补肾活血方调节ADSCs-Exos对椎间盘退变髓核细胞凋亡及ERK信号通路的影响

Bushen Huoxue Decoction regulates ADSCs-Exos to affect nucleus pulposus cell apoptosis and ERK signaling pathway in intervertebral disc degeneration

研究补肾活血方调节脂肪间充质干细胞(adipose-derived stem cells,ADSCs)-外泌体(exosome,Exos)对椎间盘髓核细胞凋亡及细胞外信号调节激酶(extracellular signal-regulated kinase,ERK)信号通路的影响。将叔丁基过氧化氢(tert-butyl hydrogen peroxide,TBHP)诱导髓核细胞(nucleus pulposus cells,NPCs)分为正常组、模型组、含药血清组、空白外泌体组、正常血清外泌体组及含药血清外泌体组。使用CCK-8检测细胞活力,EdU染色法检测细胞增殖,流式细胞术检测细胞周期与凋亡,酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)法检测白细胞介素-1β(interleukin-1β,IL-1β)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和白细胞介素-6(interleukin-6,IL-6)等炎症因子的表达,qRT-PCR法检测蛋白聚糖(aggrecan)、Ⅱ型胶原基因(collagen typeⅡalpha 1 chain,COL2A1)和ERK mRNA表达,Western blot法检测aggrecan、COL2A1、p-ERK蛋白表达。结果显示,与模型组比较,单独给予含药血清、空白外泌体及正常血清外泌体处理后,3组NPCs活性和增殖上升,G_(0)/G_(1)期细胞比例显著降低,S期细胞比例显著增加,凋亡减少,IL-1β、TNF-α和IL-6水平下降,aggrecan、COL2A1 mRNA及蛋白表达水平上升,ERK mRNA及p-ERK蛋白表达水平下降;与含药血清组、空白外泌体组及正常血清外泌体组比较,含药血清外泌体处理后,NPCs活性和增殖进一步上升,G_(0)/G_(1)期细胞比例进一步降低,S期细胞比例进一步增加,凋亡进一步减少,IL-1β、TNF-α和IL-6水平进一步下降,aggrecan、COL2A1 mRNA及蛋白水平显著上升,ERK mRNA及p-ERK蛋白表达水平显著下降。结论证实,补肾活血方含药血清处理后ADSCs分泌的Exos能够促进退变NPCs的增殖,抑制凋亡和炎症因子的表达,促进蛋白多糖、胶原生成,进而延缓椎间盘退变,其机制与调节ERK信号通路有关。

This study aims to investigate the effects of Bushen Huoxue Decoction regulating adipose-derived stem cells(ADSCs)-exosomes(Exos)on the apoptosis of intervertebral disc nucleus pulposus cells(NPCs)and extracellular signal-regulated kinase(ERK)signaling pathway.Tert-butyl hydrogen peroxide(TBHP)-induced NPCs were divided into control,model,drug-containing serum,blank Exos,normal serum Exos,and drug-containing serum Exos groups.Cell viability and proliferation were examined by the CCK-8 assay and EdU staining,respectively.The cell cycle and apoptosis were evaluated by flow cytometry.Enzyme-linked immunosorbent assay was employed to measure the levels of interleukin(IL)-1β,tumor necrosis factor(TNF)-α,and IL-6.The mRNA levels of aggrecan,collagen typeⅡalpha 1 chain(COL2A1),and ERK were determined by qRT-PCR,and the protein levels of aggrecan,COL2A1,and p-ERK were determined by Western blot.The results showed that compared with the model group,the treatments with drug-containing serum,blank Exos,and normal serum Exos enhanced the viability and proliferation of NPCs,decreased the proportion of cells in the G_(0)/G_(1)phase,increased the proportion of cells in the S phase,reduced apoptosis,lowered the levels of IL-1β,TNF-α,and IL-6,up-regulated the mRNA and protein levels of aggrecan and COL2A1,and down-regulated the mRNA level of ERK and the protein level of p-ERK.Compared with the drug-containing serum,blank Exos,and normal serum Exos groups,the treatment with drug-containing serum Exos enhanced the viability and proliferation of NPCs,decreased the proportion of cells in the G_(0)/G_(1)phase,increased the cells in the S phase,reduced apoptosis,lowered the levels of IL-1β,TNF-α,and IL-6,up-regulated the mRNA and protein levels of aggrecan and COL2A1,and down-regulated the mRNA level of ERK and the protein level of p-ERK.The results confirmed that the Exos secreted by ADSCs after treatment with Bushen Huoxue Decoction-containing serum promoted the proliferation of degenerated NPCs,inhibited apoptosis and the expression of inflammatory mediators,and promoted the production of proteoglycans and collagen,thus delaying the progression of intervertebral disc degeneration,the mechanism of which was related to the regulation of the ERK signaling pathway.