雷帕霉素-mTOR途径诱导自噬对抑制软骨细胞凋亡的影响

Effect of rapamycin-mTOR pathway-induced autophagy on inhibition of chondrocyte apoptosis

目的探讨雷帕霉素(rapamycin, RAPA)诱导软骨细胞自噬抑制骨关节炎(osteoarthritis, OA)进展的可能性。方法体外分离培养软骨细胞,应用地塞米松(dexamethasone, DEX)诱导凋亡,然后用RAPA干预。将正常软骨细胞和OA软骨细胞分别分为3组:BSA组、DEX-BSA组、RAPA+DEX-BSA组。MTT法检测软骨细胞活性,GFP-RFP-LC3腺病毒、Western-Blot和实时荧光定量PCR(qRT-PCR)法检测自噬流水平和自噬相关蛋白LC3、Beclin1和ULK1蛋白及基因表达。qRT-PCR和ELISA法检测IL-1、TNF-α浓度。Annexin V-FITC/PI法及TUNEL法检测凋亡。结果 Western-Blot显示无论正常和OA软骨细胞,与DEX-BSA组比较,RAPA+DEX-BSA组总LC3B、LC3B-Ⅱ、Beclin1和ULK1表达显著增加[正常软骨细胞DEX-BSA组分别为(1.477±0.0297)、(0.727±0.0231)、(0.240±0.0063)、(0.261±0.008),正常软骨细胞RAPA+DEX-BSA组分别为(3.648±0.0408)、(2.020±0.012)、(0.900±0.0284)、(0.604±0.0119)(P<0.01);OA软骨细胞DEX-BSA组分别为(3.085±0.081)、(1.637±0.0503)、(0.586±0.0033)、(0.446±0.0188),OA软骨细胞RAPA+DEX-BSA组分别为(5.579±0.1920)、(3.17±0.0844)、(0.970±0.0245)、(0.650±0.01),P<0.01]。qRT-PCR显示正常和OA软骨细胞,与DEX-BSA组比较,RAPA+DEX-BSA组LC3表达显著增高[正常软骨细胞DEX-BSA组(1.00±0.050),正常软骨细胞RAPA+DEX-BSA组(1.74±0.073)(P<0.01);OA软骨细胞DEX-BSA组(2.04±0.098),OA软骨细胞RAPA+DEX-BSA组(2.46±0.12),P<0.01],TNF-α和IL-1表达显著下降[正常软骨细胞DEX-BSA组分别为(1.34±0.074),(1.60±0.099),正常软骨细胞RAPA+DEX-BSA组分别为(1.00±0.028),(1.27±0.042),P<0.01;OA软骨细胞DEX-BSA组分别为(1.54±0.033),(3.08±0.167),OA软骨细胞RAPA+DEX-BSA组分别为(1.34±0.052),(1.05±0.022),P<0.01]。ELISA法显示正常和OA软骨细胞,与DEX-BSA组比较,RAPA+DEX-BSA组TNF-α和IL-1显著下降[正常软骨细胞DEX-BSA组分别为(68.71±1.78)pg/ml,(226.13±5.93)pg/ml,正常软骨细胞RAPA+DEX-BSA组分别为(57.75±1.68)pg/ml,(189.75±4.10)pg/ml,P<0.01;OA软骨细胞DEX-BSA组分别为(95.07±2.25)pg/ml,(260.91±7.61)pg/ml,OA软骨细胞RAPA+DEX-BSA组分别为(79.55±2.48)pg/ml,(214.61±8.96)pg/ml,P<0.01]。Annexin V-FITC/PI法显示,RAPA+DEX-BSA组正常软骨和OA软骨细胞凋亡较DEX-BSA组显著降低[正常软骨细胞RAPA+DEX-BSA组和DEX-BSA组分别为(21.02±0.76)、(26.02±0.864),P<0.01;OA软骨细胞RAPA+DEX-BSA组和DEX-BSA组分别为(27.98±0.811)、(34.96±0.68),P<0.01]。结论 RAPA诱导软骨细胞自噬,通过降低TNF-α和IL-1的表达、抑制软骨细胞凋亡进而减轻DEX对软骨细胞的OA样损伤进程。

