基于HSP70信号通路探究三七总皂苷对宫颈癌MPO活性及放疗敏感性的作用机制

周涛琪; 陈俊; 周欣; 庞林荣; 杨玮丽; 徐永

doi:10.16766/j.cnki.issn.1674-4152.002401

基于HSP70信号通路探究三七总皂苷对宫颈癌MPO活性及放疗敏感性的作用机制

doi: 10.16766/j.cnki.issn.1674-4152.002401

周涛琪¹,
陈俊¹,
周欣¹,
庞林荣¹,
杨玮丽²,
徐永^3, ,

1.
宁波大学附属人民医院放化疗中心，浙江宁波 315000
2.
宁波大学附属人民医院妇产科
3.
宁波市鄞州区第二医院临床药学部，浙江宁波 315199

基金项目:

浙江省医药卫生科技计划项目 2019RC274

详细信息

通讯作者:
徐永，E-mail: xxq889988@126.com

中图分类号: R707.33 730.55
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- 文章访问数: 173
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- PDF下载量: 4
- 被引次数: 0
出版历程
- 收稿日期: 2021-11-08
- 网络出版日期: 2022-08-20

Effect of Panax notoginseng saponins on MPO activity and radiotherapy sensitivity of cervical cancer based on the HSP70 signalling pathway

1.
Radiotherapy and Chemotherapy Center, the Affiliated People ' s Hospital of Ningbo University, Ningbo, Zhejiang 315000, China

摘要

摘要: 目的探究三七总皂苷通过调控热休克蛋白70(HSP70)信号通路对宫颈癌髓过氧化物酶(MPO)活性、放疗敏感性的作用机制。方法取对数生长放疗抵抗宫颈癌细胞株随机数字法将其分为宫颈癌组(A组，n=6)，低剂量三七总皂苷组(B组，n=6)，高剂量三七总皂苷组(C组，n=6)，金雀黄素组(D组，n=6)，另取一组HeLa细胞，向其中加入HSP70抑制剂，记为E组(n=6)，免疫组化法检测HSP70蛋白表达，分光光度计比色法检测MPO活性，放疗处理后，4', 6-二脒基-2-苯基吲哚染色法、四甲基偶氮唑盐法及小室法检测宫颈癌细胞活性。结果 A、B、C、D组细胞中HSP70蛋白表达分别为3.51±0.13、2.33±0.12、1.25±0.11、1.26±0.12，组间比较差异有统计学意义(F=21.090，P＜0.001)。与A组相比，B、C、D组细胞中HSP70蛋白表达、MPO活性、增殖率、细胞侵袭数量明显降低(均P＜0.05)，且C组比B组降低明显(均P＜0.05)，D组与C组相比差异无统计学意义(均P＞0.05)。与A组相比，B、C、D组细胞凋亡率明显升高(均P＜0.05)，且C组比B组升高明显(P＜0.05)，D组与C组相比差异无统计学意义(P＞0.05)；与A组相比，B、C、D、E组细胞中Bax蛋白表达明显升高(均P＜0.05)，Bcl-2显著降低(均P＜0.05)，且C组比B组变化明显(均P＜0.05)，E组与D、C组相比差异无统计学意义(均P＞0.05)。结论三七总皂苷可显著降低宫颈癌MPO活性，提高其放疗敏感性，作用机制可能与负向调控HSP70信号通路有关。
- 热休克蛋白70信号通路 /
- 三七总皂苷 /
- 宫颈癌 /
- 髓过氧化物酶活性 /
- 放疗敏感性
Abstract: Objective To explore the effect of Panax notoginseng saponins (PNS) on myeloperoxidase (MPO) activity and radiotherapy sensitivity of cervical cancer based on the heat shock protein 70 (HSP70) signalling pathway. Methods The exponential growth against cervical cancer cell lines were randomly divided into cervical cancer radiotherapy (group A, n=6), low doses of PNS group (group B, n=6), high dose PNS group (group C, n=6), gold finches flavin group (group D, n=6). Another group of HeLa cells was taken and HSP70 inhibitor was added, which was recorded as group E (n=6). The expression of HSP70 protein was detected by immunohistochemistry, and MPO activity was detected by using a spectrophotometer. After radiotherapy, the activity of cervical cancer cells was detected by DAPI staining, MTT assay and chamber assay. Results The expression levels of HSP70 protein in groups A, B, C and D were 3.51±0.13, 2.33±0.12, 1.25±0.11 and 1.26±0.12, respectively, and the differences were statistically significant (F=21.090, P < 0.001). The expression of HSP70 protein, MPO activity, proliferation rate and invasion number of cells in groups B, C and D were significantly decreased (all P < 0.05), and those in group C were significantly lower than those in group B (all P < 0.05). However, there was no statistically significant difference between group D and group C (all P>0.05). Compared with group A, the apoptosis rate of groups B, C and D was significantly increased (all P < 0.05), and the apoptosis rate of group C was significantly higher than that of group B (all P < 0.05). Moreover, there was no statistically significant difference between group D and group C (P>0.05). Compared with group A, Bax protein expression in groups B, C, D and E was significantly increased (all P < 0.05), and Bcl-2 protein expression was significantly decreased (all P < 0.05). The expression of these proteins in group C was significantly changed compared with that in group B (all P < 0.05). No statistically significant difference was observed between groups E, D and C (all P>0.05). Conclusion Panax notoginseng saponins can significantly reduce MPO activity and improve radiotherapy sensitivity of cervical cancer, and the mechanism may be related to the negative regulation of the HSP70 signalling pathway.
- HSP70 signalling pathway /
- Panax notoginseng saponins /
- Cervical cancer /
- Myeloperoxidase activity /
- Radiotherapy sensitivity

