中国病理生理杂志
中國病理生理雜誌
중국병리생리잡지
Chinese Journal of Pathophysiology
2015年
9期
1667-1672
,共6页
戴兵%徐俐%金海东%蔡宁宇%陈辉%李斌%蔡建武%潘骏
戴兵%徐俐%金海東%蔡寧宇%陳輝%李斌%蔡建武%潘駿
대병%서리%금해동%채저우%진휘%리빈%채건무%반준
橄榄苦苷%白细胞介素1β%一氧化氮%骨关节炎
橄欖苦苷%白細胞介素1β%一氧化氮%骨關節炎
감람고감%백세포개소1β%일양화담%골관절염
Oleuropein%Interleukin-1β%Nitric oxide%Osteoarthritis
目的:研究橄榄苦苷对白细胞介素1β( IL-1β)诱导的SD大鼠关节软骨细胞的影响。方法:采用酶两步顺序消化法消化SD大鼠关节软骨分离细胞,体外培养软骨细胞,光学显微镜观察细胞形态,阿尔新蓝染色及Ⅱ型胶原免疫组化法对软骨细胞进行鉴定。橄榄苦苷对软骨细胞的细胞毒性采用CCK-8实验进行评估。取第3代软骨细胞,分别用浓度为10、50或100μmol/L的橄榄苦苷预先处理,随后用IL-1β刺激细胞24 h,所生成的NO和前列腺素E2( PGE2)的量分别用格里斯重氮化反应和酶联反应吸附实验进行评估。基质金属蛋白酶( MMP)-1和MMP-13 mRNA的表达利用实时荧光定量PCR进行定量检测。采用免疫印迹实验分析诱导型一氧化氮合酶(iNOS)、环氧化酶2(COX-2)和活化NF-κB的蛋白表达。结果:软骨细胞在不同浓度橄榄苦苷培养24 h后,其生存能力与对照组相比无明显差异。橄榄苦苷可以显著降低IL-1β刺激下软骨细胞MMP-1和MMP-13 mRNA的表达及NO、PGE2的生成。橄榄苦苷通过减少IκB蛋白的降解从而抑制IL-1β介导的NF-κB通路的活化。结论:橄榄苦苷可通过NF-κB信号转导通路调控炎症的发生发展,进一步明确了橄榄苦苷对关节炎防治作用的分子机制,为骨关节炎的免疫治疗提供了重要的基础理论依据。
目的:研究橄欖苦苷對白細胞介素1β( IL-1β)誘導的SD大鼠關節軟骨細胞的影響。方法:採用酶兩步順序消化法消化SD大鼠關節軟骨分離細胞,體外培養軟骨細胞,光學顯微鏡觀察細胞形態,阿爾新藍染色及Ⅱ型膠原免疫組化法對軟骨細胞進行鑒定。橄欖苦苷對軟骨細胞的細胞毒性採用CCK-8實驗進行評估。取第3代軟骨細胞,分彆用濃度為10、50或100μmol/L的橄欖苦苷預先處理,隨後用IL-1β刺激細胞24 h,所生成的NO和前列腺素E2( PGE2)的量分彆用格裏斯重氮化反應和酶聯反應吸附實驗進行評估。基質金屬蛋白酶( MMP)-1和MMP-13 mRNA的錶達利用實時熒光定量PCR進行定量檢測。採用免疫印跡實驗分析誘導型一氧化氮閤酶(iNOS)、環氧化酶2(COX-2)和活化NF-κB的蛋白錶達。結果:軟骨細胞在不同濃度橄欖苦苷培養24 h後,其生存能力與對照組相比無明顯差異。橄欖苦苷可以顯著降低IL-1β刺激下軟骨細胞MMP-1和MMP-13 mRNA的錶達及NO、PGE2的生成。橄欖苦苷通過減少IκB蛋白的降解從而抑製IL-1β介導的NF-κB通路的活化。結論:橄欖苦苷可通過NF-κB信號轉導通路調控炎癥的髮生髮展,進一步明確瞭橄欖苦苷對關節炎防治作用的分子機製,為骨關節炎的免疫治療提供瞭重要的基礎理論依據。
목적:연구감람고감대백세포개소1β( IL-1β)유도적SD대서관절연골세포적영향。방법:채용매량보순서소화법소화SD대서관절연골분리세포,체외배양연골세포,광학현미경관찰세포형태,아이신람염색급Ⅱ형효원면역조화법대연골세포진행감정。감람고감대연골세포적세포독성채용CCK-8실험진행평고。취제3대연골세포,분별용농도위10、50혹100μmol/L적감람고감예선처리,수후용IL-1β자격세포24 h,소생성적NO화전렬선소E2( PGE2)적량분별용격리사중담화반응화매련반응흡부실험진행평고。기질금속단백매( MMP)-1화MMP-13 mRNA적표체이용실시형광정량PCR진행정량검측。채용면역인적실험분석유도형일양화담합매(iNOS)、배양화매2(COX-2)화활화NF-κB적단백표체。결과:연골세포재불동농도감람고감배양24 h후,기생존능력여대조조상비무명현차이。감람고감가이현저강저IL-1β자격하연골세포MMP-1화MMP-13 mRNA적표체급NO、PGE2적생성。감람고감통과감소IκB단백적강해종이억제IL-1β개도적NF-κB통로적활화。결론:감람고감가통과NF-κB신호전도통로조공염증적발생발전,진일보명학료감람고감대관절염방치작용적분자궤제,위골관절염적면역치료제공료중요적기출이론의거。
AIM:To investigate the effect of oleuropein on interleukin-1β( IL-1β)-induced SD rat articular chondrocytes .METHODS:The SD rat articular chondrocytes were isolated by 2 step enzyme digestions .The chondrocytes were cultured in vitro.Inverted microscopic observation was performed during the culture .Alcian blue staining and type II collagen immunohistochemical staining were used to identify the chondrocytes .The effects of oleuropein on the viability of chondrocytes were determined by CCK-8 assay.The cells in 3rd passage were pretreated with oleuropein at 10, 50 or 100 μmol/L and subsequently stimulated with IL-1βat 10 μg/L for 24 h.Production of prostaglandin E 2 ( PGE2 ) and ni-tric oxide (NO) were evaluated by the Griess reaction and an enzyme linked immunosorbent assay (ELISA).The mRNA expression of matrix metalloproteinase ( MMP)-1 and MMP-13 was measured by real-time PCR.The protein levels of in-ducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and nuclear factor-kappa B (NF-κB) were detected by Western blotting .RESULTS:The cell viability of chondrocytes was not significantly impaired by treating with oleuropein at concentration of 10, 50 or 100μmol/L for 24 h compared with control group .Pretreatment with oleuropein significantly in-hibited the production of PGE 2 and NO induced by IL-1β.Oleuropein also significantly decreased the IL-1β-stimulated MMP-1 and MMP-13 mRNA expression in articular chondrocytes .Pretreatment with oleuropein inhibited the IL-1β-media-ted activation of NF-κB by suppressing the degradation of its inhibitory protein IκBαin the cytoplasm .CONCLUSION:Oleuropein inhibits IL-1β-induced inflammatory gene expression by suppressing NF-κB activation at the transcriptional le-vel, suggesting a new mechanism for the anti-inflammatory effects of oleuropein as a novel agent on treating with osteoarthri-tis.