黄芪-莪术-重楼配伍激活PERK/eIF2α/ATF4信号通路介导抑制结直肠癌生长转移作用研究

张慧兰; 郭文晖; 苏婷婷; 陈思; 余倩慧; 殷启航; 万林鹭; 王旭; 唐德才

doi:10.14148/j.issn.1672-0482.2024.0240

黄芪-莪术-重楼配伍激活PERK/eIF2α/ATF4信号通路介导抑制结直肠癌生长转移作用研究

doi: 10.14148/j.issn.1672-0482.2024.0240

南京中医药大学中医学院, 江苏南京 210023

基金项目:

国家自然科学基金面上项目 82274116

江苏省中医药科技发展专项 2020ZX01

江苏省研究生科研创新计划项目 KYCX22_1879

江苏省研究生实践创新计划项目 SJCX23_0858

详细信息

作者简介:
张慧兰, 女，硕士研究生, E-mail: zhl88881998@163.com

通讯作者:
唐德才, 男，教授, 博士生导师, 主要从事中药药效、配伍机制及应用研究, E-mail: 290022@njucm.edu.cn

中图分类号: R285.5
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- 文章访问数: 142
- HTML全文浏览量: 21
- PDF下载量: 32
- 被引次数: 0
出版历程
- 收稿日期: 2023-10-09
- 网络出版日期: 2024-03-15
- 发布日期: 2024-03-10

Astragalus Mongholicus Bunge-Curcuma Aromatica-Paridis Rhizoma Inhibits Metastasis of Colon Cancer via the PERK/eIF2α/ATF4 Signaling Pathway

School of Chinese Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China

摘要

摘要: 目的探讨黄芪-莪术-重楼(芪-术-蚤)配伍通过激活PERK/eIF2α/ATF4信号通路介导内质网应激(ER stress)抗结直肠癌生长、转移相关作用。方法 BALB/c雄性小鼠30只随机分为假手术组、模型组、5-氟尿嘧啶(5-FU)组(25 mg·kg^-1)、芪-术-蚤高剂量组(5.85 g·kg^-1)、芪-术-蚤低剂量组(2.925 g·kg^-1)，每组6只, 构建结直肠癌原位移植瘤小鼠模型, 给药15 d后, 通过肿瘤体积大小变化评估芪-术-蚤配伍对肿瘤生长的干预效果; 通过HE染色评估芪-术-蚤角药配伍干预后小鼠肝脏组织、肿瘤组织变化情况。采用ELISA法检测丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)水平变化; Western blot检测PERK/eIF2α/ATF4信号通路和上皮细胞-间充质转化(EMT)相关蛋白表达。结果与模型组相比, 芪-术-蚤高剂量组肿瘤体积明显减小(P＜0.000 1)、肝脾转移灶较不明显, 肝组织和肿瘤组织病理染色结果产生变化, 下调氧化应激相关指标GSH-Px、SOD水平, 上调MDA水平, 上调ER stress相关蛋白磷酸化蛋白激酶R样内质网激酶(p-PERK)、磷酸化真核启动因子2α(p-eIF2α)、转录激活因子4(ATF4)等的表达, 上调上皮型钙黏蛋白(E-cadherin)、下调神经型钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)、锌指转录因子(Snail)的蛋白表达水平, 抑制EMT过程。结论芪-术-蚤角药配伍可能通过激活PERK/eIF2α/ATF4通路诱导持续的ER stress, 影响结直肠癌EMT过程, 从而抑制结直肠癌的生长转移。
- 芪-术-蚤 /
- 角药 /
- 内质网应激 /
- 上皮细胞-间充质转化 /
- 结直肠癌
Abstract: OBJECTIVE To investigate the mechanism of anti-colorectal cancer growth and metastasis-related effects of Astragalus mongholicus Bunge-Curcuma aromatica-Paridis Rhizoma (Qi-Zhu-Zao) pairing through PERK/eIF2α/ATF4 signaling pathway mediating endoplasmic reticulum stress. METHODS Twenty-four BALB/c male mice were randomly divided into sham-operated group, model group, 5-FU (5-fluorouracil) group (25 mg·kg^-1), and Qi-Zhu-Zao high dose group (5.85 g·kg^-1), Qi-Zhu-Zao low dose group(2.925 g·kg^-1) (n=6) to construct a mouse model of colorectal cancer in situ transplantation tumor, and the intervention effect of Qi-Zhu-Zao combination on tumor growth was assessed by the change of tumor volume size after 15 days of administration; the intervention effect of Qi-Zhu-Zao combination on tumor growth was assessed by H&E. Pathological staining was used to evaluate the effect of Qi-Zhu-Zao combination on the liver and tumor tissues of mice. The changes of MDA, SOD and GSH-Px levels were detected by enzyme-linked immunosorbent assay (ELISA); the expression of PERK/eIF2α/ATF4 signaling pathway and EMT-related proteins were detected by protein immunoblotting (Western blot). RESULTS Compared with the model group, the tumor volume was significantly reduced (P < 0.000 1), liver and spleen metastases were less pronounced in the Qi-Zhu-Zao high-dose group, and histopathological staining results of liver tissue and tumor produced changes in oxidative stress indicators SOD, MDA, and GSH-Px, upregulation of ER stress-related proteins p-PERK, p-IF2α, and ATF4, etc., upregulated the protein expression levels of E-Cadherin, downregulated N-Cadherin, Vimentin, and Snail, and inhibited the EMT process (P < 0.01 or P < 0.05). CONCLUSION In this paper, we investigated the regulatory mechanism related to the inhibition of colorectal cancer growth and metastasis by the combination of Qi-Zhu-Zao trigonal medicine, and demonstrated that it may inhibit the growth and metastasis of colorectal cancer by activating the PERK/eIF2α/ATF4 pathway to induce sustained ER stress and affect the EMT process of colorectal cancer.
- Qi-Zhu-Zao /
- trigonal medicine /
- endoplasmic reticulum stress /
- epithelial cell-mesenchymal transformation /
- colorectal cancer

