和厚朴酚调控Nrf2/ARE通路对胃癌细胞的顺铂化疗敏感性的影响

doi:10.3877/cma.j.issn.2095-1221.2023.04.002

目的

探讨和厚朴酚调节核转录相关因子2 （Nrf2）/抗氧化反应元件（ARE）通路对胃癌细胞顺铂耐药的影响。

方法

体外培养人胃癌顺铂耐药细胞SGC7901/DDP，采用5 μg/mL、15 μg/mL和厚朴酚及15 μg/mL和厚朴酚联合1 mmol/L Nrf2/ARE通路激活剂富马酸二甲酯（DMF）进行干预；MTT法检测细胞增殖；划痕实验、Transwell小室检测细胞迁移、侵袭；流式细胞仪检测细胞凋亡；Western blot检测细胞Nrf2、血红素加氧酶1 （HO-1）、P-糖蛋白（P-gp）、多药耐药相关蛋白1 （MRP1）、增殖细胞核抗原（PCNA）、基质金属蛋白酶2 （MMP2）、基质金属蛋白酶9 （MMP9）、半胱氨酸蛋白酶3 （caspase-3）和活化的caspase-3 （cleaved caspase-3）的表达；构建SGC7901/DDP移植瘤裸鼠模型，随机分为对照组、低剂量和厚朴酚组、高剂量和厚朴酚组、Nrf2/ARE通路激活剂组，分组处理后检测其肿瘤体积与重量。单因素方差分析和Tukey检验用于多组间的比较。

结果

与对照组比较，低、高剂量和厚朴酚组OD₄₉₀值、划痕愈合率[（31.24 ± 2.23）%、（25.36 ± 2.06）%比（48.01 ± 2.13）%]、侵袭率[（38.61 ± 2.24）%、（29.57 ± 2.41）%比（52.36 ± 2.13）%]、肿瘤体积与质量、HO-1、P-gp、MRP1、PCNA、MMP2、MMP9、核Nrf2蛋白表达均降低（P均< 0.05），凋亡率[（27.24 ± 2.24）%、（38.56 ± 2.31）%比（17.31 ± 3.12）%]、caspase-3和cleaved caspase-3蛋白表达升高（P < 0.05）；与高剂量和厚朴酚组比较，Nrf2/ARE通路激活剂组的OD₄₉₀值、划痕愈合率[（37.71 ± 2.17）%比（25.36 ± 2.06）%]、侵袭率[（43.53 ± 2.37）%比（29.57 ± 2.41）%]、肿瘤体积与质量、HO-1、P-gp、MRP1、PCNA、MMP2、MMP9和核Nrf2蛋白表达均升高（P < 0.05），凋亡率[（21.48 ± 2.16）%比（38.56 ± 2.31）%]、caspase-3和cleaved caspase-3蛋白表达降低（P均< 0.05）。

结论

和厚朴酚可能通过调控Nrf2/ARE信号通路进而影响胃癌耐药细胞株SGC7901/DDP的顺铂化疗敏感性。

关键词: 和厚朴酚, Nrf2/ARE, 胃癌, 顺铂耐药

Objective

To investigate the influence of honokiol on cisplatin resistance in gastric cancer cells by regulating the nuclear transcription-related factor 2 (Nrf2) /antioxidant response element (ARE) pathway.

Methods

Human drug-resistant gastric cancer cell line SGC7901/DDP was cultured in vitro and treated with 5, 15 μg/mL honokiol, and 15 μg/mL honokiol + 1 mmol/L Nrf2/ARE pathway activator dimethyl fumarate (DMF) ; MTT assay was applied to detect cell proliferation; scratch assay and Transwell chamber were used to detect cell migration and invasion respectively; flow cytometry was used to detect apoptosis; and Western blot was applied to detect the expression of cellular Nrf2, heme oxygenase 1 (HO-1) , P-glycoprotein (P-gp) , multidrug resistance-related protein 1 (MRP1) , proliferating cell nuclear antigen (PCNA) , matrix metalloproteinase 2 (MMP2) , matrix metalloproteinase 9 (MMP9) , and cysteine protease 3 (caspase-3) and cleaved caspase-3; the SGC7901/DDP transplanted tumor nude mouse model has been established, and then randomly divide them into a control group, low-dose honokiol group, high-dose honokiol group, and Nrf2/ARE pathway activator group. The tumor volume and weight were detected after grouping processing. One-way ANOVA and Tukey tests were used to compare differences between groups.

