摘要
炎性小体(inflammasome)作为炎症反应的中心环节,与包括肿瘤在内的很多疾病密切相关。半胱氨酸天冬氨酸蛋白酶-1(cysteinyl aspartate specific proteinase-1,Caspase-1)常以炎性小体为活化支架,促进白细胞介素-1β(interleukin-1β,IL-1β)等炎症因子的成熟与释放。前期证实,健脾益气方(Jianpi Yiqi decoction,JYD)可通过调节肝癌细胞中的Caspase-1水平来抑制肝癌细胞的增殖与侵袭,并诱导部分肝癌细胞凋亡,但该方是否通过调节Caspase-1而影响炎性小体活化及其相关炎症因子的表达尚不清楚。本研究基于核苷酸结合寡聚化结构域样受体蛋白3(NOD-like receptor protein 3,NLRP3)炎性小体活化通路,用低、中和高剂量的健脾益气方(5.25、10.5、21.0 g/kg)分别干预经二乙基亚硝胺(N-nitrosodiethylamine,DEN)诱导的肝癌大鼠,同时以Caspase-1抑制剂(VX-765)为阳性对照,采用HE染色观察大鼠肝组织病理变化,用比色法测定大鼠血清谷丙转氨酶(alanine aminotransferase,ALT)、谷草转氨酶(aspartate aminotransferase,AST)、谷氨酰转移酶(gamma-glutamyl transpeptidase,GGT)和α-L-岩藻糖苷酶(alpha-L-fucosidase,AFU)及肝组织Caspase-1酶活性;用酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测大鼠血清白细胞介素-1β(interleukin-1β,IL-1β)、白细胞介素-18(interleukin-18,IL-18)、核因子κB蛋白(nuclear factor kappa-light-chain-enhancer of activated B cells,NF-κB)、高迁移率族蛋白B1(high mobility group box 1,HMGB-1)、钙结合蛋白(calprotectin)S100A8和S100A9的水平;用蛋白免疫印迹法(Western blot)检测大鼠肝组织NLRP3、凋亡相关斑点样蛋白(apoptosis-associated speck-like protein containing CARD,ASC)和Caspase-1蛋白前体(pro-Caspase-1)表达水平。结果显示:与正常组相比,模型组大鼠肝组织出现众多大小不一的灰白色癌变结节、大量炎性细胞浸润和病理性核分裂,血清ALT、AST、GGT、AFU、IL-1β、IL-18、NF-κB、HMGB-1、S100A8和S100A9的水平,肝组织NLRP3、ASC和pro-Caspase-1蛋白水平及Caspase-1酶活性均明显上调(P<0.01);与模型组相比,中、高剂量的健脾益气方和VX-765的干预均可明显减轻大鼠肝组织相关病理变化、降低上述指标的表达水平(P<0.05或P<0.01);健脾益气方高剂量组与阳性对照组(VX-765)相比,血清ALT、AST、GGT、AFU、NF-κB、HMGB-1、S100A8、S100A9水平和肝组织ASC蛋白水平的组间差异均不具有统计学意义(P>0.05)。以上结果表明,健脾益气方可通过抑制Caspase-1使NLRP3炎性小体的激活及相关炎症因子的表达下调,具有抑制肝癌大鼠肝功能恶化和炎症程度加剧的作用。
As the central link of inflammatory response,inflammasome is closely related to many diseases including tumor.The activation of cysteinyl aspartate specific proteinase-1(Caspase-1)depends on the inflammasome,and then can promote the maturation and release of inflammatory factors such as interleukin-1β(IL-1β).Our previous study has confirmed that Jianpi Yiqi decoction(JYD)can inhibit the proliferation and invasion of hepatic carcinoma(HCC)cells and induce apoptosis of some HCC cells by regulating the level of Caspase-1 protein in HCC cells.However,whether this decoction can inhibit the activation of inflammasome and the expression of related inflammatory factors by down-regulating Caspase-1 remains unclear.In this study,based on the NOD-like receptor protein 3(NLRP3)inflammasome activation pathway,JYD was administrated to the diethylnitrosamine(DEN)-induced liver cancer rats at low,medium and high doses(5.25,10.5,21.0 g/kg)respectively.Caspase-1 inhibitor(VX-765)was used as positive control.Hematoxylin-eosin(HE)was used to detect the pathological changes of liver tissues in rats.The colorimetric method was used to mea-sure the enzyme activity of alanine aminotransferase(ALT),aspartate aminotransferase(AST),gamma-glutamyl transpep-tidase(GGT)and alpha-L-fucosidase(AFU)in serum as well as Caspase-1 in liver.Enzyme linked immunosorbent assay(ELISA)was used to detect the level of serum interleukin-1β(IL-1β),interleukin-18(IL-18),nuclear factor kappa-light-chain-enhancer of activated B cells(NF-κB),high mobility group box 1(HMGB-1),Calprotectin S100 A8 and S100 A9.Western blot was used to detect the expression of NLRP3,apoptosis-associated speck-like protein containing CARD(ASC),and pro-Caspase-1 in liver.As the results,compared with the control group,there were multiple gray-white cancerous nodules,infiltration of inflammatory cells and abnormal mitosis in the liver tissues of the model rats.Meanwhile,the level of serum ALT,AST,GGT,AFU,IL-1β,IL-18,NF-κB,HMGB-1,S100 A8 and S100 A9,the expression level of NLRP3,ASC and pro-Caspase-1 in liver as well as the enzyme activity of Caspase-1 in liver were significantly increased(P<0.01)in the model rats.Compare to the model group,the medium-dose,high-dose of JYD and VX-765 treatments could significantly lessen the pathological changes of the liver tissue,decline the serum level and expression of the above corresponding molecules(P<0.05 or P<0.01).There were no significant differences between the high-dose of JYD group(HJYD)and the VX-765 group in the level of serum ALT,AST,GGT,AFU,NF-κB,HMGB-1,S100 A8 and S100 A9 as well as the expression level of ASC in liver(P>0.05).In summary,JYD can ameliorate the severity of the liver injury and inflammation in rat of DEN-induced liver cancer via down-regulating the activation of NLRP3 inflammasome as the result of the Caspase-1 inhibition.
作者
乐宁
杨琪
音金萍
岳紫晨
王超
卓少元
Le Ning;Yang Qi;Yin Jinping;Yue Zichen;Wang Chao;Zhuo Shaoyuan(School of Basic Medical Sciences,Guangxi University of Chinese Medicine,Nanning,530200)
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2022年第6期1350-1359,共10页
Genomics and Applied Biology
基金
国家自然科学基金地区科学基金项目(81660775、81860824)
广西中医药大学“桂派杏林拔尖人才”项目(2022C004)共同资助
作者简介
通信作者:卓少元,zhuosy@gxtcmu.edu.cn
