| 周磊,黄琦,王淼,王珍珍.丹栀逍遥散通过调节Notch1-Jagged1通路影响线粒体自噬从而抑制喉鳞状细胞癌恶性进展的机制研究[J].浙江中西医结合杂志,2026,36(1): |
| 丹栀逍遥散通过调节Notch1-Jagged1通路影响线粒体自噬从而抑制喉鳞状细胞癌恶性进展的机制研究 |
| The mechanism of Danzhi Xiaoyao Powder influencing mitochondrial autophagy to inhibit malignant progression of laryngeal squamous cell carcinoma by regulating Notch1-Jagged1 signaling pathway |
| 投稿时间：2025-06-11 修订日期：2025-11-09 |
| DOI： |
| 中文关键词: 喉鳞状细胞癌 丹栀逍遥散 线粒体自噬 Notch1 Jagged1 |
| 英文关键词:Laryngeal squamous cell carcinoma Danzhi Xiaoyao powder mitophagy Notch1 Jagged1 |
| 基金项目:浙江省中医药科技计划(2024ZL952),浙江省医药卫生科技计划(2024KY289),宁波市卫生健康青年技术骨干人才培养专项(第五期) |
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| 中文摘要: |
| 目的：探究丹栀逍遥散（Danzhi Xiaoyao Powder，DXP）对喉鳞状细胞癌（larynx squamous cell carcinoma ，LSCC）恶性进展的影响及其具体分子机制。
方法：体外实验：人 LSCC 细胞系AMC-HN-8细胞随机分为阴性对照（Control）组、正常大鼠血清（NS）组、低剂量（10% DXP）含药血清（DS-L）组、高剂量（20% DXP）含药血清（DS-H）组、DS-H+线粒体分裂抑制剂1（Mdivi-1）组、DS-H+对照空质粒（oe-NC）组和DS-H+ Notch1过表达质粒（oe-Notch1）组。使用高、低剂量DXP含药血清处理LSCC细胞，加入Mdivi-1或转染oe-Notch1及oe-NC干预LSCC细胞。体内实验：将Balb/c裸鼠随机分为对照（Control，生理盐水）组、DXP 低剂量（DXP-L，6 g/kg）组和DXP 高剂量（DXP-H，12 g/kg）组，每组6只。将AMC-HN-8细胞接种至小鼠皮下构建小鼠LSCC皮下瘤模型，使用高、低剂量DXP灌胃干预。使用细胞计数试剂盒8（CCK-8）检测细胞活性；流式细胞术检测细胞凋亡水平；跨膜（Transwell）侵袭实验和细胞划痕实验检测细胞侵袭和迁移能力；免疫荧光检测细胞线粒体自噬水平；蛋白免疫印迹（Western Blot，WB）检测细胞和小鼠皮下瘤组织中线粒体自噬和果蝇双翅边缘缺刻同源基因1（Notch1）-锯齿典型Notch配体1（Jagged1）通路相关蛋白、微管相关蛋白轻链3 Ⅱ与Ⅰ的比值（LC3 Ⅱ/Ⅰ）、泛素结合蛋白p62（p62）、线粒体内膜转位酶23（Tim23）、E3泛素-蛋白连接酶（PARKIN）、Notch1、Jagged1的表达。
结果：体外实验：与NS组比较， DS-L、DS-H组细胞活性[（0.73±0.04）、（0.56±0.07）比（0.96±0.09），P＜0.05]显著下降，凋亡率[（11.26±1.06）%、（18.92±0.77）% 比（5.18±0.35）%，P＜0.05]显著上升，细胞侵袭[（36.00±3.61）、（15.67±1.53）比（86.33±3.79），P＜0.05]和迁移能力[（52.48±2.30）%、（24.07±2.07）% 比（77.67±2.13）%，P＜0.05]显著下降，线粒体自噬水平[（23.17±0.95）、（26.70±1.08）比（12.93±0.74），P＜0.05]上升，LC3 Ⅱ/I[（2.55±0.08）、（3.21±0.09）比（0.81±0.02），P＜0.05]和PARKIN[（0.58±0.07）、（0.88±0.06）比（0.30±0.01），P＜0.05]表达显著升高，p62[（0.56±0.03）、（0.42±0.03）比（1.03±0.05），P＜0.05]和Tim23[（0.43±0.02）、（0.26±0.06）比（0.90±0.06），P＜0.05]表达显著下降， Notch1[（0.43±0.12）、（0.17±0.03）比（0.92±0.03），P＜0.05]和Jagged1[（0.37±0.05）、（0.07±0.05）比（0.90±0.01），P＜0.05]表达显著下降，呈剂量依赖关系。与DS-H组比较，DS-H+Mdivi-1组细胞活性[（0.71±0.05）比（0.56±0.07），P＜0.05]显著升高，凋亡率[（11.23±0.18）%比（18.92±0.77）%，P＜0.05]显著降低，细胞侵袭[（31.67±2.52）比（15.67±1.53），P＜0.05]和迁移 [（40.66±0.69）%比（24.07±2.07）%，P＜0.05] 能力显著升高，LC3 Ⅱ/Ⅰ[（0.21±0.03）比（0.60±0.01），P＜0.05]、PARKIN[（0.82±0.01）比（1.04±0.03），P＜0.05]表达降低，p62[（0.55±0.03）比（0.34±0.02），P＜0.05]、Tim23[（0.71±0.01）比（0.53±0.02），P＜0.05]表达升高。与DS-H+ oe-NC组比较，DS-H+ oe-Notch1组细胞活性 [（0.74±0.03）比（0.62±0.03），P＜0.05]显著升高，凋亡率[（9.97±0.15）%比（17.95±0.84）%，P＜0.05]显著降低，细胞侵袭[（34.00±2.00）比（14.67±0.47），P＜0.05]和迁移 [（46.22±2.99）%比（25.74±1.25）%，P＜0.05] 能力显著升高， LC3 Ⅱ/Ⅰ[（0.22±0.02）比（0.42±0.02），P＜0.05]、PARKIN[（0.78±0.04）比（1.07±0.04），P＜0.05]显著降低，p62[（0.82±0.01）比（0.63±0.02），P＜0.05]，Tim23[（0.44±0.02）比（0.19±0.01），P＜0.05]显著升高。