低强度脉冲超声协同冬凌草甲素通过激活PIEZO1诱导胰腺癌细胞铁死亡的机制

doi:10.12122/j.issn.1673-4254.2025.10.12

摘要/Abstract

摘要：

目的基于铁死亡探讨冬凌草甲素（Ori）对胰腺癌的影响，以及低强度脉冲超声（LIPUS）在协同增效中的作用机制。方法体外实验首先分为对照组、不同浓度的Ori组，通过CCK-8法检测不同浓度Ori对PANC-1增殖活力的影响。其次实验分为对照组、Ori组，通过流式细胞术检测细胞内Fe²⁺、ROS含量，试剂盒检测细胞内二醛（MDA）、而谷胱甘肽（GSH）和三磷酸腺苷（ATP）浓度，Western blotting检测细胞内GPX4、Nrf2、HO-1蛋白表达。随后分为对照组、铁死亡抑制剂组（Fer-1）、Ori组以及Ori联合Fer-1组，通过CCK-8法检测各组对PANC-1增殖活力的影响。最后分为对照组、Ori组、LIPUS组及联合组，通过Western blotting检测细胞内PIEZO1蛋白表达。体内实验构建C57BL/6J小鼠胰腺癌模型，实验分组分为对照组、Ori组、LIPUS组及联合组，通过检测皮下移植瘤小鼠肿瘤的苏木精-伊红染色（HE）、免疫组织化学Ki67染色评估肿瘤组织病理变化及细胞增殖活性，Western blotting及免疫荧光染色（IF）检测小鼠肿瘤内GPX4表达。结果与对照组相比，Ori可以抑制PANC-1细胞增殖（P<0.001），细胞内Fe²⁺、ROS、MDA升高（P<0.05），GSH和ATP下降（P<0.001）。与对照组相比，Ori组GPX4蛋白表达下降（P<0.01），HO-1、Nrf2蛋白表达升高（P<0.05）。与Ori组相比，Ori联合LIPUS组细胞活力下降（P<0.01）。比较各组皮下移植瘤小鼠肿瘤发现，与Ori组相比，Ori联合LIPUS组进一步抑制肿瘤的生长（P<0.01）。Ki67染色表明联合组相较于Ori组细胞增殖活性更弱。与Ori组相比，联合组GPX4蛋白表达及荧光强度均更低（P<0.05）。与对照组相比，LIPUS组和Ori组PIEZO1蛋白表达均升高（P<0.005），与Ori组相比，联合组PIEZO1蛋白表达升高更为显著（P<0.01）。结论 LIPUS协同Ori通过激活PIEZO1，通过Nrf2/HO-1/GPX4通路促进胰腺癌细胞铁死亡。

关键词: 低强度脉冲超声, 冬凌草甲素, 铁死亡, PIEZO1, 胰腺癌

Abstract:

Objective To evaluate the inhibitory effect of oridonin against proliferation of pancreatic cancer cells and the mechanism underlying the synergistic effect of low-intensity pulsed ultrasound (LIPUS). Methods PANC-1 cells treated with different concentrations of oridonin were examined for changes in cell proliferation using CCK-8 assay and in MDA, GSH and ATP levels using flow cytometry. The protein expressions of GPX4, Nrf2 and HO-1 in the treated cells were detected with Western blotting. The effect of Fer-1, a ferroptosis inhibitor, on proliferation of oridonin-treated cells were assessed, and the effects of oridonin combined with LIPUS on PIEZO1 protein expression was evalauted using Western blotting. A C57BL/6J mouse model bearing pancreatic cancer cell xenograft was established and treated with oridonin, LIPUS, or both, and the histological changes in the tumor tissues and tumor cell proliferation were examined with HE staining and immunohistochemistry for Ki67; the changes in GPX4 expression in the tumor tissues were detected using Western blotting and immunofluorescence staining. Results In PANC-1 cells, oridonin treatment significantly inhibited cell proliferation, increased intracellular Fe²⁺, ROS, and MDA levels, and decreased GSH and ATP levels. Oridonin also resulted in lowered GPX4 and increased HO-1 and Nrf2 protein expression levels in the cells. The combined treatment with LIPUS signficiantly enhanced the inhibitory effect of oridonin on PANC-1 cell viability in vitro and on xenograft growth in the mouse models, resulting also in more obvious reduction of the intensity of Ki67 staining and GPX4 protein expression and more pronounced increase of PIEZO1 protein expression in the tumor tissues in the mouse models. Conclusion LIPUS enhances the effect of oridonin to promote ferroptosis of pancreatic cancer cells by activating PIEZO1 through the Nrf2/HO-1/GPX4 pathway.

