南方医科大学学报 ›› 2025, Vol. 45 ›› Issue (11): 2309-2319.doi: 10.12122/j.issn.1673-4254.2025.11.03
陈一镠1(
), 马民1(
), 苏燃1, 朱寅宾1, 冯晴1, 罗嘉丽1, 冯伟峰2(
), 颜显欣1(
)
收稿日期:2025-06-26
出版日期:2025-11-20
发布日期:2025-11-28
通讯作者:
冯伟峰,颜显欣
E-mail:cyl9708@163.com;tmamin@jnu.edu.cn;fwf2000ok@sina.com;871655006@qq.com
作者简介:陈一镠,在读博士研究生,E-mail:cyl9708@163.com基金资助:
Yiliu CHEN1(
), Min MA1(
), Ran SU1, Yinbin ZHU1, Qing FENG1, Jiali LUO1, Weifeng FENG2(
), Xianxin YAN1(
)
Received:2025-06-26
Online:2025-11-20
Published:2025-11-28
Contact:
Weifeng FENG, Xianxin YAN
E-mail:cyl9708@163.com;tmamin@jnu.edu.cn;fwf2000ok@sina.com;871655006@qq.com
Supported by:摘要:
目的 探讨理冲消癥颗粒通过调控ANT3介导的线粒体凋亡通路增敏顺铂治疗卵巢癌的分子机制。 方法 LC-MS检测理冲消癥颗粒入血成分。采用皮下异种移植瘤模型,将BALB/c-nud小鼠随机分为3组(8只/组):Tumor组(仅接种肿瘤)、DDP组(每周腹腔注射1次0.1 mL的5 mg/kg的顺铂)、DDP_LCXZ组(在顺铂基础上按照15 g·kg-1·d-1灌胃0.2 mL的理冲消癥颗粒)。在实验过程中动态监测肿瘤体积、质量及小鼠体质量,并用HE染色观察肿瘤和肾脏的病理学变化。同时,采用RNA-seq筛选差异基因并行KEGG分析,电镜用于观察线粒体结构变化,Western blotting检测线粒体凋亡相关蛋白的表达。 结果 LC-MS共检测出218个成分为理冲消癥颗粒的入血成分;与Tumor组比较,DDP组与DDP_LCXZ联合用药组的肿瘤体积分别减少60.3%和72.6%(P<0.01)。转录组学结果显示,ANT3在DDP组与DDP_LCXZ联合用药组中的表达水平较对照组上调(P<0.05)。分子对接后发现理冲消癥颗粒的主要活性成分与ANT3的结合能均<-6 kcal mol。电镜显示,与Tumor组相比,DDP组肿瘤细胞线粒体肿胀及外膜损伤程度增加,DDP+LCXZ组该现象更为明显。Western blotting结果显示,DDP组和DDP+LCXZ组中BAX、ANT3、cleaved-caspase-3、cleaved-caspase-9表达水平升高,而BCL-2表达水平降低(P<0.05)。 结论 LCXZ通过上调ANT3,促进线粒体功能失衡并激活凋亡信号,从而增强顺铂对卵巢癌的抑瘤效应。
陈一镠, 马民, 苏燃, 朱寅宾, 冯晴, 罗嘉丽, 冯伟峰, 颜显欣. 理冲消癥颗粒通过上调ANT3介导的线粒体凋亡增强小鼠卵巢癌移植瘤对顺铂的敏感性[J]. 南方医科大学学报, 2025, 45(11): 2309-2319.
Yiliu CHEN, Min MA, Ran SU, Yinbin ZHU, Qing FENG, Jiali LUO, Weifeng FENG, Xianxin YAN. Lichong Xiaozheng Granules enhances cisplatin sensitivity of ovarian cancer xenografts in rats by regulating adenine nucleotide translocator 3-mediated mitochondrial apoptosis[J]. Journal of Southern Medical University, 2025, 45(11): 2309-2319.
图1 理冲消癥颗粒的 HPLC-UV 指纹图谱及代表性成分质谱解析
Fig.1 HPLC-UV fingerprints and mass spectrometric analysis of Lichong Xiaozheng Granules (LCXZ). A, B: HPLC-UV fingerprint of LCXZ at 210 nm and 254 nm, respectively. C: High-resolution mass spectrometric fragmentation pathway of paeoniflorin.
