Sodium new houttuyfonate inhibits hepatocellular carcinoma progression through USP22-mediated SIRT1 deubiquitination regulating autophagy
ZHANG Xuexi, DONG Changhong, ZI Chen, SUN Xiangjun
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doi: 10.7501/j.issn.0253-2670.2026.04.015
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摘要
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目的 探究新鱼腥草素钠（sodium new houttuyfonate，SNH）通过泛素特异性肽酶22（ubiquitin-specific peptidase 22，USP22）介导的沉默调节蛋白1（silent mating type information regulation 2 homolog 1，SIRT1）去泛素化调控自噬抑制肝细胞癌进展的作用机制。方法 人肝癌HepG2细胞给予SNH或索拉非尼处理24 h后，采用CCK-8法检测细胞活力，流式细胞术检测细胞凋亡，Transwell实验检测细胞侵袭能力。质粒转染诱导USP22过表达，免疫共沉淀（co-immunoprecipitation，Co-IP）验证USP22和SIRT1之间的相互作用，Western blotting检测USP22、SIRT1蛋白表达和SIRT1泛素化水平。使用SIRT1激活剂处理后，通过mRFP-GFP-LC3双荧光标记、三磷酸腺苷（adenosine triphosphate，ATP）水平测定、乳酸生成测定以及USP22、SIRT1、微管相关蛋白轻链3（microtubule-associated protein light chain 3，LC3）-Ⅱ/Ⅰ、p62的蛋白表达分析评估自噬活性。在体内实验中，40只BALB/c-nu裸鼠异位移植HepG2细胞，给予SNH或索拉非尼干预12 d，每3天监测肿瘤体积和质量。对肿瘤组织进行苏木素-伊红（hematoxylin-eosin，HE）染色、TUNEL染色，采用免疫组化法检测核增殖抗原Ki67、USP22和SIRT1的蛋白表达。结果 SNH呈剂量相关性地抑制HepG2细胞活力和侵袭（P＜0.01、0.001），诱导细胞凋亡（P＜0.001），其中高剂量SNH的作用与索拉非尼相当。Co-IP证实了USP22-SIRT1蛋白相互作用，高剂量SNH显著降低USP22-SIRT1蛋白相互作用（P＜0.01）。高剂量SNH显著下调USP22和SIRT1蛋白表达（P＜0.01、0.001），增加SIRT1的泛素化（P＜0.001），以上作用被USP22过表达逆转（P＜0.05、0.001）。SNH抑制HepG2细胞自噬，表现为GFP/mRFP荧光信号增强（P＜0.01），ATP水平降低（P＜0.001），乳酸水平升高（P＜0.001），p62表达增加以及USP22、SIRT1、LC3-II/I蛋白表达水平降低（P＜0.05、0.001）。SIRT1激活部分抵消了SNH的自噬抑制作用（P＜0.05、0.01、0.001）。在体内，高剂量SNH显著降低肿瘤体积、质量和恶性程度（P＜0.001），诱导肿瘤细胞凋亡以及Ki67、USP22和SIRT1表达降低（P＜0.001）。结论 SNH调控USP22表达介导SIRT1去泛素化来抑制自噬，从而抑制肝细胞癌的进展，为肝细胞癌的治疗提供了潜在策略。
关键词
新鱼腥草素钠
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肝细胞癌
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自噬
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USP22
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SIRT1
Abstract
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Objective To investigate the mechanism by which sodium new houttuyfonate (SNH) inhibits hepatocellular carcinoma (HCC) progression through autophagy regulation via ubiquitin-specific peptidase 22 (USP22)-mediated silent mating type information regulation 2 homolog 1 (SIRT1) deubiquitination. Methods Human liver cancer HepG2 cells were treated with SNH or sorafenib for 24 h, cell viability was detected by CCK-8 assay, cell apoptosis was detected by flow cytometry, and cell invasion ability was detected by Transwell assay. After plasmid transfection induced overexpression of USP22, the USP22-SIRT1 interaction was verified by co-immunoprecipitation (Co-IP). USP22 and SIRT1 protein expressions, as well as SIRT1 ubiquitination were detected by Western blotting. After treatment with SIRT1 activator, autophagy activity was evaluated through mRFP-GFP-LC3 dual fluorescence labeling, adenosine triphosphate (ATP) level measurement, lactate production assay, and analysis of USP22, SIRT1, microtubule-associated protein light chain 3 (LC3)-II/I and p62 protein expressions. For in vivo experiments, 40 BALB/c-nu nude mice were eutectopically transplanted with HepG2 cells and intervened with SNH or sorafenib for 12 d. Tumor volume and weight were monitored every 3 d. Hematoxylin-eosin (HE) staining and TUNEL staining were performed on tumor tissues, and immunohistochemistry was used to detect the protein expressions of nuclear proliferation antigens Ki67, USP22 and SIRT1. Results SNH dose-dependently suppressed HepG2 cell viability and invasion (P < 0.01, 0.001), induced cell apoptosis (P < 0.001). The effect of high-dose SNH was comparable to sorafenib. Co-IP confirmed USP22-SIRT1 protein interaction, which was significantly reduced by high-dose SNH (P < 0.01). High-dose SNH significantly down-regulated USP22 and SIRT1 protein expressions (P < 0.01, 0.001), increased SIRT1 ubiquitination (P < 0.001). These effects were reversed by USP22 overexpression (P < 0.05, 0.001). SNH inhibited autophagy in HepG2 cells, manifested by enhanced GFP/mRFP fluorescence signal (P < 0.01), decreased ATP level (P < 0.001), increased lactate level (P < 0.001), increased p62 expression and decreased expression levels of USP22, SIRT1 and LC3-II/I proteins (P < 0.05, 0.001). SIRT1 activation partially counteracted the autophagic inhibition of SNH (P < 0.05, 0.01, 0.001). In vivo, high-dose SNH significantly reduced tumor volume, weight and malignancy degree (P < 0.001), induced tumor cell apoptosis and decreased Ki67, USP22, and SIRT1 expressions (P < 0.001). Conclusion SNH inhibits HCC progression by suppressing autophagy through USP22-mediated regulation of SIRT1 deubiquitination, providing a potential therapeutic strategy for HCC.
ZHANG Xuexi, DONG Changhong, ZI Chen, SUN Xiangjun.
Sodium new houttuyfonate inhibits hepatocellular carcinoma progression through USP22-mediated SIRT1 deubiquitination regulating autophagy[J].
Chinese Traditional and Herbal Drugs,
2026
, 57
(4)
: 1366
-1376
.
DOI: 10.7501/j.issn.0253-2670.2026.04.015
基金
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山东省自然科学基金资助项目 (ZR2020QH041)
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