Key words: low-intensity pulsed ultrasound, oridonin, ferroptosis, piezo1, pancreatic cancer

孙陛航, 郭煜君, 祁玉麟, 姚丹, 陈文直, 陈念芝. 低强度脉冲超声协同冬凌草甲素通过激活PIEZO1诱导胰腺癌细胞铁死亡的机制[J]. 南方医科大学学报, 2025, 45(10): 2160-2170.

Bihang SUN, Yujun GUO, Yulin QI, Dan YAO, Wenzhi CHEN, Nianzhi CHEN. Low-intensity pulsed ultrasound and oridonin synergistically induce ferroptosis of pancreatic cancer cells by activating PIEZO1 via the Nrf2/HO-1/GPX4 pathway[J]. Journal of Southern Medical University, 2025, 45(10): 2160-2170.

图/表 13

图1 不同Ori浓度对PANC-1活力的影响

Fig.1 Effect of different concentrations of oridoni (Ori) on proliferation of PANC-1 cells (n=6).

图2 Ori处理后PANC-1细胞内Fe2+水平

Fig.2 Effect of Ori on intracellular Fe2+ level in PANC-1 cells (n=3). ***P<0.001 vs Control.

图3 Ori处理后PANC-1细胞内ROS水平

Fig.3 Effect of Ori on intracellular ROS level (n=3). **P<0.01 vs Control.

图4 Ori联合Fer-1对PANC-1活力的影响

Fig.4 Effect of Ori combined with Fer-1 on proliferation of PANC-1 cells (n=6). ****P<0.001 vs Control, ***P<0.005 vs Ori Group.

图5 Ori处理后PANC-1细胞内MDA、GSH、ATP表达

Fig.5 Effect of Ori on intracellular MDA, GSH, and ATP levels in PANC-1 cells (n=3). *P<0.05, ***P<0.001 vs Control.

图6 Ori处理后PANC-1细胞内GPX4、Nrf2、HO-1蛋白表达

Fig.6 Effect of Ori on intracellular GPX4, Nrf2, and HO-1 expressions in PANC-1 cells (n=3). **P<0.001, ***P<0.005 vs Control.

图7 Ori处理后PANC-1细胞内C11-BODIPY标记荧光强度

Fig.7 Fluorescence intensity of C11-BODIPY labeling in PANC-1 cells after Ori treatment (Original magnification: ×40) (n=3).

图8 Ori联合LIPUS对PANC-1细胞活力影响

Fig.8 Effect of Ori combined with LIPUS on proliferation of PANC-1 cells (n=6). **P<0.01 vs Ori-20 μmol/L.

图9 Ori联合LIPUS处理后小鼠肿瘤内GPX4表达

Fig.9 Effect of Ori combined with LIPUS on intra-tumoral GPX4 expression in the mouse models (n=3). *P<0.05 vs Control,***P<0.005 vs Ori.

图11 Ori联合LIPUS对胰腺癌小鼠体质量的影响

Fig.11 Effect of Ori combined with LIPUS on body weight of the mouse models bearing pancreatic cancer xenograft (n=8).

图12 Ori联合LIPUS对胰腺癌小鼠肿瘤体积及体质量影响

Fig.12 Effect of LIPUS combined with Ori on tumor volume and weight in the tumor-bearing mice (n=8). ****P<0.001 vs Control, **P<0.01 vs Ori.

图14 LIPUS处理后PANC-1细胞内Fe2+水平

Fig.14 Effect of LIPUS on intracellular Fe2+ level (n=3). *P<0.05, **P<0.01 vs Control.

图15 Ori联合LIPUS处理后PANC-1细胞内PIEZO1蛋白表达

Fig.15 Effect of Ori combined with LIPUS on intracellular PIEZO1 expression (n=3). **P<0.005 vs Control; ***P<0.001 vs Ori.

