摘要
目的研究羧甲基壳聚糖（CMCS）对一氧化氮诱导大鼠软骨细胞凋亡的保护作用，并探索P13K／Akt信号转导通路在该过程中的作用及其机制。方法体外培养大鼠膝关节软骨细胞，通过Ⅱ型胶原免疫组织化学染色鉴定软骨细胞；3mmol，L硝普钠诱导软骨细胞构建凋亡模型；实验分为空白对照组、硝普钠诱导凋亡组、硝普钠＋不同浓度CMCS处理组、硝普衲＋CMCS＋P13K抑制剂Wortmannin处理组。通过细胞增殖检测试剂盒（CCK-8）对软骨细胞增殖活性进行检测，流式细胞技术检测软骨细胞凋亡率，荧光定量PCR检测软骨细胞内MMP-13及基质金属蛋白酶组织抑制物（TIMP）．1mRNA的表达水平，通过蛋白印迹法检测软骨细胞内Akt及p-Akt表达水平，采用单因素方差分析进行统计学处理。结果3mmo札硝普钠可以抑制软骨细胞增殖活性（吸光度值0．221±0．023），与对照组（0．736±0．032）比较降低70％（F=8．203，P=-0．021），诱导软骨细胞发生早期凋亡，早期凋亡率为（68．8±5．2）％；增加软骨细胞内MMP-13mRNA表达，降低TIMP-1mRNA表达；加入CMCS后软骨细胞增殖活性有所增加，软骨细胞凋亡降低，凋亡率降低为（14．7±2．3）％；并且CMCS可以激活硝普钠诱导软骨细胞内Akt激活，增加P-Akt蛋白表达水平，CMCS可以恢复硝普钠诱导软骨细胞内MMP-13mRNA增加及TIMP-1mRNA降低。结论CMCS对硝普钠诱导软骨细胞凋亡具有一定的保护作用，P13K／Akt信号通路的激活在该过程中起重要作用。
Objective To investigate the protective effects of carboxymethylated chitosan （CMCS） on nitric oxide （NO） induced apoptosis in rat chondrocytes, and the probable roles and mechanisms of PI3K/Akt signaling pathway in these process. Methods Rat knee articular cartilage was used as the source of chon- drocytes, the ceils were identified by immunohistochemical staining against collagen type I1, odium nitro- prusside （SNP, 3 mmol/L） was used to establish the apoptotic models of chondrocytes. Ceils were divided into four groups： the control group, the SNP-induced group, the SNP＋CMCS treated group, the SNP＋CMCS＋PI3K inhibitor Wortmannin treated group. Cell proliferation were assessed by cell proliferation assay kit （CCK-8）, the apoptotic rate of chondrocytes was determined by FCM with Annexin V-FITC/PI double staining, the expression levels of MMP-13 and TIMP-1 mRNA were detected by real-time polymerase chain reaction （PCR） analysis, the expression of Akt and p-Akt protein levels was detected by Western blotting analysis. One-way analysis of variance （ANOVA） statistical analysis was used to calculate the data. Results Three mmol/L SNP can inhibit proliferation （0.221±0.023）, and the proliferation was reduced by 70% compared with the control group （0.736± 0.032, F=8.203, P=0.021）; and the induced apoptosis in cultured chondrecytes could be observed. The apoptotic rate was （68.8＋5.2）%. Increased MMP-3 and decreased TIMP-1 mRNA expression were observed in SNP-induced cells. After adding CMCS to SNP-induced chondrocytes, the proliferation was increased while apoptotic rate was decreased, the apoptotic rate decreased to （14.7±2.3）%. CMCS could promote the activation of p-Akt in SNP-indueed chondrocytes and restore SNP-induced MMP-13 and T1MP-1 mRNA expression. Conehtsion CMCS could protect apoptosis in SNP induced chon-drocytes via activation of PI3K/Akt pathwaY.
出处
《中华风湿病学杂志》
CAS
CSCD
北大核心
2015年第3期170-175,I0002,共7页
Chinese Journal of Rheumatology
基金
国家自然科学基金（81301056,30801166）