在体内实验中，与Control比较，DXP-H、DXP-L组小鼠皮下瘤组织体积[(585.75±73.37) mm3、(759.64±57.32) mm3比(920.09±98.58) mm3，P＜0.05]和质量[（0.59±0.09）g、（0.73±0.07）g比（0.95±0.13）g，P＜0.05]显著下降， LC3 Ⅱ/Ⅰ[（0.82±0.09）、（0.56±0.10）比（0.25±0.05），P＜0.05]和PARKIN[（0.80 ± 0.11）、（0.60 ± 0.10）比（0.26 ± 0.04），P＜0.05]表达升高，p62[ (0.26 ± 0.09)、(0.55 ± 0.15)比(1.02 ± 0.16)，P＜0.05]和Tim23[（0.17 ± 0.07）、（0.40 ± 0.05）比（1.01 ± 0.13），P＜0.05]表达下降， Notch1[（0.25 ± 0.07）、（0.57 ± 0.10）比（1.06 ± 0.24），P＜0.05]、Jagged1[（0.37 ± 0.06）、（0.60 ± 0.10）比（1.02 ± 0.19），P＜0.05]表达下降。
结论：丹栀逍遥散能够通过调节Notch1-Jagged1通路诱导LSCC线粒体自噬从而抑制其恶性进展。
关键词：喉鳞状细胞癌；丹栀逍遥散；线粒体自噬；Notch1；Jagged1 |
| 英文摘要: |
| Objective: To explore the effect of Danzhi Xiaoyao Powder (DXP) on malignant progression of laryngeal squamous cell carcinoma (LSCC) and its molecular mechanism.
Methods: In vitro experiments: The human LSCC cell line AMC-HN-8 cells were randomly divided into negative control (Control) group, normal rat serum (NS) group, low-dose (10% DXP) drug-containing serum (DS-L) group, high-dose (20% DXP) drug-containing serum (DS-H) group, DS-H + mitochondrial fission inhibitor 1 (Mdivi-1) group, DS-H + control empty plasmid (oe-NC) group, and DS-H + Notch1 overexpression plasmid (oe-Notch1) group. High and low-dose DXP drug-containing serum, Mdivi-1, and oe-Notch1 and oe-NC were used to intervene in LSCC cells. In vivo experiments: Balb/c nude mice were randomly divided into control (normal saline) group, low-dose DXP (DXP-L, 6 g/kg) group, and high-dose DXP (DXP-H, 12 g/kg) group, with 6 mice in each group. AMC-HN-8 cells were inoculated subcutaneously into mice to establish mouse LSCC subcutaneous tumor model, and mice were intervened with high and low doses of DXP by gavage. Cell Counting Kit-8 (CCK-8) was used to detect cell viability; flow cytometry was used to detect the level of cell apoptosis; transmembrane (Transwell) invasion and cell scratch assays were used to detect cell invasion and migration abilities; immunofluorescence was used to detect the level of mitochondrial autophagy in cells; Western Blot (WB) was used to detect the expression of mitochondrial autophagy and Notch1-Jagged1 pathway-related proteins, including the ratio of microtubule-associated protein light chain 3 II to I (LC3 II/I), ubiquitin-binding protein p62 (p62), mitochondrial inner membrane translocase 23 (Tim23), E3 ubiquitin-protein ligase (PARKIN), Notch1, and Jagged1 in cells and mouse subcutaneous tumor tissues.