| No. | Mass-to-charge ratio (m/z) | Formula | Mass Error (ppm) | Retention time (min) |
|---|---|---|---|---|
| M0001 | 387.1132 | C12H22O11 | -3.69 | 0.89 |
| M0002 | 170.0210 | C7H6O5 | -3.02 | 1.87 |
| M0003 | 329.0869 | C14H18O9 | -2.67 | 3.13 |
| M0004 | 183.0293 | C8H8O5 | -3.03 | 3.90 |
| M0005 | 356.1097 | C16H20O9 | -2.96 | 4.04 |
| M0006 | 496.1569 | C23H28O12 | -2.36 | 4.15 |
| M0007 | 457.1572 | C20H27NO11 | -2.76 | 4.29 |
| M0008 | 431.1910 | C19H30O8 | -3.31 | 4.51 |
| M0009 | 481.1692 | C23H28O11 | -2.65 | 4.62 |
| M0010 | 295.1044 | C14H17NO6 | -4.10 | 4.67 |
| M0011 | 480.1618 | C23H28O11 | -2.89 | 4.71 |
| M0012 | 446.1200 | C22H22O10 | -2.88 | 4.88 |
| M0013 | 428.1671 | C20H28O10 | -2.70 | 4.97 |
| M0014 | 580.2141 | C28H36O13 | -2.51 | 5.12 |
| M0015 | 433.1130 | C21H22O10 | -2.46 | 5.25 |
| M0016 | 463.1221 | C22H22O11 | -3.07 | 5.26 |
| M0017 | 441.1756 | C20H28O8 | -2.57 | 5.41 |
| M0018 | 507.1491 | C23H26O10 | -3.62 | 5.58 |
| M0019 | 161.0593 | C10H10O3 | -2.29 | 5.71 |
| M0020 | 507.1493 | C23H26O10 | -3.22 | 5.90 |
表1 理冲消癥颗粒中鉴定出的含量Top 20的入血化合物的信息
Tab.1 Information on the top 20 compounds of Lichong Xiaozheng Granules (LCXZ) detected in medicated mouse serum
| No. | Mass-to-charge ratio (m/z) | Formula | Mass Error (ppm) | Retention time (min) |
|---|---|---|---|---|
| M0001 | 387.1132 | C12H22O11 | -3.69 | 0.89 |
| M0002 | 170.0210 | C7H6O5 | -3.02 | 1.87 |
| M0003 | 329.0869 | C14H18O9 | -2.67 | 3.13 |
| M0004 | 183.0293 | C8H8O5 | -3.03 | 3.90 |
| M0005 | 356.1097 | C16H20O9 | -2.96 | 4.04 |
| M0006 | 496.1569 | C23H28O12 | -2.36 | 4.15 |
| M0007 | 457.1572 | C20H27NO11 | -2.76 | 4.29 |
| M0008 | 431.1910 | C19H30O8 | -3.31 | 4.51 |
| M0009 | 481.1692 | C23H28O11 | -2.65 | 4.62 |
| M0010 | 295.1044 | C14H17NO6 | -4.10 | 4.67 |
| M0011 | 480.1618 | C23H28O11 | -2.89 | 4.71 |
| M0012 | 446.1200 | C22H22O10 | -2.88 | 4.88 |
| M0013 | 428.1671 | C20H28O10 | -2.70 | 4.97 |
| M0014 | 580.2141 | C28H36O13 | -2.51 | 5.12 |
| M0015 | 433.1130 | C21H22O10 | -2.46 | 5.25 |
| M0016 | 463.1221 | C22H22O11 | -3.07 | 5.26 |
| M0017 | 441.1756 | C20H28O8 | -2.57 | 5.41 |
| M0018 | 507.1491 | C23H26O10 | -3.62 | 5.58 |
| M0019 | 161.0593 | C10H10O3 | -2.29 | 5.71 |
| M0020 | 507.1493 | C23H26O10 | -3.22 | 5.90 |
图2 理冲消癥颗粒协同顺铂抑制卵巢癌的增殖
Fig.2 LCXZ synergizes with cisplatin to inhibit ovarian cancer proliferation in nude mice. A: Measurement of tumor volume in the mice. B: Gross observation of the dissected tumors. C: Measurement of tumor weight. D: HE/Ki67 staining of the tumors,Arrows indicate widened intercellular spaces (Original magnification: ×400). *P<0.05 vs Tumor group, #P<0.05 vs DDP group (n=8).
图3 理冲消癥颗粒缓解顺铂的毒性作用
Fig.3 LCXZ alleviates the toxic effect of cisplatin. A: Body weight changes of the tumor-bearing mice. B: Gross observation of the spleen of the mice. C: HE staining of renal tissues of the mice (×400). D: SOD content in the kidneys of the mice. *P<0.05, **P<0.01 vs Tumor group; #P<0.05 vs DDP group (n=8).
图4 理冲消癥颗粒协同顺铂治疗靶点的转录组学分析
Fig.4 Transcriptomic analysis of therapeutic targets of LCXZ combined with cisplatin. A: Principal component analysis. B: Volcano plot of the differential genes of DDP vs Tumor. C: Volcano plot of the differential genes of DDP_LCXZ vs Tumor. D: Volcano plot of the differential genes of DDP_LCXZ vs DDP. E: Venn diagram of the differential genes.
图5 理冲消癥颗粒协同顺铂治疗靶点的KEGG富集分析
Fig.5 KEGG enrichment analysis of the therapeutic targets of LCXZ combined with cisplatin. A: KEGG enrichment analysis of DDP vs Tumor. B: KEGG enrichment analysis of DDP_LCXZ vs Tumor. C: KEGG enrichment analysis of DDP_LCXZ vs DDP
图6 理冲消癥颗粒主要成分的分子对接
Fig.6 Molecular docking of the main constituents of LCXZ. A: Paeoniflorin-ANT3 docking. B: L-amygdalin-ANT3 docking. C: Turanose-ANT3 docking. D: Benzoyl paeoniflorin-ANT3 docking.
图7 理冲消癥颗粒协同顺铂导致肿瘤细胞线粒体孔过度开放
Fig.7 LCXZ combined with cisplatin causes excessive opening of the mitochondrial pores in the tumor cells.( Left: scale bar=2 μm; Right: scale bar=1 μm. Labels: N:nucleus; M: mitochondria; ER:endoplasmic reticulum; ASS: autophagolysosomal structure).
图8 理冲消癥颗粒介导的线粒体凋亡通路增敏顺铂
Fig.8 LCXZ -mediated mitochondrial apoptosis pathway sensitizes cisplatin. A: Representitative protein bands in Westen blotting. B-F: Relative protein expression levels of ANT3, BAX, cleaved caspase-3, cleaved caspase-9 and BCL-2, respectively. *P<0.05, **P<0.01 vs Tumor group (n=3).
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