Objective  To investigate the mechanism of Cistanche phenylethanol glycosides (CPhGs) on innate immune function, tumor growth and tumor cell apoptosis in mice bearing drug-resistant osteosarcoma.

Methods  A drug-resistant strain of human osteosarcoma MG-63 cell was established by the gradient concentration method, and a drug-resistant osteosarcoma loaded tumor model of nude mice were constructed (36  mice, 6 mice in each group). The model group was given daily gavage of saline; the methotrexate (MTX) group was injected intraperitoneally with MTX (20 mg/kg) every three days; the combined group was intraperitoneally injected with MTX every three days and given a medium-dose concentration of CPhGs (250  mg/ kg) by gavage daily; the CPhGs high-, medium- and low-dose groups were respectively given daily gavage of high (500 mg/kg), medium (250 mg/kg) and low (125 mg/kg) dose concentrations of CPhGs. The changes in the size of osteosarcoma tumors in nude mice were observed and the average tumor volume of each group was plotted over time to calculate the tumor inhibition rate. The serum levels of IL-2 and TNF-α in nude mice were measured by ELISA. The apoptosis of tumor tissue cells was detected by flow cytometry. The expression of apoptosis-related proteins Bcl-2 and Bax, as well as the expression changes of p-AKT, p-mTOR and p-PI3K were examined in tumor tissues by Western blot.

Results  Compared with the model group, the tumor tissue volume and weight were significantly decreased in all experimental groups (P<0.001). The serum levels of IL-2 and TNF-α in nude mice were significantly higher in each CPhGs group and the combined group than those in both the model group and MTX group (P<0.01). The expression level of apoptosis protein Bax was significantly higher in each test group compared with Methotrexate (MTX) group (P<0.001), while the expression level of Bcl-2 was significantly reduced in the high- and medium- dose CPhGs groups and the combined group (P<0.001). Compared with the model group and the MTX group, the apoptosis rates of the tumor tissue were significantly increased in each CPhGs group and the combination group, and the levels of p-AKT, p-mTOR and p-PI3K were significantly decreased (P<0.05).

Conclusion  CPhGs significantly improved the immune function in nude mice bearing drug-resistant osteosarcoma, and inhibited tumor growth by promoting tumor cell apoptosis, which was mediated by inhibiting the expression of PI3K/AKT/mTOR signaling pathway and modulating the expression of Bax and Bcl-2 apoptotic proteins.

1.Zhang C, Hu J, Zhu K, et al. Survival, complications and functional outcomes of cemented megaprostheses for high-grade osteosarcoma around the knee[J]. Int Orthop, 2018, 42(4): 927-938. DOI: 10.1007/s00264-018-3770-9.

2.Liu Q, Xu R, Xu X, et al. Characteristics and significance of T lymphocyte subsets in peripheral blood of osteosarcoma mice[J]. Transl Cancer Res, 2022, 11(6): 1503-1509. DOI: 10.21037/tcr-22-264.

3.吴蔚, 景豆豆, 曹理, 等. 骨肉瘤免疫治疗的现状和前景[J]. 肿瘤防治研究, 2022, 49(7): 721-726. [Wu  W, Jing DD, Cao L, et al. Current status and prospects of immunotherapy for osteosarcoma[J]. Cancer Research on Prevention and Treatment, 2022, 49(7): 721-726.] DOI: 10.3971/j.issn.1000-8578.2022.21.1281.

4.王成志, 刘一帆, 张晓青, 等. 黄芪甲苷调控免疫细胞抗肿瘤作用机制研究进展[J]. 中华中医药学刊, 2024, 42(11): 145-149. [Wang CZ, Liu YF, Zhang XQ, et al. Research progress on mechanism of astragaloside Ⅳ regulating anti-tumor action of immune cells[J]. Chinese Archives of Traditional Chinese Medicine, 2024, 42(11): 145-149.] DOI: 10.13193/j.issn.1673-7717.2024.11.029.

5.Zhang J, Yu XH, Yan YG, et al. PI3K/Akt signaling in osteosarcoma[J]. Clin Chim Acta, 2015, 444: 182-192. DOI: 10.1016/j.cca.2014.12.041.

