骨炎消方通过PI3K/Akt/mTOR信号通路对骨关节炎细胞自噬的影响

Effect of Guyanxiao prescription on autophagy of osteoarthritis cells through PI3K/Akt/mTOR

摘要:

目的 通过PI3K/Akt/mTOR信号通路探究骨炎消方对骨关节细胞自噬调节的作用机制。方法 构建软骨细胞炎症模型，将细胞随机分为6组：空白组（兔软骨细胞+完全培养基）、模型组（兔软骨细胞+10 μg/mL LPS培养基）、阳性组（兔软骨细胞+LPS+D-氨基葡萄糖150 μmol/L）、骨炎消低剂量组（兔软骨细胞+ LPS+1 μg/mL骨炎消）、骨炎消中剂量组（兔软骨细胞+LPS+10 μg/mL骨炎消）、骨炎消高剂量组（兔软骨细胞+ LPS+100 μg/mL骨炎消）。CCK-8法检测细胞增殖；酶联免疫吸附试验检测细胞上清液中自噬因子ATG5、ATG12水平；实时荧光定量聚合酶链反应检测细胞PI3K、Akt、mTOR基因相对表达量；Western blotting检测PI3K、Akt、mTOR、Beclin-1蛋白相对表达量。结果 空白组、模型组及不同浓度组软骨细胞存活率比较，差异有统计学意义（P <0.05）。与空白组比较，模型组、阳性组及骨炎消高、中、低组软骨细胞ATG5和ATG12水平均降低（P <0.05）；与模型组比较，阳性组和骨炎消高、中、低组软骨细胞ATG5和ATG12水平均升高（P <0.05）。与模型组相比，骨炎消高、中、低组软骨细胞中PI3K、Akt、mTOR mRNA相对表达量均降低（P <0.05）。与空白组相比，模型组软骨细胞中PI3K、Akt、mTOR蛋白相对表达量均升高（P <0.05），Beclin-1蛋白相对表达量降低（P <0.05）；与模型组比较，阳性组和骨炎消高、中剂量组的PI3K、Akt、mTOR蛋白相对表达降低（P <0.05），阳性组和骨炎消高、中、低组Beclin-1蛋白相对表达量升高（P <0.05）。结论 骨炎消方可有效防治骨关节炎，其作用机制可能是通过抑制PI3K/Akt/mTOR信号通路，引起自噬在一定程度上调，促进软骨细胞增殖分化，从而减缓骨关节炎的进展。

Abstract:

Objective To investigate the mechanism of action of Guyanxiao Fang on the regulation of autophagy in bone and joint cells through the PI3K/Akt/mTOR signaling pathway.Methods A chondrocyte inflammation model was constructed, and cells were randomly divided into 6 groups: blank group (rabbit chondrocytes + complete culture medium), model group (rabbit chondrocytes + 10 μg/mL LPS culture medium), positive group (rabbit chondrocytes + LPS + 150 μmol/L D-glucosamine), low-dose Guyanxiao group (rabbit chondrocytes + LPS + 1 μg/mL Guyanxiao), medium-dose Guyanxiao group (rabbit chondrocytes + LPS + 10 μg/mL Guyanxiao), and high-dose Guyanxiao group (rabbit chondrocytes + LPS + 100 μg/mL Guyanxiao). Cell proliferation was detected by the CCK-8 method; the levels of autophagy factors ATG5 and ATG12 in cell supernatants were detected by enzyme-linked immunosorbent assay; the relative expression levels of PI3K, Akt, and mTOR genes in cells were detected by real-time fluorescence quantitative polymerase chain reaction; Western blotting was used to detect the relative expression levels of PI3K, Akt, mTOR, and Beclin-1 proteins.Results The differences in cell viability among the blank group, model group, and different concentration groups were statistically significant (P < 0.05). Compared with the blank group, the levels of ATG5 and ATG12 in chondrocytes of the model group, positive group, and high, medium, and low-dose Guyanxiao groups were all decreased (P < 0.05); compared with the model group, the levels of ATG5 and ATG12 in chondrocytes of the positive group and high, medium, and low-dose Guyanxiao groups were all increased (P < 0.05). Compared with the model group, the relative expression levels of PI3K, Akt, and mTOR mRNA in chondrocytes of the high, medium, and low-dose Guyanxiao groups were all decreased (P < 0.05). Compared with the blank group, the relative expression levels of PI3K, Akt, and mTOR proteins in chondrocytes of the model group were all increased (P < 0.05), while the relative expression level of Beclin-1 protein was decreased (P < 0.05); compared with the model group, the relative expression levels of PI3K, Akt, and mTOR proteins in chondrocytes of the positive group and high, medium-dose Guyanxiao groups were decreased (P < 0.05), and the relative expression levels of Beclin-1 protein in chondrocytes of the positive group and high, medium, and low-dose Guyanxiao groups were increased (P < 0.05).Conclusion Guyanxiao Fang can effectively prevent and treat osteoarthritis. Its mechanism of action may be through inhibiting the PI3K/Akt/mTOR signaling pathway, causing autophagy to be regulated to a certain extent, promoting chondrocyte proliferation and differentiation, thereby slowing down the development of osteoarthritis.