Results

Compared with the control group, the OD₄₉₀ value, scratch healing rate [ (31.24 ± 2.23) %, (25.36 ± 2.06) % vs (48.01 ± 2.13) %], invasion rate [ (38.61 ± 2.24) %, (29.57 ± 2.41) % vs (52.36 ± 2.13) %], tumor volume and mass, protein expressions of HO-1, P-gp, MRP1, PCNA, MMP2, MMP9 and nuclear Nrf2 were decreased in low and high-dose honokiol groups (all P < 0.05) , the apoptosis rate [ (27.24 ± 2.24) %, (38.56 ± 2.31) % vs (17.31 ± 3.12) %] and the protein expression of caspase-3 and cleaved caspase-3 were increased (all P < 0.05) ; compared with high-dose honokiol group, the OD₄₉₀ value, scratch healing rate [ (37.71 ± 2.17) % vs (25.36 ± 2.06) %], invasion rate [ (43.53 ± 2.37) % vs (29.57 ± 2.41) %], tumor volume and mass, protein expressions of HO-1, P-gp, MRP1, PCNA, MMP2, MMP9 and nuclear Nrf2 in the Nrf2/HO-1 pathway activator group were increased (all P < 0.05) , the apoptosis rate [ (21.48 ± 2.16) % vs (38.56 ± 2.31) %] and the protein expression of caspase-3 and cleaved caspase-3 were decreased (all P < 0.05) .

Conclusion

Honokiol may affect the cisplatin chemosensitivity of drug-resistant gastric cancer cell line SGC7901/DDP by regulating the Nrf2/ARE signaling pathway.

Key words: Honokiol, Nrf2/ARE, Gastric cancer, Cisplatin resistance

图1 SGC7901/DDP细胞OD₄₉₀值比较注：与对照比较，^aP < 0.05；与低剂量和厚朴酚比较，^bP < 0.05；与高剂量和厚朴酚比较，^cP < 0.05；n = 6

图2 SGC7901/DDP细胞划痕愈合率比较注：与对照比较，^aP < 0.05；与低剂量和厚朴酚比较，^bP < 0.05；与高剂量和厚朴酚比较，^cP < 0.05。n = 6

图3 划痕实验检测SGC7901/DDP细胞迁移注：低、高剂量和厚朴酚能够抑制细胞迁移，Nrf2/ARE通路激活剂恢复了高剂量和厚朴酚对细胞迁移的抑制作用

图4 SGC7901/DDP细胞侵袭率比较注：与对照比较，^aP < 0.05；与低剂量和厚朴酚组比较，^bP < 0.05；与高剂量和厚朴酚组比较，^cP < 0.05；n = 6

图5 Transwell小室实验检测SGC7901/DDP细胞侵袭（结晶紫染色，× 400）注：a ~ d图分别为对照、低剂量和厚朴酚、高剂量和厚朴酚和Nrf2/ARE通路激活剂组细胞侵袭，可见低、高剂量和厚朴酚可抑制细胞侵袭，Nrf2/ARE通路激活剂恢复了高剂量和厚朴酚对细胞侵袭的抑制作用

图6 SGC7901/DDP细胞凋亡的代表性流式细胞术检测结果注：a ~ d图分别为对照、低剂量和厚朴酚、高剂量和厚朴酚和Nrf2/ARE通路激活剂组，可见低、高剂量和厚朴酚可促进细胞凋亡，Nrf2/ARE通路激活剂恢复了高剂量和厚朴酚对细胞凋亡的促进作用

图7 SGC7901/DDP细胞凋亡率比较注：与对照比较，^aP < 0.05；与低剂量和厚朴酚组比较，^bP < 0.05；与高剂量和厚朴酚组比较，^cP < 0.05；n = 6

图8 Western blot检测SGC7901/DDP细胞中Nrf2、HO-1、PCNA蛋白表达注：1为对照，2为低剂量和厚朴酚，3为高剂量和厚朴酚，4为Nrf2/ARE通路激活剂

图9 Western blot检测MMP2、MMP9、caspase-3、cleaved caspase-3蛋白表达注：1为对照，2为低剂量和厚朴酚，3为高剂量和厚朴酚，4为Nrf2/ARE通路激活剂

表1 SGC7901/DDP细胞中Nrf2、HO-1、PCNA、MMP2、MMP9、caspase-3蛋白表达比较（ ± s，n = 6）

分组	PCNA	MMP2	MMP9	核Nrf2	HO-1	caspase-3	cleaved caspase-3
对照	1.16 ± 0.13	1.44 ± 0.12	1.32 ± 0.16	1.21 ± 0.14	1.35 ± 0.21	1.02 ± 0.08	0.82 ± 0.05
低剂量和厚朴酚	0.57 ± 0.10^a	0.75 ± 0.10^a	0.62 ± 0.07^a	0.72 ± 0.11^a	0.81 ± 0.13^a	1.43 ± 0.15^a	1.11 ± 0.12^a
高剂量和厚朴酚	0.38 ± 0.07^ab	0.55 ± 0.08^ab	0.44 ± 0.06^ab	0.53 ± 0.07^ab	0.52 ± 0.11^ab	1.91 ± 0.16^ab	1.53 ± 0.14^ab
Nrf2/ARE通路激活剂	0.79 ± 0.06^c	1.06 ± 0.11^c	1.03 ± 0.04^c	0.94 ± 0.13^c	1.07 ± 0.15^c	1.22 ± 0.07^c	0.90 ± 0.06^c
F值	76.045	86.326	106.280	38.430	31.655	58.949	60.449
P值	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001