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图 1 各组细胞HSP70免疫组化结果(免疫组化染色，×200)

Figure 1. Immunohistochemical results of HSP70 cells in each group (immunohistochemical staining, ×200)

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图 2 各组细胞凋亡检测(DAPI染色，×200)

Figure 2. Apoptosis detection of each group (DAPI staining, ×200)

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图 3 各组细胞侵袭检测(结晶紫染色，×200)

Figure 3. Cell invasion detection in each group (crystal violet staining, ×200)

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图 4 各组细胞Bax及Bcl-2蛋白表达

Figure 4. Expression of Bax and Bcl-2 proteins in cells of each group

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表 1 仪器及设备一览表

Table 1. List of instruments and equipment

仪器及设备	型号	生产厂家
电子显微镜	DMI6000	上海辅泽商贸有限公司
紫外分光光度计	UV-1750	上海佐明机械设备贸易有限公司
低温冷冻离心机	Medifriger-BL-S	北京金恒祥仪器有限公司
高能直线加速器	Turebeam	美国Varian公司
倒置荧光显微镜	BM4000	上海玉研科学仪器有限公司
Transwell小室	3422	上海朗喜工业科技有限公司

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表 2 试剂及材料一览表

Table 2. List of reagents and materials

药品	货号	生产厂家
宫颈癌细胞HeLa	YS131C	上海雅吉生物科技有限公司
胎牛血清	RY-F22	兰州荣晔生物科技有限责任公司
DMEM培养基	A31600-500 mL	上海吉至生化科技有限公司
三七总皂苷	YT1892	北京伊塔生物科技有限公司
金雀异黄素	SCPT-30516	上海赛可锐生物科技有限公司
免疫组化试剂盒	YDDEF001	上海羽哚生物科技有限公司
HSP70抗体	FNab04050	武汉菲恩生物科技有限公司
Bax抗体	FNab00811	武汉菲恩生物科技有限公司
Bcl-2抗体	FNab00839	武汉菲恩生物科技有限公司
DAB显色试剂盒	YT8204	北京伊塔生物科技有限公司
MPO试剂盒	YS02047B	上海雅吉生物科技有限公司
DAPI染液	JKMK1511A	上海经科化学科技有限公司
多聚甲醛	158127	Sigma-Aldrich LLC.公司
结晶紫	D3276-100 mg	上海宝曼生物科技有限公司
PBS	SY3154	北京伊塔生物科技有限公司
注：PBS为磷酸缓冲盐溶液(phosphate buffered saline)。

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参考文献(22)

