;再利用蛋白免疫印跡法檢測軟骨細胞中基質(zhì)金屬蛋白酶MMP-3、MMP-9,和化學因子COX-2的表達水平;及BAI對細胞質(zhì)和細胞核內(nèi)NF-κB p65和phospho-NF-κB p65表達水平的影響。應用免疫熒光法檢測BAI對軟骨細胞NF-κB通路中p65核轉(zhuǎn)位的影響。實驗結(jié)果表明不同濃度的BAI干預并沒有對軟骨細胞造成損傷。BAI能夠抑制由IL-1β誘導的OA軟骨細胞中MMP-3、MMP-9的表達,并調(diào)控了軟骨細胞中COX-2的表達下調(diào);抑制細胞質(zhì)中p65的磷酸化及核轉(zhuǎn)位。綜上所述,本實驗通過對IL-1β誘導的OA軟骨細胞的研究,證實了BAI可以抑制OA軟骨細胞的凋亡,阻斷軟骨基質(zhì)的降解。BAI通過遏制p65的磷酸化及核轉(zhuǎn)位而阻斷NF-κB信號通路的激活,從而實現(xiàn)其對軟骨細胞的保護機制。為今后臨床上應用BAI治療OA提供了理論依據(jù)。
[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.
【學位授予單位】：東北農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S857.16
本文編號：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.
【學位授予單位】：東北農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S857.16
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