Results: In vitro experiments: Compared with the NS group, the cell viability in the DS-L and DS-H groups [(0.73±0.04), (0.56±0.07) vs. (0.96±0.09), P<0.05] significantly decreased, the apoptosis rate [(11.26±1.06)%, (18.92±0.77)% vs. (5.18±0.35)%, P<0.05] significantly increased, the cell invasion [(36.00±3.61), (15.67±1.53) vs. (86.33±3.79), P<0.05] and migration ability [(52.48±2.30)%, (24.07±2.07)% vs. (77.67±2.13)%, P<0.05] significantly decreased, the level of mitochondrial autophagy [(23.17±3.61), (26.70±1.08) vs. (12.93±0.74), P<0.05] increased, the expression of LC3Ⅱ/Ⅰ [(2.55±0.08), (3.21±0.09) vs. (0.81±0.02), P<0.05] and PARKIN [(0.58±0.07), (0.88±0.06) vs. (0.30±0.01), P<0.05] significantly increased, the expression of p62 [(0.56±0.03), (0.42±0.03) vs. (1.03±0.05), P<0.05] and Tim23 [(0.43±0.02), (0.26±0.06) vs. (0.90±0.06), P<0.05] significantly decreased, and the expression of Notch1 [(0.43±0.12), (0.17±0.03) vs. (0.92±0.03), P<0.05] and Jagged1 [(0.37±0.05), (0.07±0.05) vs. (0.90±0.01), P<0.05] significantly decreased, showing a dose-dependent relationship. Compared with the DS-H group, the cell viability in the DS-H + Mdivi-1 group [(0.71±0.05) vs. (0.56±0.07), P<0.05] significantly increased, the apoptosis rate [(11.23±0.18)% vs. (18.92±0.77)%, P<0.05] significantly decreased, the cell invasion [(31.67±2.52) vs. (15.67±1.53), P<0.05] and migration [(40.66±0.69)% vs. (24.07±2.07)%, P<0.05] ability significantly increased, the expression of LC3 Ⅱ/Ⅰ [(0.21±0.03) vs. (0.60±0.01), P<0.05] and PARKIN [(0.82±0.01) vs. (1.04±0.03), P<0.05] decreased, and the expression of p62 [(0.55±0.03) vs. (0.34±0.02), P<0.05] and Tim23 [(0.71±0.01) vs. (0.53±0.02), P<0.05] increased. Compared with the DS-H+ oe-NC group, the cell viability in the DS-H+ oe-Notch1 group was significantly increased [(0.74±0.03) vs. (0.62±0.03), P<0.05], the apoptosis rate was significantly decreased [(9.97±0.15)% vs. (17.95±0.84)%, P<0.05], the cell invasion [(34.00±2.00) vs. (14.67±0.47), P<0.05] and migration [(46.22±2.99)% vs. (25.74±1.25)%, P<0.05] abilities were significantly enhanced, and the expression of LC3 Ⅱ/Ⅰ [(0.22±0.02) vs. (0.42±0.02), P<0.05] and PARKIN [(0.78±0.04) vs. (1.07±0.04), P<0.05] were significantly decreased, while the expression of p62 [(0.82±0.01) vs. (0.63±0.02), P<0.05] and Tim23 [(0.44±0.02) vs. (0.19±0.01), P<0.05] were significantly increased. In vivo experiments: Compared with the Control group, the subcutaneous tumor tissue volume [(585.75±73.37) mm3, (759.64±57.32) mm3 vs. (920.09±98.58) mm3, P<0.05] and mass [(0.59±0.09) g, (0.73±0.07) g vs. (0.95±0.13) g, P<0.05] in the DXP-H and DXP-L groups were significantly reduced, the expression of LC3 Ⅱ/Ⅰ [(0.82±0.09), (0.56±0.10) vs. (0.25±0.05), P<0.05] and PARKIN [(0.80±0.11), (0.60±0.10) vs. (0.26±0.04), P<0.05] was increased, while the expression of p62 [(0.26±0.09), (0.55±0.15) vs. (1.02±0.16), P<0.05] and Tim23 [(0.17±0.07), (0.40±0.05) vs. (1.01±0.13), P<0.05] was decreased, and the expression of Notch1 [(0.25±0.07), (0.57±0.10) vs. (1.06±0.24), P<0.05] and Jagged1 [(0.37±0.06), (0.60±0.10) vs. (1.02±0.19), P<0.05] was decreased.
Conclusion: DXP can significantly inhibit the malignant progression of LSCC. Moreover, mitochondrial autophagy of LSCC may be induced through Notch1-Jagged1 signaling pathway to inhibit its progression.
Keywords: Laryngeal squamous cell carcinoma; Danzhi Xiaoyao powder; mitophagy; Notch1; Jagged1 |
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