6.胡琼, 由淑萍, 刘涛, 等. 肉苁蓉苯乙醇总苷抗肝癌作用的实验研究[J]. 癌变·畸变·突变, 2018, 30(3): 194-199. [Hu Q, You SP, Liu T, et al. An investigation on the anti-liver cancer effect of cistanche[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2018, 30(3): 194-199.] DOI: 10.3969/j.issn.1004-616x.2018.03.006.

7.齐鑫鑫, 由淑萍, 何转霞, 等. 肉苁蓉苯乙醇苷对HepG2肝癌细胞增殖和凋亡的影响[J].新疆医科大学学报, 2021, 44(9): 1041-1047. [Qi XX, You SP, He ZX, et al. Effect of Cistache deserticola phenylethanol glycoside on proliferaion and apoptosis of HepG2 cells in vitro[J]. Journal of Xinjiang Medical University, 2021, 44(9): 1041-1047.] DOI: 10.3639/j.issn.1009-5551.2021.09.012.

8.Panaampon J, Sasamoto K, Kariya R, et al. Establishment of nude mice lacking NK cells and their application for human tumor xenografts[J]. Asian Pac J Cancer Prev, 22(4): 1069-1074. DOI: 10.31557/APJCP.2021.22.4.1069.

9.于春波, 孙鹏, 张小伟, 等. 胰腺癌裸鼠皮下异种移植动物模型建立研究[J]. 现代生物医学进展, 2012, 12(9): 1648-1650. [Yu CB, Sun P, Zhang XW, et al. The animal model study of pancreatic cancer xenograft in nude mice[J]. Progress in Modern Biomedicine, 2012, 12(9): 1648-1650.] DOI: 10.13241/j.cnki.pmb.2012.09.017.

10.徐小茹, 王楠, 孙濛濛, 等. 针刺单穴与腧穴配伍对骨肉瘤模型小鼠的效应差异研究[J]. 中华中医药杂志, 2020, 35(5): 2271-2276. [Xu XR, Wang N, Sun MM, et al. Different effects in osteosarcoma model mice with acupuncture at single point and compatibility of acupoints[J]. China Journal of Traditional Chinese Medicine and Pharmacy, 2020, 35(5): 2271-2276.] DOI: 10.13241/j.cnki.pmb.2012.09.017.

11.张洪泉, 张爱香, 堵年生, 等. 肉苁蓉对小白鼠免疫功能的影响[J]. 中西医结合杂志, 1988, (12): 736-737, 710. [Zhang HQ, Zhang AX, Du NS, et al. Effects of Cistanche deserticola on immune function of mice[J]. Chinese Journal of Integrated Traditional and Western Medicine, 1988, (12): 736-737, 710.] https://www.cnki.com.cn/Article/CJFDTotal-ZZXJ198812019.htm.

12.张洪泉, 许爱华, 李电东. 肉苁蓉多糖对肿瘤和荷瘤小鼠的药理作用[J]. 中国中西医结合杂志, 1997, (S1): 155-156, 294. [Zhang HQ, Xu AH, Li DD. Pharmacological effects of Cistanche polysaccharides on tumors and tumor-bearing mice[J]. Chinese Journal of Integrated Traditional and Western Medicine, 1997, (S1): 155-156, 294.] https://kns.cnki.net/kcms2/article/abstract?v=mg_qcPBBapnmsDqon66yc8-zI2VqwJgixywPNSnAl0qboPnZqB4FNuT0bsHSSvs0Dn72KJt__SvxLZeQ8vJicy96v2o-S-Wc-bp5ClislhRETaTYT6KGBMKP3_EreGXg3-BH52Itk0rqWiNksajNxzld06uvdfRzCDj-utLfgfANyySxJMHM-g==&uniplatform=NZKPT&language=CHS

13.薛海燕, 焦婵媛, 姚军. 肉苁蓉总苷药理作用的研究现状 [J]. 中国临床药理学杂志, 2018, 34(4): 486-488. [Xue  HY, Jiao CY, Yao J. Current status of pharmacological effects of glycosides of Cistanches Herba[J]. The Chinese Journal of Clinical Pharmacology, 2018, 34(4): 486-488.] DOI: 10.13699/j.cnki.1001-6821.2018.04.026.