[1]	汪瑶, 王春兰, 俞惠艳, 等. 宫颈癌术后带导尿管出院患者自我管理能力的影响因素研究[J]. 中华全科医学, 2021, 19(10): 1772-1774. doi: 10.16766/j.cnki.issn.1674-4152.002164 WANG Y, WANG C L, YU H Y, et al. Studies on the influencing factors of self-management of discharged patients with urinary catheters after cervical cancer surgery[J]. Chinese Journal of General Practice, 2021, 19(10): 1772-1774. doi: 10.16766/j.cnki.issn.1674-4152.002164
[2]	马欢, 张贤雨, 张飞, 等. miR-193b靶向调节cyclin D1对宫颈癌细胞放疗敏感性的影响[J]. 中华内分泌外科杂志, 2020, 14(6): 482-486. doi: 10.3760/cma.j.cn.115807-20201007-00306 MA H, ZHANG X Y, ZHANG F, et al. Effect of miR-193b on radiosensitivity of cervical cancer cells by targeting cyclin D1[J]. Chinese Journal of Endocrine Surgery, 2020, 14(6): 482-486. doi: 10.3760/cma.j.cn.115807-20201007-00306
[3]	王军, 袁纯辉, 孙红, 等. 高危型人乳头瘤病毒16 E6蛋白通过外泌体传递热休克蛋白70调控巨噬细胞介导宫颈癌细胞增殖, 侵袭和迁移[J]. 中华微生物学和免疫学杂志, 2020, 40(7): 538-546. doi: 10.3760/cma.j.cn112309-20190822-00267 WANG J, YUAN C H, SUN H, et al. High-risk human papillomavirus 16 E6 protein regulates tumor-associated macrophages through exosomal heat shock protein 70 to promote cervical cancer cell C33A proliferation, invasion and migration[J]. Chinese Journal of Microbiology and Immunology, 2020, 40(7): 538-546. doi: 10.3760/cma.j.cn112309-20190822-00267
[4]	ZEINDLER J, ANGEHRN F, DROESER R, et al. Infiltration by myeloperoxidase-positive neutrophils is an independent prognostic factor in breast cancer[J]. Breast Cancer Res Treat, 2019, 177(3): 581-589. doi: 10.1007/s10549-019-05336-3
[5]	魏剑锋, 许泽波, 王伟, 等. 三七总皂苷对肝癌细胞株HepG2的钙离子平衡和生长的影响[J]. 中华实验外科杂志, 2020, 37(10): 1815-1818. doi: 10.3760/cma.j.cn421213-20200122-01033 WEI J F, XU Z B, WANG W, et al. Effects of PANAX Notoginseng saponins on calcium balance and growth of hepatocellular carcinoma cell line HepG2[J]. Chinese Journal of Experimental Surgery, 2020, 37(10): 1815-1818. doi: 10.3760/cma.j.cn421213-20200122-01033
[6]	吕红青, 张琳, 倪镌. 帕唑帕尼联合放疗对宫颈癌的治疗效果及对血清VEGFR水平的影响[J]. 中华全科医学, 2020, 18(6): 934-937. doi: 10.16766/j.cnki.issn.1674-4152.001396 LYU H Q, ZHANG L, NI J. Therapeutic effect of pazopanib combined with radiotherapy on cervical cancer and its effect on serum levels of vascular endothelial growth factor receptor[J]. Chinese Journal of General Practice, 2020, 18(6): 934-937. doi: 10.16766/j.cnki.issn.1674-4152.001396
[7]	姚黎超, 王伟, 武伦, 等. 三七总皂苷对人胰腺癌PANC-1细胞增殖, 凋亡的影响[J]. 肿瘤药学, 2019, 9(6): 870-874. doi: 10.3969/j.issn.2095-1264.2019.06.008 YAO L C, WANG W, WU L, et al. Effects of PANAX Notoginseng saponins on proliferation and apoptosis of human pancreatic cancer PANC-1 cells[J]. Anti-tumor Pharmacy, 2019, 9(6): 870-874. doi: 10.3969/j.issn.2095-1264.2019.06.008
[8]	张芳, 王雅莉, 李萌萌, 等. 金雀异黄素对白介素-6诱导下SKOV3人卵巢癌细胞的抑制作用[J]. 毒理学杂志, 2019, 33(1): 12-17. https://www.cnki.com.cn/Article/CJFDTOTAL-WSDL201901002.htm ZHANG F, WANG Y L, LI M M, et al. Inhibition of genistein on interleukin-6-induced human ovarian cancer SKOV3 cells[J]. Journal of Toxicology, 2019, 33(1): 12-17. https://www.cnki.com.cn/Article/CJFDTOTAL-WSDL201901002.htm
[9]	CHEN L, CHEN M Y, SHAO L, et al. PANAX Notoginseng saponins prevent colitis-associated colorectal cancer development: The role of gut microbiota[J]. Chin J Nat Med, 2020, 18(7): 500-507.
[10]	CHENG Y, BAO D, CHEN X, et al. Microwave-triggered/HSP-targeted gold nano-system for triple-negative breast cancer photothermal therapy[J]. Int J Pharm, 2021, 593: 120162. DOI: 10.1016/j.ijpharm.2020.120162.
