摘要
目的探讨圣愈汤对白细胞介素-1β(Interleukin-1β,IL-1β)处理髓核细胞凋亡及炎症反应的影响及其潜在作用机制。方法将25只雄性SD大鼠分别给予生理盐水,低、中、高剂量圣愈汤(9 g/kg、18 g/kg和36 g/kg)以及阳性药物(尼美舒利分散片,0.18 mg/kg)灌胃,连续给药14 d,收集血清。(1)分离培养原代髓核细胞,10 ng/ml的IL-1β处理构建髓核细胞退变模型。圣愈汤含药血清和阳性药物含药血清处理髓核细胞24 h,细胞计数试剂盒(CCK-8)检测细胞活力,末端标记法(Terminal-deoxynucleotidyl transferase mediated nick end labeling,TUNEL)和比色法分别检测细胞凋亡和胱天蛋白酶3(caspase-3)活性,酶联免疫吸附实验(Enzyme-linked immunosorbent assay,ELISA)检测IL-6和肿瘤坏死因子α(Tumor necrosis factorα,TNF-α)水平。免疫印迹法检测Toll样受体4(Toll-like receptor 4,TLR4)、核转录因子κB(Nuclear factor kappa B,NF-κB)。(2)将细胞分为对照组、模型组(经10 ng/ml的IL-1β处理)、圣愈汤含药血清高剂量组(经10 ng/ml的IL-1β和高剂量圣愈汤含药血清处理)、Kdo2脂质(KLA)组(经TLR4激动剂KLA、IL-1β和高剂量圣愈汤含药血清处理)。免疫印迹检测TLR4、NF-κB和cleaved caspase-3蛋白表达,免疫荧光检测TNF-α和IL-6表达。结果与对照组比较,模型组髓核细胞活力降低,髓核细胞凋亡率、Caspase-3活性以及TNF-α和IL-6水平升高,差异有统计学意义(P<0.05,P<0.01)。模型组髓核细胞中TLR4和NF-κB蛋白水平均较对照组明显升高,差异有统计学意义(P<0.01)。与模型组比较,各剂量圣愈汤含药血清组和阳性药物组髓核细胞活力升高,细胞凋亡率和Caspase-3活性降低,TNF-α和IL-6水平减少,TLR4与NF-κB蛋白水平降低,差异有统计学意义(P<0.01)。KLA有效减弱圣愈汤含药血清对IL-1β处理髓核细胞TLR4、NF-κB、cleaved caspase-3、TNF-α和IL-1β蛋白表达的抑制作用,差异有统计学意义(P<0.01)。结论圣愈汤能够抑制退
Objective To study the effect of Shengyu Decoction on apoptosis and inflammatory response of interleukin-1β(IL-1β)-treated nucleus pulposus cells and its potential mechanism of action.Methods 25 SD male rats were given saline,Shengyu Decoction in low,medium,and high doses(9,18,and 36 g/kg),and positive drug(nimesulide dispersible tablets,0.18 mg/kg)by gavage separately,and serum was collected after 14 days of continuous administration.①Primary nucleus pulposus cells were isolated and cultured,and a nucleus pulposus cells degeneration model was established by treatment with 10 ng/mL IL-1β.Nucleus pulposus cells were treated with Shengyu Decoction-containing serum and positive drug-containing serum for 24 h.CCK-8 was used to detect cell viability,and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL)and colorimetric assay were employed for detecting apoptosis and caspase-3 activity,respectively.Enzyme-linked immunosorbent assay(ELISA)was performed to detect IL-6 and tumor necrosis factorα(TNF-α)levels.Toll-like receptor 4(TLR4)and nuclear factor kappa B(NF-κB)were detected by Western blot(WB).②The cells were divided into a control group,a model group(treated with 10 ng/mL IL-1β),a high-dose group of Shengyu Decoction-containing serum(treated with 10 ng/mL IL-1βand high-dose Shengyu Decoction-containing serum),and a KLA group(treated with the TLR4 agonist KLA,IL-1β,and high-dose Shengyu Decoction-containing serum).TLR4,NF-κB,and cleaved caspase-3 protein expression was detected by WB,and TNF-αand IL-6 expression was detected by immunofluorescence(IF).Results Compared to the control group,the model group showed reduced viability of nucleus pulposus cells,elevated apoptosis rates,caspase-3 activity,and levels of TNF-αand IL-6 of nucleus pulposus cells with significant differences(P<0.05,P<0.01).TLR4 and NF-κB protein levels in nucleus pulposus cells were significantly higher in the model group than in the control group(P<0.01).Compared with the model group,the low-,medium-,an
作者
郭闯豪
张诚
刘汝银
岳宗进
贾鑫瑜
GUO Chuang-hao;ZHANG Cheng;LIU Ru-yin;YUE Zong-jin;JIA Xin-yu(Henan University of Chinese Medicine,Zhengzhou Henan 450046;Henan Province Hospital of TCM,Zhengzhou Henan 450002)
出处
《世界中西医结合杂志》
2024年第10期1987-1993,2016,共8页
World Journal of Integrated Traditional and Western Medicine
基金
河南省中医药科学研究专项课题(2021JDZX2074,2022ZY1092,2024ZY2074)。
