罗哌卡因对白细胞介素-1β诱导的软骨细胞损伤的保护作用探究

Protective effect of ropivacaine on interleukin-1β-induced chondrocyte injury

目的 基于腺苷一磷酸活化蛋白激酶(AMPK)/哺乳动物西罗莫司靶蛋白(mTOR)通路探究罗哌卡因对白细胞介素-1β(IL-1β)诱导的软骨细胞损伤的保护作用。方法 对软骨细胞进行培养,将软骨细胞随机分为对照组、模型组和低、中、高剂量实验组。对照组常规培养,模型组用含10 ng·mL^(-1)IL-1β的培养基刺激细胞24 h,低、中、高剂量实验组在模型组的基础上,用25,50,100 mg·L^(-1)的罗哌卡因进行处理。用细胞计数法-8(CCK-8)检测各组细胞的增殖情况;用流式细胞术检测细胞凋亡情况;用蛋白质印迹(Western blot)法检测细胞凋亡相关蛋白、细胞外基质降解相关蛋白及细胞中磷酸化AMPK(p-AMPK)和磷酸化mTOR(p-mTOR)蛋白表达的影响。结果 与对照组比较,模型组24,48,72 h OD值降低;与模型组相比,中、高剂量实验组24,48,72 h OD值均明显升高(均P<0.05)。对照组、模型组和低、中、高剂量实验组细胞凋亡率分别为(4.24±0.64)%,(18.13±1.53)%,(14.93±0.52)%,(11.19±0.74)%和(7.67±1.19)%。与对照组相比,模型组的凋亡蛋白B细胞淋巴瘤/白血病-2(Bcl-2)、细胞外基质降解蛋白COL2A1及ACAN蛋白表达水平均明显降低,Bcl-2相关X蛋白(Bax)、裂解的胱天蛋白酶-3(Cl-caspase-3)蛋白水平、基质金属蛋白酶13(MMP13)及ADAMTS5蛋白水平均明显升高(均P<0.05);与模型组相比,中、高剂量实验组的凋亡蛋白Bcl-2、细胞外基质降解蛋白COL2A1及ACAN蛋白表达水平均明显升高,Bax、Cl-caspase-3蛋白水平、MMP13及ADAMTS5蛋白水平均明显降低(均P<0.05)。结论 基于AMPK/mTOR通路,罗哌卡因对IL-1β诱导的软骨细胞损伤具有一定的保护作用。

Objective To investigate the protective effect of ropivacaine on interleukin-1β(IL-1β) induced chondrocyte injury based on the adenosine monophosphate activated protein kinase(AMPK)/mammalin target of rapamycin(mTOR) pathway. Methods The chondrocytes were cultured and randomly divided into control group, model group and low, medium, high dose experimental groups. Control group was cultured routinely, model group was stimulated with medium containing 10 ng·mL^(-1)IL-1β for 24 h, low, medium, high dose experimental groups were treated with 25, 50 and 100 mg·L^(-1)ropivacaine on the basis of model group. Cell proliferation was detected by cell counting kit-8(CCK-8). Cell apoptosis was detected by flow cytometry. Western blot was used to detect the expression of apoptosis related proteins extracellular matrix degradation related proteins and phosphorylation-AMPK(p-AMPK) and phosphorylation-m TOR(p-m TOR) protein expression in cells. Results Compared with control group,the OD of model group at 24,48,72 h were decreased;compared with model gorup,the OD of medium,high dose experimental groups at 24,48,72 h were increased(all P < 0. 05). The apoptosis rates of control group,model group and low,medium,high dose experimental groups were(4. 24 ± 0. 64) %,(18. 13 ± 1. 53) %,(14. 93 ± 0. 52) %,(11. 19 ± 0. 74) % and(7. 67 ± 1. 19) %,respectively. Compared with control group,the expression levels of apoptotic protein B-cell lymphoma/leukemia-2(Bcl-2),extracellular matrix degrading protein COL2A1 and ACAN protein in model group were decreased. The levels of Bcl-2 associated X protein(Bax),Cl-Caspase-3 protein,matrix metalloproteinase13(MMP13) and ADAMTS5 protein were increased(all P < 0. 05). Compared with model group,the protein expression levels of apoptosis protein Bcl-2,extracellular matrix degradation protein COL2A1 and ACAN were increased in medium,high dose experimental groups,while the protein levels of Bax and Cl-Caspase-3,MMP13and ADAMTS5 were decreased(all P < 0. 05). Conclusion Ropivacaine has a protective effect on IL-1β induced chondrocyte injury based on AMPK/m TOR pathway.