[11]	段智, 李代强, 陈辉, 等. 宫颈癌患者人乳头瘤病毒感染和HSP70表达及Treg细胞分布研究[J]. 中华医院感染学杂志, 2020, 30(5): 729-733. https://www.cnki.com.cn/Article/CJFDTOTAL-ZHYY202005022.htm DUAN Z, LI D Q, CHEN H, et al. Heat shock protein 70 and regulatory T cells in cervical cancer patients with human papillomavirus infection[J]. Chinese Journal of Nosocomiology, 2020, 30(5): 729-733. https://www.cnki.com.cn/Article/CJFDTOTAL-ZHYY202005022.htm
[12]	苏玉强, 范晓英, 秦艳, 等. 三七总皂苷调节丙泊酚所致大鼠认知功能损伤的实验研究[J]. 海南医学院学报, 2019, 25(10): 739-743. https://www.cnki.com.cn/Article/CJFDTOTAL-HNYY201910006.htm SU Y Q, FAN X Y, QIN Y, et al. Effects of PANAX Notoginseng saponins on apoptosis, inflammation and oxidative stress in rats with propofol-induced cognitive dysfunction[J]. Journal of Hainan Medical University, 2019, 25(10): 739-743. https://www.cnki.com.cn/Article/CJFDTOTAL-HNYY201910006.htm
[13]	柳俊, 陈军, 鲍晨, 等. HSP70、eIF4E、DNMT1在宫颈癌中的表达及意义[J]. 现代生物医学进展, 2020, 20(20): 3988-3991. https://www.cnki.com.cn/Article/CJFDTOTAL-SWCX202020041.htm LIU J, CHEN J, BAO C, et al. Expression and significance of HSP70, eIF4E and DNMT1 in cervical cancer[J]. Progress in Modern Biomedicine, 2020, 20(20): 3988-3991. https://www.cnki.com.cn/Article/CJFDTOTAL-SWCX202020041.htm
[14]	YANG W J, WANG M Y, PAN F Z, et al. Association between MPO-463G>A polymorphism and cancer risk: Evidence from 60 case-control studies[J]. World J Surg Oncol, 2017, 15(1): 144. doi: 10.1186/s12957-017-1183-7
[15]	梁海清, 李一然, 张淑燕, 等. 髓过氧化物酶与结直肠癌相关性的研究进展[J]. 中国医师杂志, 2021, 23(6): 958-960, 963. doi: 10.3760/cma.j.cn431274-20200523-00655 LIANG H Q, LI Y R, ZHANG S Y, et al. Research and development of correlation between myeloperoxidaseand colorectal cancer[J]. Journal of Chinese Physician, 2021, 23(6): 958-960, 963. doi: 10.3760/cma.j.cn431274-20200523-00655
[16]	SHI X, LI B, YUAN Y, et al. The possible association between the presence of an MPO-463 G>A (rs2333227) polymorphism and cervical cancer risk[J]. Pathol Res Pract, 2018, 214(8): 1142-1148. doi: 10.1016/j.prp.2018.05.018
[17]	ROSZAK A, LUTKOWSKA A, LIANERI M, et al. Involvement of myeloperoxidase gene polymorphism 463G>A in development of cervical squamous cell carcinoma[J]. Int J Biol Markers, 2016, 31(4): e440-e445. doi: 10.5301/jbm.5000212
[18]	王雯智, 郑蕾, 李虹. 宫颈癌中miR-15b, DCLK1的表达变化及与放疗敏感性的关系[J]. 河北医药, 2020, 42(3): 330-334. doi: 10.3969/j.issn.1002-7386.2020.03.002 WANG W Z, ZHENG L, LI H. The correlation between the expression changes of MiR-15b, DCLK1 and radiotherapy sensitivity in patients with cervical cancer[J]. Hebei Medical Journal, 2020, 42(3): 330-334. doi: 10.3969/j.issn.1002-7386.2020.03.002
[19]	万莉, 比丽克孜·艾克木, 刘娜. 辛伐他汀通过调控Chk1基因的表达对宫颈癌细胞增殖, 凋亡以及放疗敏感性的影响[J]. 现代肿瘤医学, 2020, 28(18): 3138-3144. doi: 10.3969/j.issn.1672-4992.2020.18.009 WAN L, BILIZI A, LIU N. Effect of simvastatin on proliferation, apoptosis and radiosensitivity of cervical cancer cells by regulating the expression of Chk1 gene[J]. Journal of Modern Oncology, 2020, 28(18): 3138-3144. doi: 10.3969/j.issn.1672-4992.2020.18.009
[20]	白桦. miR-451靶向MIF调控宫颈癌细胞生物学功能并通过外泌体释放影响其化疗敏感性[D]. 太原: 山西医科大学, 2020. BAI H. The targeting of miR-451 with MIF regulates the biological function of cervical cancer cells and affects its chemosensitivity through exosome release[D]. Taiyuan: Shanxi Medical University, 2020.
[21]	邹存华. 三七总皂苷抑制Hela细胞增殖的实验研究[D]. 青岛: 青岛大学, 2012. ZOU C H. Effect of total saponins of Panax Notoginseng on the proliferation of Hela cells[D]. Qingdao: Qingdao University, 2012.
[22]	YAO L C, WU L, WANG W, et al. Panax notoginseng saponins promote cell death and chemosensitivity in pancreatic cancer through the apoptosis and autophagy pathways[J]. Anticancer Agents Med Chem, 2021, 21(13): 1680-1688. doi: 10.2174/1871520620999201110191459