摘要
目的探究异槲皮素(Qct)通过调控巨噬细胞极化和肿瘤细胞自噬对肝癌的抑制作用。方法采用佛波酯和白细胞介素-4诱导人单核细胞株THP-1肿瘤相关巨噬细胞(TAM)极化,分别以5、10、15、20 ng/mL Qct处理诱导后的THP-1细胞。CCK-8法检测细胞活性,流式细胞术检测CD86和CD206表达,qRT-PCR检测诱导型一氧化氮合酶(iNOS)、肿瘤坏死因子-α(TNF-α)、转化生长因子-β(TGF-β)、精氨酸酶-1(Arg-1)的mRNA表达量。将肝癌SMMC-7721细胞随机分为4组并进行相应处理:对照组(细胞培养液)、Qct组(含20 ng/mL Qct细胞培养液)、TAM(TAM培养液上清)、Qct+TAM组(20 ng/mL Qct共培养的TAM的培养液上清)。CCK-8法检测各组细胞活力,Transwell小室评价细胞迁移与侵袭能力,Annexin V-FITC/PI双染法检测细胞凋亡,Western blot检测Beclin-1蛋白、p62泛素化蛋白及微管相关蛋白1轻链3BⅡ型(LC3-Ⅱ)/微管相关蛋白1轻链3Ⅰ型(LC3-Ⅰ)蛋白表达水平,免疫荧光染色检测自噬标志物LC3表达,透射电子显微镜检测自噬体形成。结果与对照组比较,不同浓度Qct处理TAM后其存活率未发生变化(P>0.05),CD86表达及iNOS、TNF-α的mRNA表达量升高(P<0.01),而CD206表达及TGF-β、Arg-1的mRNA表达量降低(P<0.01)。对照组、Qct组和TAM组SMMC-7721细胞存活率,迁移数目与侵袭数目,细胞凋亡率,Beclin-1、p62蛋白表达,LC3-Ⅱ/LC3-Ⅰ比值,自噬体数目差异均无统计学意义(P>0.05);与TAM组比较,Qct+TAM组SMMC-7721细胞存活率下降(P<0.01),迁移数目与侵袭数目减少(P<0.01),细胞凋亡率增加(P<0.01),LC3-Ⅱ/LC3-Ⅰ比值降低(P<0.01),p62蛋白表达增加(P<0.01),Beclin-1蛋白表达下降(P<0.01),细胞内自噬体减少(P<0.01)。结论Qct调控TAM的M1型极化,进而抑制肝癌细胞增殖、迁移与侵袭能力,促进其凋亡,其机制可能与调控细胞自噬相关。
Objective To investigate the inhibitory effects of isoquercetin(Qct)on hepatocellular carcinoma(HCC)through the regulation of macrophage polarization and tumor cell autophagy.Methods Human monocytic cell line THP-1 was induced to polarize into tumor-associated macrophage(TAM)using phorbol ester and interleukin-4.The induced THP-1 cells were then treated with Qct at concentrations of 5,10,15,and 20 ng/mL respectively.Cell viability was assessed by CCK-8 assay,and the expressions of CD86 and CD206 was determined by flow cytometry.The mRNA expression levels of inducible nitric oxide synthase(iNOS),tumor necrosis factor-α(TNF-α),transforming growth factor-β(TGF-β),and arginase-1(Arg-1)were measured by qRT-PCR.Hepatocellular carcinoma SMMC-7721 cells were randomly divided into four groups and treated accordingly:control group(cell culture medium),Qct group(cell culture medium containing 20 ng/mL Qct),TAM group(supernatant from TAM culture),and Qct+TAM group(supernatant from TAM culture co-cultured with 20 ng/mL Qct).Cell viability in each group was evaluated using the CCK-8 assay,while cell migration and invasion capabilities were assessed using Transwell chambers.Apoptosis was examined by Annexin V-FITC/PI double staining,and the protein expression levels of Beclin-1,p62 ubiquitinated protein,and microtubule-associated protein 1 light chain 3B type II(LC3-Ⅱ)/microtubule-associated protein 1 light chain 3 type I(LC3-I)were measured by Western blot.Autophagy marker LC3 expression was checked by immunofluorescence staining,and autophagosome formation was observed using transmission electron microscopy.Results Compared with the control group,the survival rate of TAMs treated with different concentrations of Qct remained unchanged(P>0.05).The expression of CD86 and the relative mRNA expression levels of iNOS and TNF-αincreased(P<0.01),while the expression of CD206 and the relative mRNA expression levels of TGF-βand Arg-1 decreased(P<0.01).No statistically significant differences were observed in the cell survival rate,migration count,invasion count,apoptosis rate,Beclin-1 and p62 protein expressions,LC3-Ⅱ/LC3-I ratio,or autophagosome count of SMMC-7721 cells among the control,Qct,and TAM groups(P>0.05).Compared with the TAM group,the Qct+TAM group exhibited a decrease in SMMC-7721 cell survival rate(P<0.01),a reduction in migration and invasion counts(P<0.01),an increase in apoptosis rate(P<0.01),a decrease in the LC3-Ⅱ/LC3-I ratio(P<0.01),an increase in p62 protein expression(P<0.01),a decrease in Beclin-1 protein expression(P<0.01),and a reduction in intracellular autophagosomes(P<0.01).Conclusion Qct regulates the M1 polarization of TAMs,thereby inhibiting the proliferation,migration,and invasion capabilities of hepatocellular carcinoma cells while promoting their apoptosis.The underlying mechanism may be related to the regulation of cellular autophagy.
作者
龚裕伟
井鑫淼
苏朴承
黄俊达
吕璐璐
GONG Yuwei;JING Xinmiao;SU Pucheng;HUANG Junda;LYU Lulu(Huazhong University of Science and Technology,Wuhan,Hubei 430074,China;Southern Medical University,Guangzhou,Guangdong 510000,China;South China University of Technology,Guangzhou,Guangdong 510000,China;Heyuan Biotechnology(Tianjin)Co.,Ltd.,Tianjin 300384,China)
出处
《湖南中医药大学学报》
2025年第7期1240-1248,共9页
Journal of Hunan University of Chinese Medicine
基金
天津市科技计划项目(24ZYCGSY00420)。
关键词
肝癌
异槲皮素
巨噬细胞极化
肿瘤相关巨噬细胞
自噬
M1/M2极化
hepatocellular carcinoma
isoquercetin
macrophage polarization
tumor-associated macrophage
autophagy
M1/M2 polarization
