摘要
目的:探究四君子汤通过调控肝癌癌旁组织核因子-κB p65(NF-κB p65)的O-连接的β-N-乙酰葡糖胺(O-Glc NAc)糖基化修饰,干预肝癌发展的分子机制。方法:建立原位肝癌小鼠模型,将24只C57BL/6小鼠,随机分为4组,每组6只,分别为正常组、模型组、四君子汤低剂量组(10 g·kg^(-1))、四君子汤高剂量组(25 g·kg^(-1)),通过蛋白免疫印迹法(Western blot)检测肝癌癌旁组织中O-Glc NAc糖基化水平和p65的磷酸化修饰水平;免疫沉淀法(IP)检测p65的O-Glc NAc糖基化修饰水平;实时荧光定量聚合酶链式反应(Real-time PCR)检测癌旁组织中白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、转化生长因子-β_(1)(TGF-β_(1))、血管内皮生长因子A(VEGFA)、基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)m RNA表达水平;观察各组小鼠肿瘤数量,称量肝脏质量;检测各组小鼠的旷场运动情况和抓力,分析小鼠的气盛衰度。结果:与正常组小鼠肝组织比较,模型组小鼠癌旁组织中O-Glc NAc糖基化水平显著下降(P<0.01);p65的O-Glc NAc糖基化修饰水平显著下降(P<0.01);p65的磷酸化水平显著升高(P<0.01);IL-6、TNF-α、TGF-β_(1)、VEGFA、MMP-2、MMP-9 m RNA水平显著升高(P<0.01);肝脏质量显著升高(P<0.01);小鼠抓力、旷场水平跨格数和垂直站立次数显著下降(P<0.01),气盛衰度显著下降(P<0.01)。与模型组小鼠比较,四君子汤低剂量组和四君子汤高剂量组小鼠癌旁组织中O-Glc NAc糖基化水平明显升高(P<0.05,P<0.01);p65的O-Glc NAc糖基化修饰水平明显上调(P<0.05,P<0.01);p65的磷酸化水平显著下降(P<0.01);四君子汤低剂量组IL-6、TGF-β_(1)、VEGFA m RNA水平明显降低(P<0.05,P<0.01);四君子汤高剂量组中IL-6、TNF-α、TGF-β_(1)、VEGFA、MMP-2、MMP-9 m RNA水平显著降低(P<0.01),直径>3 mm肿瘤数量显著减少(P<0.01),小鼠肝脏质量明显降低(P<0.05),小鼠抓力、旷场水平跨格数和垂直站立次数明显升高(P<0.05,P<0.01),气盛衰度显著升高(P<0.01)。结论:四君子汤可以抑制小鼠原位肝癌的进展,该作用可能是通过增加肝癌癌旁组织中O-Glc NAc糖基化水平,增加癌旁组织中p65的O-Glc NAc糖基化修饰,抑制p65的磷酸化修饰,最终抑制下游IL-6、TNF-α、TGF-β_(1)、VEGFA、MMP-2、MMP-9的表达而实现。
Objective:To investigate the molecular mechanism by which Si Junzitang in intervening in the development of hepatocellular carcinoma(HCC)by regulating the O-linkedβ-N-acetylglucosamine modification(O-GlcNAcylation)of nuclear factor kappa-B(NF-κB)p65 in the paracancerous tissues.Method:The orthotopic liver cancer mouse model was established.Twenty-four C57BL/6 mice were randomly divided into four groups:Normal group,model group,Si Junzitang low-dose group(10 g·kg^(-1)),and Si Junzitang high-dose group(25 g·kg^(-1)),with 6 mice in each group.The O-GlcNAcylation level and phosphorylation modification level of p65 in the paracancerous tissues were detected using Western blot.The O-GlcNAcylation of p65 was assessed using immunoprecipitation(IP).The mRNA expression of interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),transforming growth factor-β_(1)(TGF-β_(1)),vascular endothelial growth factor A(VEGFA),matrix metalloproteinase-2(MMP-2),matrix metalloproteinase-9(MMP-9)in the paracancerous tissues was detected using real-time quantitative polymerase chain reaction(Real-time PCR).The tumor number,liver weight,locomotor activity,grip strength,and Qi status of the mice were observed and analyzed.Result:Compared with the normal group,the model group showed a significant decrease in O-GlcNAcylation in the paracancerous tissues(P<0.01),a significant decrease in p65 O-GlcNAcylation(P<0.01),a significant increase in p65 phosphorylation(P<0.01),significantly elevated mRNA levels of cytokines IL-6,TNF-α,TGF-β_(1),VEGFA,MMP-2,and MMP-9(P<0.01),significantly increased liver weight(P<0.01),significantly declined grip strength,number of grid crossings,and number of vertical stand-ups(P<0.01),and significantly dwindled Qi status(P<0.01).Compared with model group,the Si Junzitang low-dose and high-dose groups showed significantly increased levels of O-GlcNAcylation in the paracancerous tissues(P<0.05,P<0.01),significantly upregulated p65 O-GlcNAcylation levels(P<0.05,P<0.01),and significantly decreased p65 phosphorylation levels(P<0.01).In the Si Junzitang low-dose group,the mRNA levels of IL-6,TGF-β_(1),and VEGFA significantly decreased(P<0.05,P<0.01).In the Si Junzitang high-dose group,the mRNA levels of IL-6,TNF-α,TGF-β_(1),VEGFA,MMP-2,and MMP-9 significantly decreased(P<0.01),the number of tumors larger than 3 mm in diameter significantly decreased(P<0.01),and liver weight significantly decreased(P<0.05).Additionally,grip strength,number of grid crossings,and number of vertical stand-ups significantly increased(P<0.05,P<0.01),along with a significant increase in qi status(P<0.01).Conclusion:Si Junzitang can inhibit the progression of orthotopic HCC in mice,which may be achieved by increasing the O-GlcNAcylation level in the paracancerous tissues,enhancing the O-GlcNAcylation of p65,inhibiting the phosphorylation modification of p65,and ultimately suppressing the expression of downstream IL-6,TNF-α,TGF-β_(1),VEGFA,MMP-2,and MMP-9.
作者
李铭哲
方肇勤
张伟妃
彭佩克
贾冬威
LI Mingzhe;FANG Zhaoqin;ZHANG Weifei;PENG Peike;JIA Dongwei(School of Traditional Chinese Medicine(TCM),Shanghai University of TCM,Shanghai 201203,China;School of Acupuncture-Moxibustion and Tuina,Shanghai University of TCM,Shanghai 201203,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2024年第15期21-28,共8页
Chinese Journal of Experimental Traditional Medical Formulae
基金
上海中医药大学预算内项目(2021LK016)
国家自然科学基金项目(82004000,81873212)。
