p38丝裂原活化蛋白激酶信号通路在羧甲基壳聚糖保护一氧化氮诱导软骨细胞凋亡中的作用及机制研究

The role of p38 MAPK signal pathway in carboxymethylated chitosan protecting NO-induced chondro- cyles apoptosis and the mechanisms

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影响，并探索p38丝裂原活化蛋白激酶（MAPK）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；通过不同浓度硝普钠诱导软骨细胞凋亡；实验分为对照组、硝普钠处理组、硝普钠＋不同浓度CMCS处理组、硝普钠＋p38MAPK抑制剂SB203580处理组。通过流式细胞技术检测软骨细胞凋亡率，Hoeehst33342染色观察凋亡细胞核形态变化，罗丹明染色观察软骨细胞线粒体膜电位的改变情况，通过蛋白印迹法检测p38与磷酸化p38（p．p38）的表达。采用单因素方差分析进行统计学处理。结果1～3mmol／L硝普钠可以不同程度地诱导软骨细胞发生凋亡，随着硝普钠浓度的增加凋亡率也增加，当硝普钠浓度为3mmol／L时其凋亡率为69．8％（P〈0．05）；硝普钠可以增加软骨细胞核碎裂发生率，其核碎裂细胞数明显高于对照组；硝普钠可降低软骨细胞线粒体膜电位水平，与对照组比较差异明显；硝普钠可以增加软骨细胞内p-p38的表达，与对照组比较增加4．3倍。不同浓度CMCS可以降低硝普钠诱导软骨细胞凋亡率，分别为51．0％、29．9％、15．2％，与3mmol／L硝普钠诱导组比较差异有统计学意义（P〈0．05）；不同浓度CMCS可以降低硝普钠诱导软骨细胞核碎裂的数量；CMCS可以增加硝普钠诱导凋亡软骨细胞线粒体膜电位；CMCS可降低硝普钠诱导凋亡软骨细胞内p-p38表达水平。结论CMCS对硝普钠诱导软骨细胞凋亡具有一定的保护作用，是通过抑制p38MAPK信号通路活性来完成的。

Objective To study the effects of carboxymethylated chitosan （CMCS） to nitric oxide （NO）-induced apoptosis on rat chondrocytes, and explore p38MAPK signal transduetion pathway in the process and its mechanism. Methods The rat articular cartilage cells were cultured in vitro, collagen type-2 （collagen-2） immunohistochemical staining was used to identify the cartilage cells. The model of chondrecyte apoptosis was built by different concentrations of sodium nitroprusside （SNP） induction. The cells were divided into the control group, the SNP treated group SNP＋CMCS treated group, and the SNP＋p38 MAPK inhibitor SB203580 treated group. The apoptotic rate of chondroeytes was calculated by FCM, apoptotic nuclei was identified by Hoechst33342 stain, the mitochondrial membrane potential changes was detected by Rhodamine123 （Rho123） stain, the expression of p38 and p-p38 were detected by Western blotting analysis. Results 1-3 mmol/L SNP could induce chondrocyte apoptosis, the apoptotic rate was increased with the SNP increasing, the most obvious apoptosis was occurred in 3 mmol/L SNP treated chondrocytes, which was 69.8% （P〈0.05）. SNP could increase the nuclear fragmentation of chondrocytes, the cells with nuclear fragmentation was significantly higher than that in the control group. SNP could reduce mitochondrial membrane potential in chondrocytes, which decreased significantly compared with the control group. SNP could increase the p-p38expression in chondrocytes, which was 4.3 times compared to the control group. CMCS of different concen- trations could reduce the apoptotic rate of SNP-induced chondrocytes, which was 51.0%, 29.9% and 15.2%, which was decreased significantly （P〈0.05） when compared with 3 mmol/L SNP induced group, CMCS decre- ased the cells number of SNP-induced nuclear fragmentation. CMCS increased the mitochondrial membrane potential in SNP-induced chondrocytes. CMCS reduced the expression levels of p-p38 in SNP-induced chondrocytes. Conclusion CMCS has protective effect on SNP-induced apoptosis of chondrocytes. This process is completed by inhibiting the activity of p38 MAPK signal pathway.