表1 SGC7901/DDP细胞中Nrf2、HO-1、PCNA、MMP2、MMP9、caspase-3蛋白表达比较（ ± s，n = 6）

分组	PCNA	MMP2	MMP9	核Nrf2	HO-1	caspase-3	cleaved caspase-3
对照	1.16 ± 0.13	1.44 ± 0.12	1.32 ± 0.16	1.21 ± 0.14	1.35 ± 0.21	1.02 ± 0.08	0.82 ± 0.05
低剂量和厚朴酚	0.57 ± 0.10^a	0.75 ± 0.10^a	0.62 ± 0.07^a	0.72 ± 0.11^a	0.81 ± 0.13^a	1.43 ± 0.15^a	1.11 ± 0.12^a
高剂量和厚朴酚	0.38 ± 0.07^ab	0.55 ± 0.08^ab	0.44 ± 0.06^ab	0.53 ± 0.07^ab	0.52 ± 0.11^ab	1.91 ± 0.16^ab	1.53 ± 0.14^ab
Nrf2/ARE通路激活剂	0.79 ± 0.06^c	1.06 ± 0.11^c	1.03 ± 0.04^c	0.94 ± 0.13^c	1.07 ± 0.15^c	1.22 ± 0.07^c	0.90 ± 0.06^c
F值	76.045	86.326	106.280	38.430	31.655	58.949	60.449
P值	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001

图10 SGC7901/DDP细胞中P-gp、MRP1耐药蛋白表达注：a图为SGC7901/DDP细胞耐药蛋白表达的统计学分析；b图为Western blot检测SGC7901/DDP细胞耐药蛋白相对表达；1为对照，2为低剂量和厚朴酚，3为高剂量和厚朴酚，4为Nrf2/ARE通路激活剂；与对照比较，^aP < 0.05；与低剂量和厚朴酚组比较，^bP < 0.05；与高剂量和厚朴酚组比较，^cP < 0.05

图11 各组移植瘤裸鼠肿瘤生长情况注：a图为各组移植瘤裸鼠肿瘤大小图片（比例尺：5 mm），b图为各组移植瘤裸鼠肿瘤体积，c图为各组移植瘤裸鼠肿瘤重量；与对照比较，^aP < 0.05；与低剂量和厚朴酚组比较，^bP < 0.05；与高剂量和厚朴酚组比较，^cP < 0.05

