黄连素对IL-1β诱导炎性退变软骨细胞增殖的影响及其分子机制  

Effect of berberine on proliferation of chondrocytes with inflammatory degeneration caused by IL-1β

作　　者：黄文 马石庭 姚军[2] HUANG Wen;MA Shiting;YAO Jun(Department of Bone Joint and Sports Medicine,Hezhou People's Hospital,Hezhou 542800,China;不详)

机构地区：[1]贺州市人民医院骨关节与运动医学科,广西贺州542800 [2]广西医科大学第一附属医院骨关节外科

出　　处：《山东医药》2021年第23期28-32,共5页Shandong Medical Journal

基　　金：广西壮族自治区中医药管理局自筹经费科研项目(GZZC2020339)。

摘　　要：目的观察黄连素对白细胞介素1β(IL-1β)诱导炎性退变软骨细胞增殖的影响,并探讨其可能的作用机制。方法采用酶消化法提取小鼠膝关节软骨细胞,通过镜下观察及甲苯胺蓝染色鉴定证实。使用IL-1β构建软骨细胞炎性退变模型,采用MTT法检测不同浓度黄连素对软骨细胞活性的影响,筛选出对软骨细胞活性有积极作用的低、高两个黄连素浓度(6.25×10^(-3)g/L、0.05 g/L)。将软骨细胞随机分为空白组(不采取任何干预)、模型组(仅加入10 ng/mL IL-1β干预)、黄连素低剂量组(10 ng/mL IL-1β+6.25×10^(-3)g/L黄连素干预)、黄连素高剂量组(10ng/mL IL-1β+0.05 g/L黄连素干预),分组处理后继续培养2 d。采用FDA荧光染色检测细胞增殖活性(活细胞数),番红O染色检测软骨细胞分泌功能(染色面积),Real-time PCR法检测软骨细胞特异性相关基因[蛋白聚糖(Acan)、Ⅱ型胶原1(Col2a1)]及炎症相关基因[基质金属蛋白酶13(MMP-13)、环氧合酶2(COX-2)]表达,Western blotting法检测转化生长因子β(TGF-β)/Smad2/3信号通路相关蛋白(TGF-β_(1)、Smad2、Smad3)表达。结果模型组、空白组、黄连素低剂量组、黄连素高剂量组活细胞数分别为(212.50±17.68)、(375.00±35.35)、(530.00±42.43)、(650.00±70.71)个,组间两两比较P均<0.05。模型组、空白组、黄连素低剂量组、黄连素高剂量组染色面积分别为24.50%±4.95%、44.50%±6.36%、65.00%±7.07%、77.5%±3.53%,组间两两比较P均<0.05。模型组、空白组、黄连素低剂量组、黄连素高剂量组软骨细胞Acan、Col2a1 mRNA及TGF-β_(1)、Smad2、Smad3蛋白表达均依次升高,MMP-13、COX-2 mRNA表达均依次降低,组间两两比较P均<0.05。结论黄连素可促进IL-1β诱导炎性退变软骨细胞的增殖及活性,其机制可能与抑制炎症反应及激活TGF-β/Smad2/3信号通路有关。Objective To observe the effect of berberine on the proliferation of chondrocytes with inflammatory degeneration induced by interleukin-1β(IL-1β)and to investigate its possible mechanism.Methods The mouse knee joint chondrocytes were extracted by enzyme digestion method.The chondrocytes were identified by microscope and toluidine blue staining.The inflammatory degeneration models of chondrocytes were induced by IL-1β.The effect of berberine on the proliferation of chondrocytes treated with IL-1βwas evaluated by MTT assay.The minimum and maximum concentrations of berberine(6.25×10^(-3)g/L,0.05 g/L)that had positive effect on chondrocyte activity were selected for subsequent experiments.The chondrocytes were randomly divided into four groups:the blank group(no intervention),model group(only 10 ng/mL IL-1βintervention),low-dose berberine group(10 ng/mL IL-1β+6.25×10^(-3) g/L berberine intervention),and high-dose berberine group(10 ng/mL IL-1β+0.05 g/L berberine intervention);cells in each group were cultured for two days.Subsequently,FDA fluorescent staining was used to detect cell proliferation activity(number of viable cells),and Safranin O staining was used to detect chondrocyte secretion function(stained area).Real-time PCR was used to detect the expression of chondrocyte-specific related genes[proteoglycan(Acan),type Ⅱ collagen 1(Col2a1)]and inflammation-related genes[matrix metalloproteinase 13(MMP-13),cyclooxygenase 2(COX-2)].The protein expression levels of transforming growth factorβ(TGF-β)/Smad2/3 signaling pathway[TGF-β_(1),Smad2,Smad3]were detected by Western blotting.Results The numbers of living cells in the model group,blank group,low-dose berberine group and high-dose berberine group were 212.50±17.68,375.00±35.35,530.00±42.43,and 650.00±70.71,respectively.The staining area of the model group,blank group,low-dose berberine group and high-dose berberine group was 24.50%±4.95%,44.50%±6.36%,65.00%±7.07%,and 77.5%±3.53%,respectively,with statistically significant difference(all P<0.

关 键 词：黄连素 IL-1Β 软骨细胞 炎性退变 骨关节炎 TGF-β/Smad2/3信号通路 

分 类 号：R684.3[医药卫生—骨科学]