14.唐颖, 武建强. 肉苁蓉苯乙醇苷的抗肿瘤作用[J]. 生物技术进展, 2023, 13(3): 399-405. [Tang Y, Wu JQ. Anti-tumor effects of phenylethanoid glycosides deprived from Cistanche deserticola[J]. Current Biotechnology, 2023, 13(3): 399-405.] DOI: 10.19586/j.2095-2341.2023.0040.

15.许伟, 丁聚贤, 谢兴文, 等. 肉苁蓉对化疗耐药骨肉瘤细胞的逆转作用及MRP1、P53表达的影响[J]. 时珍国医国药, 2021, 32(3): 551-554. [Xu W, Ding JX, Xie XW, et al. Effect of Cistanche deserticola on chemotherapy-resistant osteosarcoma cells and expression of MRP1 and P53[J]. Journal of Li-shizhen Traditional Chinese Medicine, 2021, 32(3): 551-554.] DOI: 10.3969/j.issn.1008-0805.2021.03.11.

16.候晓甜, 刘涛, 苏德奇. 肉苁蓉苯乙醇总苷对H22荷瘤小鼠皮下移植瘤抑制作用的实验研究[J]. 毒理学杂志, 2021, 35(3): 231-235, 240. [Hou XT, Liu T, Su DQ. Anti-tumor effect of phenylethanol glycosides from Cistanche deserticola on H22 tumor-bearing mice[J]. Journal of Toxicology, 2021, 35(3): 231-235, 240.] DOI: 10.16421/j.cnki.1002-3127.2021.03.010.

17.张欣雨, 王丹, 付春雪, 等. 白细胞介素-2治疗肿瘤的研究进展[J]. 癌症进展, 2024, 22(6): 581-585, 618. [Zhang XY, Wang D, Fu CX, et al. Research progress on the treatment of tumors with interleukin-2[J]. Oncology Progress, 2024, 22(6): 581-585, 618.] DOI: 10.11877/j.issn.1672-1535.2024.22.06.01.

18.高世勇, 李丹. 肿瘤坏死因子与癌症相关研究进展[J]. 中国药理学通报, 2020, 36(9): 1209-1213. [Gao SY, Li D. Research advances in tumor necrosis factor and cancer[J]. Chinese Pharmacological Bulletin, 2020, 36(9): 1209-1213.] DOI: 10.3969/j.issn.1001-1978.2020.09.006.

19.周慧, 海广范, 张涛, 等. 癌症治疗中PI3K/AKT/mTOR通路及靶向抑制剂研究进展[J]. 中国药业, 2023, 32(5): 127-135. [Zhou H, Hai GF, Zhang T, et al. Research progress of PI3K/AKT/mTOR pathway and its targeted inhibitors in cancer treatment[J]. China Pharmaceuticals, 2023, 32(5): 127-135.] DOI: 10.3969/j.issn.1006-4931.2023.05.030.

20.刘彦娟, 曹雨虹, 匡荣. 肿瘤多药耐药发生机制及中药逆转研究进展[J]. 中国药学杂志, 2024, 59(7): 561-570. [Liu  YJ, Cao YH, Kuang R. Research progress of mechanism of tumor multidrug resistance and reversal by traditional Chinese medicine[J]. Chinese Pharmaceutical Journal, 2024, 59(7): 561-570.] DOI: 10.11669/cpj.2024.07.001.

21.杨浩东, 李宁, 谢兴文, 等. 中药单体调控PI3K/Akt/mTOR信号通路治疗骨肉瘤的研究进展[J]. 中国实验方剂学杂志, 2023, 29(3): 254-262. [Yang HD, Li N, Xie XW, et al. Chinese medicine monomers in treatment of osteosarcoma by regulating PI3K/Akt/mTOR signaling pathway: a review[J]. Chinese Journal of Experimental Traditional Medical Formulae, 2023, 29(3): 254-262.] DOI: 10.13422/j.cnki.syfjx.20221527.