21	Qi MM, Chen XJ, Bian LQ, et al. Honokiol combined with curcumin sensitizes multidrug-resistant human lung adenocarcinoma A549/DDP cells to cisplatin[J]. Exp Ther Med, 2021, 22(5):1301.
22	王丽君，王世广，邓同兴.汉黄芩苷通过抑制PI3K/Akt/Nrf2/ARE通路逆转SGC7901/cDDP细胞耐药性[J]. 生理学报，2018，70(4):397-405.
23	Liu JZ, Hu YL, Feng Y, et al. BDH2 triggers ROS-induced cell death and autophagy by promoting Nrf2 ubiquitination in gastric cancer[J]. J Exp Clin Cancer Res, 2020, 39(1):123.
24	Hong Y, Liu J, Kong W, et al. WASH regulates the oxidative stress Nrf2/ARE pathway to inhibit proliferation and promote apoptosis of HeLa cells under the action of Jolkinolide B[J]. PeerJ, 2022, 10:e13499.
25	Wang H, Wu J, Fan H, et al. The impact of catalpol on proliferation, apoptosis, migration, and oxidative stress of lung cancer cells based on Nrf2/ARE signaling[J]. Biomed Res Int, 2022, 2022:5621341. doi: 10.1155/2022/5621341.
26	Chen M, Huang SL, Zhang XQ, et al. Reversal effects of pantoprazole on multidrug resistance in human gastric adenocarcinoma cells by down-regulating the V-ATPases/mTOR/HIF-1α/P-gp and MRP1 signaling pathway in vitro and in vivo[J]. J Cell Biochem, 2012, 113(7):2474-2487.
1	Stewart A, Wu F, Chen Y. The role of gastric microbiota in gastric cancer[J]. Gut Microbes, 2020, 11(5):1220-1230.
2	Machlowska J, Baj J, Sitarz M, et al. Gastric cancer: epidemiology, risk factors, classification, genomic characteristics and treatment strategies[J]. Int J Mol Sci, 2020, 21(11): 4012.
3	Wei L, Sun J J, Zhang N S, et al. Noncoding RNAs in gastric cancer: Implications for drug resistance[J]. Mol Cancer, 2020, 19(1):62.
4	王春玲，赖小平，吴安国. 和厚朴酚与青蒿素体外抗人胃癌MGC-803细胞的作用研究[J]. 时珍国医国药, 2012, 23(2):407-410.
5	Yan Y, Xu J, Mao G. Honokiol suppression of human epidermal growth factor receptor 2 (HER2)-positive gastric cancer cell biological activity and its mechanism[J]. Med Sci Monit, 2020, 26(1):e923962.
6	Yi X, Lou L, Wang J, et al. Honokiol antagonizes doxorubicin resistance in human breast cancer via miR-188-5p/FBXW7/c-Myc pathway[J]. Cancer Chemother Pharmacol, 2021, 87(5):647-656.
7	耿苗，唐修文. Nrf2/ARE信号通路与胃癌耐药关系的研究进展[J]. 中国细胞生物学学报，2012, 34(11):1129-1133.
8	王春玲，赖小平，吴安国. 和厚朴酚与青蒿素体外抗人胃癌MGC-803细胞的作用研究[J].时珍国医国药，2012，23（2）:407-410.
9	Li P, Hu J R, Shi B Z, et al. Baicalein enhanced cisplatin sensitivity of gastric cancer cells by inducing cell apoptosis and autophagy via Akt/mTOR and Nrf2/Keap1 pathway[J]. Biochem Biophys Res Commun, 2020, 531(3):320-327.
10	韦尉元，孔凡彪，王晓通，等. RNA干扰鼠尾同源盒转录因子2基因沉默逆转人胃癌裸鼠耐药模型耐药性的研究[J]. 中华实验外科杂志，2013，30(2):357.
11	肖嘎，杨锐，党玲，等.和厚朴酚通过激活JNK信号通路强化TRAIL的抗肝细胞癌HepG2细胞作用的机制[J]. 中华肝脏病杂志，2018，26(6):441-445.
12	Liu H, Feng X D, Yang B, et al. Dimethyl fumarate suppresses hepatocellular carcinoma progression via activating SOCS3/JAK1/STAT3 signaling pathway[J]. Am J Transl Res, 2019, 11(8):4713-4725.
13	王颖，左方，韩凤梅，等.进展期胃癌腹腔镜切除术后炎性反应和疼痛因子研究[J]. 中国内镜杂志，2022，28(4):19-24.
14	Kawai A, Matsumoto H, Endou Y, et al. Repeated combined chemotherapy with cisplatin lowers carnitine levels in gastric cancer patients[J]. Ann Nutr Metab, 2017, 71(3-4):261-265.
15	Liu Y, Liu C Q, Tan T, et al. Sinomenine sensitizes human gastric cancer cells to cisplatin through negative regulation of PI3K/AKT/Wnt signaling pathway.[J]. Anticancer drugs, 2019, 30(10):983-990.
16	叶青，王瑞平，邹玺.中药逆转胃癌多药耐药作用的研究进展[J]. 中华中医药杂志，2016，31(8):3194-3197.
17	Zhao HY, Zhao DL, Jin HL, et al. Bufalin reverses intrinsic and acquired drug resistance to cisplatin through the AKT signaling pathway in gastric cancer cells[J]. Mol Med Rep, 2016, 14(2):1817-1822.
18	Huang XF, Qian J, Li LC, et al. Curcumol improves cisplatin sensitivity of human gastric cancer cells through inhibiting PI3K/AKT pathway[J]. Drug Dev Res, 2020, 81(8):217-229.
19	成旭东，陈婷，范玲，等. 和厚朴酚通过NF-κB信号通路抑制IL-1诱导的人肺癌细胞H460血管生成[J]. 中国药理学通报，2022，38(3):380-386.
20	Yan B, Peng ZY. Honokiol induces cell cycle arrest and apoptosis in human gastric carcinoma MGC-803 cell line[J]. Int J Clin Exp Med, 2015, 8(4):5454-5461.

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The effect of honokiol on the sensitivity of gastric cancer cells to cisplatin chemotherapy by regulating the Nrf2/ARE pathway

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The effect of honokiol on the sensitivity of gastric cancer cells to cisplatin chemotherapy by regulating the Nrf2/ARE pathway