【摘要】 目的：观察独活寄生汤通过p38分裂原活化蛋白激酶（p38 MAPK）/组蛋白去乙酰基酶4（HDAC4）通路对髓核（NP）细胞增殖凋亡的影响，探讨潜在作用机制。方法：制备独活寄生汤含药血清及从轻度退化的椎间盘组织中分离培养NP细胞，采用白细胞介素（IL）-1β诱导构建NP细胞损伤模型。CCK8法筛选独活寄生汤含药血清最佳作用浓度及时间，采用CCK8和流式细胞术观察对照组，空白血清组，中药血清低、中、高剂量组NP细胞增殖和凋亡的影响；药效实验研究，将NP细胞分为对照组、IL-1β组、中药血清组、IL-1β+中药血清组；Rescue实验，将NP细胞分为对照组、IL-1β组、IL-1β+中药血清组、IL-1β+中药血清+Scriptaid组，采用流式细胞术检测NP细胞的周期和凋亡，蛋白质印迹法（WB）检测p38 MAPK、p-p38 MAPK、HDAC4、增殖细胞核抗原（PCNA）、肿瘤坏死因子（TNF）-α、IL-6、Ⅱ型胶原蛋白（ColⅡ）、基质金属蛋白酶（MMP）-3、含凝血酶敏感蛋白基序的解整合素金属蛋白酶（ADAMTS）-5蛋白表达，RT-PCR检测TNF-α、IL-6、MMP-3、ADAMTS-5mRNA的表达，免疫荧光法检测ColⅡ和PCNA的荧光表达。结果：IL-1β激活p38 MAPK会降低HDAC4、CollagenⅡ和PCNA蛋白表达（P<0.01），上调TNF-α、IL-6、MMP-3和ADAMTS-5 mRNA及蛋白表达水平（P<0.01），延迟更多细胞在G₀/G₁期（P<0.01）；然而独活寄生汤含药血清上调HDAC4、ColⅡ和PCNA蛋白表达（P<0.01），降低TNF-α、IL-6、MMP-3和ADAMTS-5 mRNA及蛋白表达水平（P<0.01），诱导细胞进入S期（P<0.01）；而Rescue实验阻断HDAC4的表达ColⅡ和PCNA免疫荧光表达再次降低（P<0.01）,TNF-α、IL-6、MMP-3和ADAMTS-5 mRNA表达再次升高（P<0.05）,S期细胞再次减少（P<0.01）。结论：独活寄生汤含药血清可通过促进NP细胞增殖和p38 MAPK/HDAC4抑制炎症通路，预防NP细胞凋亡。更多还原

【Abstract】 Objective:To observe the effects of Duhuo Jisheng Decoction（独活寄生汤） on proliferation and apoptosis in nucleus pulposus（NP） cells via the p38 mitogen-activated protein kinase（p38 MAPK）/histone deacetylase 4（HDAC4） signaling pathway,and to explore its potential mechanism. Methods:Drug-containing serum of Duhuo Jisheng Decoction was prepared,and NP cells were isolated and cultured from mildly degenerated intervertebral disc tissues. The NP cell injury model was established by induction with interleukin（IL）-1 β. The CCK-8 assay was used to screen the optimal concentration and duration of action for the drug-containing serum. The CCK-8 and flow cytometry were applied to observe the effects on NP cell proliferation and apoptosis in the control group,blank serum group,and low-,medium-,and high-dose Chinese medicine serum groups. For pharmacodynamic experiments,NP cells were divided into the control group,IL-1β group,Chinese medicine serum group,and IL-1β + Chinese medicine serum group. For rescue experiments,NP cells were divided into the control group,IL-1β group,IL-1β + Chinese medicine serum group,and IL-1β + Chinese medicine serum + Scriptaid group. Flow cytometry was used to detect NP cell cycle and apoptosis. Western blotting（WB） was performed to measure the protein expression of p38 MAPK,p-p38 MAPK,HDAC4,proliferating cell nuclear antigen（PCNA）,tumor necrosis factor（TNF）-α,IL-6,collagen type Ⅱ（Col Ⅱ）,matrix metalloproteinase（MMP）-3,and a disintegrin and metalloproteinase with thrombospondin motifs（ADAMTS）-5. RT-PCR was used to detect the mRNA expression of TNF-α,IL-6,MMP-3,and ADAMTS-5. Immunofluorescence was used to detect the fluorescent expression of Col Ⅱ and PCNA. Results:IL-1β-induced activation of p38 MAPK down-regulated the protein expression of HDAC4,Col Ⅱ,and PCNA（P<0.01）,up-regulated the mRNA and protein levels of TNF-α,IL-6,MMP-3,and ADAMTS-5（P<0.01）,and arrested more cells in the G₀/G₁ phase（P<0.01）. However,drug-containing serum of Duhuo Jisheng Decoction up-regulated the protein expression of HDAC4,Col Ⅱ,and PCNA（P<0.01）,downregulated the mRNA and protein levels of TNF-α,IL-6,MMP-3,and ADAMTS-5（P<0.01）,and promoted cell entry into the S phase（P<0.01）. In rescue experiments shaved blocking HDAC4 expression decreased the immunofluorescence expression of Col Ⅱ,and PCNA again（P<0.01）,increased the mRNA expression of TNF-α,IL-6,MMP-3,and ADAMTS-5（P<0.05）,and reduced the proportion of cells in sphase（P<0.01）. Conclusions:Drug-containing serum of Duhuo Jisheng Decoction can prevent NP cell apoptosis by promoting cell proliferation and suppressing inflammation via p38 MAPK/HDAC4 pathway.更多还原