参考文献 39

[1]	易丽夏, 方涵露, 李婧怡, 等. 2022年全球和中国胰腺癌发病及死亡分析[J]. 海军军医大学学报, 2024, 45(12): 1470-7.
[2]	Zheng RS, Zhang SW, Zeng HM, et al. Cancer incidence and mortality in China, 2016[J]. J Natl Cancer Cent, 2022, 2(1): 1-9. doi：10.1016/j.jncc.2022.02.002
[3]	Halbrook CJ, Lyssiotis CA, Pasca di Magliano M, et al. Pancreatic cancer: advances and challenges[J]. Cell, 2023, 186(8): 1729-54. doi：10.1016/j.cell.2023.02.014
[4]	中国抗癌协会胰腺癌专业委员会, 虞先濬, 徐近. 中国抗癌协会胰腺癌整合诊治指南(精简版)[J]. 中国肿瘤临床, 2023, 50(10): 487-96.
[5]	Rohatgi S, Jagannathan JP, Rosenthal MH, et al. Vascular toxicity associated with chemotherapy and molecular targeted therapy: what should a radiologist know[J]? AJR Am J Roentgenol, 2014, 203(6): 1353-62. doi：10.2214/ajr.13.11967
[6]	韦天夫, 周琪, 胡凤林, 等. 慢性胰腺炎炎癌转化中医理论浅析[J]. 时珍国医国药, 2020, 31(5): 1192-4.
[7]	魏小曼, 李柳, 王俊壹, 等. 癌毒病机理论辨治胰腺癌探讨[J]. 中华中医药杂志, 2022, 37(4): 2062-5.
[8]	刘延泽, 陈士林, 马培, 等. 中草药中发现新抗癌药物的途径[J]. 现代药物与临床, 2012, 27(4): 323-37.
[9]	高世勇, 唐博琪, 魏新瑞, 等. 冬凌草的本草考证、化学成分、药理作用及其临床应用[J]. 中草药, 2023, 54(19): 6543-54.
[10]	Li X, Zhang CT, Ma W, et al. Oridonin: a review of its pharmacology, pharmacokinetics and toxicity[J]. Front Pharmacol, 2021, 12: 645824. doi：10.3389/fphar.2021.645824
[11]	Zhang CL, Wu LJ, Zuo HJ, et al. Cytochrome c release from oridonin-treated apoptotic A375-S2 cells is dependent on p53 and extracellular signal-regulated kinase activation[J]. J Pharmacol Sci, 2004, 96(2): 155-63. doi：10.1254/jphs.fpj04008x
[12]	Huang J, Wu LJ, Tashiro SI, et al. Reactive oxygen species mediate oridonin-induced HepG2 apoptosis through p53, MAPK, and mitochondrial signaling pathways[J]. J Pharmacol Sci, 2008, 107(4): 370-9. doi：10.1254/jphs.08044fp
[13]	Liu Y, Song Z, Liu YJ, et al. Identification of ferroptosis as a novel mechanism for antitumor activity of natural product derivative a2 in gastric cancer[J]. Acta Pharm Sin B, 2021, 11(6): 1513-25. doi：10.1016/j.apsb.2021.05.006
[14]	Ye SY, Hu XY, Sun SW, et al. Oridonin promotes RSL3-induced ferroptosis in breast cancer cells by regulating the oxidative stress signaling pathway JNK/Nrf2/HO-1[J]. Eur J Pharmacol, 2024, 974: 176620. doi：10.1016/j.ejphar.2024.176620
[15]	Cai MR, Fu TT, Zhu RY, et al. An iron-based metal-organic framework nanoplatform for enhanced ferroptosis and oridonin delivery as a comprehensive antitumor strategy[J]. Acta Pharm Sin B, 2024, 14(9): 4073-86. doi：10.1016/j.apsb.2024.05.015
[16]	倪琰, 郭晓冬, 王静霞, 等. 胰腺癌中医治疗评述[J]. 中国中医基础医学杂志, 2023, 29(12): 2117-22.
[17]	Yang X, Liu YQ, Wang Z, et al. Ferroptosis as a new tool for tumor suppression through lipid peroxidation[J]. Commun Biol, 2024, 7(1): 1475. doi：10.1038/s42003-024-07180-8
[18]	De Leon-Oliva D, Boaru DL, Minaya-Bravo AM, et al. Improving understanding of ferroptosis: Molecular mechanisms, connection with cellular senescence and implications for aging[J]. Heliyon, 2024, 10(21): e39684. doi：10.1016/j.heliyon.2024.e39684
[19]	Chen AQ, Huang HF, Fang SM, et al. ROS: a “booster” for chronic inflammation and tumor metastasis[J]. Biochim Biophys Acta BBA Rev Cancer, 2024, 1879(6): 189175. doi：10.1016/j.bbcan.2024.189175
[20]	Su ZY, Liu YQ, Wang L, et al. Regulation of SLC7A11 as an unconventional checkpoint in tumorigenesis through ferroptosis[J]. Genes Dis, 2024, 12(1): 101254. doi：10.1016/j.gendis.2024.101254
[21]	Zhu LP, Leng DL, Guo ZA, et al. Self-catalyzed nitric oxide nano complexes induce ferroptosis for cancer immunotherapy[J]. J Control Release, 2025, 377: 524-39. doi：10.1016/j.jconrel.2024.11.048