Objective To investigate the possibility of rapamycin(RAPA)-induced autophagy in chondrocytes inhibiting the progression of osteoarthritis(OA). Methods Chondrocytes cultured in vitro were induced apoptosis with dexamethasone(DEX), and were divided into BSA, DEX-BSA and RAPA+DEX-BSA groups respectively. The activity was detected by MTT. Autophagy levels were detected by GFP-RFP-LC3 adenovirus, Western blot, and qRT-PCR. The concentrations of IL-1 and TNF-α were measured by qRT-PCR and ELISA. Apoptosis was detected by Annexin V-FITC/PI and TUNEL. Results In normal or OA chondrocytes, Western blot showed that the total LC3 B, LC3 B-Ⅱ, Beclin1, and ULK1 expression in the RAPA+DEX-BSA group were higher than those in the DEX-BSA group[The normal chondrocytes in the DEX-BSA group respectively were(1.477±0.0297),(0.727±0.0231),(0.240±0.0063),(0.261±0.008). The normal chondrocytes in the RAPA+DEX-BSA group respectively were(3.648±0.0408),(2.020±0.012),(0.900±0.0284),(0.604±0.0119),P<0.01;The OA chondrocytes in the DEX-BSA group respectively were(3.085±0.081),(1.637±0.0503),(0.586±0.0033),(0.446±0.0188). The OA chondrocytes in the RAPA+DEX-BSA group respectively were(5.579±0.1920),(3.17±0.0844),(0.970±0.0245),(0.650±0.01),P<0.01]. qRT-PCR showed increased expression of LC3 in RAPA+DEX-BSA group compared with DEX-BSA[The normal chondrocytes in the DEX-BSA group(1.00±0.050). The normal chondrocytes in the RAPA+DEX-BSA group(1.74±0.073),P<0.01. The OA chondrocytes in the DEX-BSA group(2.04±0.098). The OA chondrocytes in theRAPA+DEX-BSA group(2.46±0.12),P<0.01], TNF-α and IL-1 decreased either in normal or OA chondrocytes[The normal chondrocytes in the DEX-BSA group respectively were(1.34±0.074),(1.60±0.099)/The normal chondrocytes in the RAPA+DEX-BSA group respectively were(1.00±0.028),(1.27±0.042),P<0.01. The OA chondrocytes in the DEX-BSA group respectively were(1.54±0.033),(3.08±0.167). The OA chondrocytes in the RAPA+DEX-BSA group respectively were(1.34±0.052),(1.05±0.022),P<0.01]. ELISA showed that TNF-α and IL-1 decreased in the RAPA+DEX-BSA group compared with DEX-BSA[The normal chondrocytes in the DEX-BSA group(68.71±1.78, 226.13±5.93)pg/ml. The normal chondrocytes in the RAPA+DEX-BSA group(57.75±1.68, 189.75±4.10)pg/ml, P<0.01;The OA chondrocytes in the DEX-BSA group(95.07±2.25, 260.91±7.61)pg/ml. The OA chondrocytes in theRAPA+DEX-BSA group(79.55±2.48, 214.61±8.96)pg/ml,P<0.01], regardless of normal or OA chondrocytes. Annexin V-FITC/PI showed that the apoptotic of the RAPA+DEX-BSA group was lower in the normal and OA chondrocytes than in the DEX-BSA group[The normal chondrocytes in RAPA+DEX-BSA and DEX-BSA group respectively were(21.02±0.76),(26.02±0.864),P<0.01. The OA chondrocytes in RAPA+DEX-BSA and DEX-BSA group respectively were(27.98±0.811),(34.96±0.68),P<0.01]. Conclusions RAPA induced autophagy in chondrocytes, which not only inhibiting the expression of TNF-α and IL-1, but also inhibiting the apoptotic and the OA-like damage of DEX on chondrocytes.