HTML全文

图 1 2021年1月—12月南京中医药大学附属医院肿瘤科临床中药处方重楼剂量统计

Figure 1. Dosage statistics of clinical Chinese medicine prescriptions in the Oncology Department of the Affiliated Hospital of Nanjing University of Chinese Medicine from January 2021 to December 2021

下载: 全尺寸图片幻灯片

图 2 芪-术-蚤角药配伍对CT26.WT原位移植模型小鼠体征影响

注：K.假手术组；M.模型组；5-FU.阳性对照组；HEZ-G.芪-术-蚤高剂量组；HEZ-D.芪-术-蚤低剂量组。A.原位移植瘤小鼠肿瘤解剖; B.原位移植瘤小鼠肝脏解剖图; C.原位移植瘤小鼠脾脏解剖图; D.小鼠肿瘤体积对比图; E.小鼠体质量变化图。与模型组相比, ^****P＜0.000 1。与5-FU组比较，^#P＜0.05, ^##P＜0.01。x±s, n=6。

Figure 2. Effect of Qi-Zhu-Zao on physical signs in mices transplantated CT26.WT

下载: 全尺寸图片幻灯片

图 3 芪-术-蚤角药配伍对CT26.WT原位移植模型小鼠肝组织病理学改变的影响

注：K.假手术组；M.模型组；5-FU.阳性对照组；HEZ-G.芪-术-蚤高剂量组；HEZ-D.芪-术-蚤低剂量组

Figure 3. Effects of Qi-Zhu-Zao on liver histopathological changes in mice transplantated CT26.WT

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图 4 芪-术-蚤角药配伍对CT26.WT原位移植模型小鼠肿瘤组织病理学改变的影响

注：K.假手术组；M.模型组；5-FU.阳性对照组；HEZ-G.芪-术-蚤高剂量组；HEZ-D.芪-术-蚤低剂量组

Figure 4. Effects of Qi-Zhu-Zao on tumor histopathological changes in mice transplantated CT26.WT

下载: 全尺寸图片幻灯片

图 5 芪-术-蚤角药对CT26.WT原位移植模型小鼠氧化应激相关指标的影响

注：K.假手术组；M.模型组；5-FU.阳性对照组；HEZ-G.芪-术-蚤高剂量组；HEZ-D.芪-术-蚤低剂量组。
A.肿瘤中相关指标水平; B.血清中相关指标水平。与M组比较, ^*P＜0.05, ^**P＜0.01，^***P＜0.001，^****P＜0.000 1;与5-FU组比较, ^#P＜0.05, ^##P＜0.01, ^####P＜0.000 1;与HEZ-G组比较, ^▲P＜0.05, ^▲▲▲▲P＜0.000 1。

Figure 5. Effects of Qi-Zhu-Zao on oxidative stress related indexes of CT26.WT mice after orthotopic transplantation

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图 6 芪-术-蚤角药对CT26.WT原位移植模型小鼠ER stress和EMT相关蛋白的影响

注：K.假手术组；M.模型组；5-FU.阳性对照组；HEZ-G.芪-术-蚤高剂量组；HEZ-D.芪-术-蚤低剂量组。
与M组比较, ^*P＜0.05, ^**P＜0.01，^***P＜0.001;与5-FU组比较, ^#P＜0.05, ^##P＜0.01;与HEZ-G组比较, ^▲P＜0.05, ^▲▲P＜0.01。

Figure 6. Effects of Qi-Zhu-Zao on ER stress and EMT-related proteins in orthotopic transplantation mice of CT26.WT

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图 7 芪-术-蚤角药配伍抗结直肠癌机制图

Figure 7. Diagram of mechanism hypothesis of Qi-Zhu-Zao (Astragalus mongholicus Bunge-Curcuma aromatica-Rhizoma Paridis) against colon cancer

下载: 全尺寸图片幻灯片

表 1 分组和给药剂量

Table 1. Grouping and dosing

组别	质量比	给药剂量	给药方式	时间/d
假手术组(K)	-	0.01 mL·g^-1	ig×qd	15
模型组(M)	-	0.01 mL·g^-1	ig×qd	15
阳性对照组(5-FU)	-	25 mg·kg^-1	ip×qd	15
芪-术-蚤高剂量组(HEZ-G)	4∶2∶3	5.85 g·kg^-1	ig×qd	15
芪-术-蚤低剂量组(HEZ-D)	4∶2∶3	2.925 g·kg^-1	ig×qd	15

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