[22]	李泽彦, 李国东, 孙硕, 等. 雷公藤红素诱导人胰腺癌PANC-1细胞铁死亡的机制研究[J]. 中国病理生理杂志, 2024, 40(6): 1062-9.
[23]	Liu X, Xu JM, Zhou J, et al. Oridonin and its derivatives for cancer treatment and overcoming therapeutic resistance[J]. Genes Dis, 2020, 8(4): 448-62. doi：10.1016/j.gendis.2020.06.010
[24]	Nomikou N, Li YS, McHale AP. Ultrasound-enhanced drug dispersion through solid tumours and its possible role in aiding ultrasound-targeted cancer chemotherapy[J]. Cancer Lett, 2010, 288(1): 94-8. doi：10.1016/j.canlet.2009.06.028
[25]	Gong YP, Wang ZG, Dong GF, et al. Low-intensity focused ultrasound mediated localized drug delivery for liver tumors in rabbits[J]. Drug Deliv, 2016, 23(7): 2280-9. doi：10.3109/10717544.2014.972528
[26]	孙亚超, 王昊, 商慧娟, 等. 低强度脉冲超声研究现状及趋势的可视化分析[J]. 联勤军事医学, 2024, 38(1): 75-84.
[27]	王敏丹, 刘莉, 崔夏莲, 等. 冬凌草甲素对食管鳞状细胞癌细胞内谷胱甘肽代谢网络影响的研究[J]. 上海中医药杂志, 2024, 58(7): 77-82, 94.
[28]	Ru Q, Li YS, Chen L, et al. Iron homeostasis and ferroptosis in human diseases: mechanisms and therapeutic prospects[J]. Signal Transduct Target Ther, 2024, 9(1): 271. doi：10.1038/s41392-024-01969-z
[29]	Kwon MY, Park E, Lee SJ, et al. Heme oxygenase-1 accelerates erastin-induced ferroptotic cell death[J]. Oncotarget, 2015, 6(27): 24393-403. doi：10.18632/oncotarget.5162
[30]	O’Rourke SA, Shanley LC, Dunne A. The Nrf2-HO-1 system and inflammaging[J]. Front Immunol, 2024, 15: 1457010. doi：10.3389/fimmu.2024.1457010
[31]	Zhu YT, Zhang JC, Liu QN, et al. Semen Cuscutae-Fructus Lycii attenuates Tripterygium glycosides-induced spermatogenesis dysfunction by inhibiting oxidative stress-mediated ferroptosis via the Nrf2/HO-1 pathway[J]. Phytomedicine, 2024, 135: 156221. doi：10.1016/j.phymed.2024.156221
[32]	Du HF, Wu JW, Zhu YS, et al. Fucoxanthin induces ferroptosis in cancer cells via downregulation of the Nrf2/HO-1/GPX4 pathway[J]. Molecules, 2024, 29(12): 2832. doi：10.3390/molecules29122832
[33]	Acheta J, Stephens SBZ, Belin S, et al. Therapeutic low-intensity ultrasound for peripheral nerve regeneration - a schwann cell perspective[J]. Front Cell Neurosci, 2022, 15: 812588. doi：10.3389/fncel.2021.812588
[34]	Xu MS, Wang L, Wu SM, et al. Review on experimental study and clinical application of low-intensity pulsed ultrasound in inflammation[J]. Quant Imaging Med Surg, 2021, 11(1): 443-62. doi：10.21037/qims-20-680
[35]	Tardoski S, Gineyts E, Ngo J, et al. Low-intensity ultrasound promotes clathrin-dependent endocytosis for drug penetration into tumor cells[J]. Ultrasound Med Biol, 2015, 41(10): 2740-54. doi：10.1016/j.ultrasmedbio.2015.06.006
[36]	Lentacker I, De Cock I, Deckers R, et al. Understanding ultrasound induced sonoporation: definitions and underlying mechanisms[J]. Adv Drug Deliv Rev, 2014, 72: 49-64. doi：10.1016/j.addr.2013.11.008
[37]	Jin JQ, Fan ZF, Long YL, et al. Matrine induces ferroptosis in cervical cancer through activation of piezo1 channel[J]. Phytomedicine, 2024, 122: 155165. doi：10.1016/j.phymed.2023.155165
[38]	Wang SY, Li WW, Zhang PF, et al. Mechanical overloading induces GPX4-regulated chondrocyte ferroptosis in osteoarthritis via Piezo1 channel facilitated calcium influx[J]. J Adv Res, 2022, 41: 63-75. doi：10.1016/j.jare.2022.01.004
[39]	Xiang ZQ, Zhang PF, Jia CW, et al. Piezo1 channel exaggerates ferroptosis of nucleus pulposus cells by mediating mechanical stress-induced iron influx[J]. Bone Res, 2024, 12(1): 20. doi：10.1038/s41413-024-00317-9