切换至 "中华医学电子期刊资源库"

中华脑科疾病与康复杂志(电子版) ›› 2021, Vol. 11 ›› Issue (05) : 285 -293. doi: 10.3877/cma.j.issn.2095-123X.2021.05.007

基础研究

毛蕊花糖苷上调SHP1表达抑制STAT3磷酸化治疗胶质母细胞瘤的分子机制研究
贾伟强1, 王兆涛2, 徐如祥1,()   
  1. 1. 610072 成都,四川省医学科学院·四川省人民医院神经外科
    2. 510260 广州,广州医科大学附属第二医院神经外科
  • 收稿日期:2021-07-05 出版日期:2021-10-15
  • 通信作者: 徐如祥

Molecular mechanism of verbascoside in the treatment of glioblastoma by inhibiting STAT3 phosphorylation by upregulating SHP-1 expression

Weiqiang Jia1, Zhaotao Wang2, Ruxiang Xu1,()   

  1. 1. Department of Neurosurgery, Sichuan Academy of Medical Sciences and Sichuan Provincial People’s Hospital, Chengdu 610072, China
    2. Department of Neurosurgery, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, China
  • Received:2021-07-05 Published:2021-10-15
  • Corresponding author: Ruxiang Xu
引用本文:

贾伟强, 王兆涛, 徐如祥. 毛蕊花糖苷上调SHP1表达抑制STAT3磷酸化治疗胶质母细胞瘤的分子机制研究[J]. 中华脑科疾病与康复杂志(电子版), 2021, 11(05): 285-293.

Weiqiang Jia, Zhaotao Wang, Ruxiang Xu. Molecular mechanism of verbascoside in the treatment of glioblastoma by inhibiting STAT3 phosphorylation by upregulating SHP-1 expression[J]. Chinese Journal of Brain Diseases and Rehabilitation(Electronic Edition), 2021, 11(05): 285-293.

目的

验证毛蕊花糖苷(VB)能否对胶质母细胞瘤产生作用并探究其相关分子机制。

方法

细胞增殖实验检测VB对胶质母细胞瘤细胞增殖的影响。流式细胞术检测细胞凋亡,迁移实验检测胶质母细胞瘤的细胞侵袭迁移能力。利用裸鼠成瘤实验在体内验证VB的抑制肿瘤能力。Western blot检测相关分子机制。

结果

VB处理后,胶质母细胞瘤细胞增殖、迁移、侵袭能力减弱,凋亡增加。Western blot发现相关信号通路酪氨酸磷酸酶1(SHP-1)表达增加,磷酸化信号转导及转录激活子(STAT3)表达下调,下游相关促凋亡基因表达增加,抑制凋亡基因、促进细胞增殖存活、迁移基因表达减少。在裸鼠移植肿瘤模型中,与对照组相比,VB处理后能减小肿瘤体积(P<0.05)。同时,VB能增强传统化学治疗药替莫唑胺的抗胶质母细胞瘤的作用。

结论

VB能通过上调SHP-1的表达抑制STAT3的磷酸化进而产生抗胶质母细胞瘤的作用。

Objective

To confirm the effects of verbascoside (VB) on glioblastoma and explore the potential molecular mechanism.

Methods

The Thiazolyl blue assay was used to test cell proliferation, flow cytometry was used to detect cell apoptosis, and a transwell assay was used for cell migration and invasion. The tumor formation experiment in nude mice was used to verify the tumor inhibition ability of VB in vivo. Western blot analysis was employed to determine the protein expression of related genes.

Results

After VB treatment, the proliferation, migration and invasion of glioblastoma cells decreased, and apoptosis increased. Western blot showed that the expression of related signal pathway Src-homology domain 2-containing protein tyrosine phosphatase 1 (SHP-1) increased, the expression of phosphorylated signal transduction and transcriptional activator (STAT3) down-regulated, the expression of downstream related Pro apoptotic genes increased, and the expression of genes that inhibit apoptotic genes, promote cell proliferation, survival and migration decreased. In nude mice transplanted tumor model, compared with the control group, VB treatment could reduce the tumor volume (P<0.05). At the same time, VB can enhance the anti-glioblastoma effect of temozolomide, a traditional chemotherapeutic drug.

Conclusion

VB can inhibit the phosphorylation of STAT3 by up regulating the expression of SHP-1, and then produce the effect of anti-glioblastoma

图1 VB对胶质母细胞瘤细胞增殖能力的影响不同浓度VB(0、10、20、40、60、80、100 μmol/L)在不同时间(24、48、72 h)分别对U87(A)、A172(B)、T98G(C)、U373(D)、SHG-44(E)和U251(F)细胞抑制率的影响;在同一时间点与上一药物浓度的存活率比较,aP<0.05;在同一药物浓度与上一作用时间点的存活率比较,bP<0.05;VB:毛蕊花糖苷
图2 SHP-1 siRNA转染对于VB抑制U87胶质母细胞瘤增殖作用的影响A:倒置荧光显微镜下观察U87和U251细胞的SHP-1 siRNA转染效率,2 μL invitrogen lipofectamin2000+3 μL FAM-siRNA(浓度20 pm/μL)的比例加入细胞转染24 h后,分别于普通光镜视野下(明场)和荧光镜视野下(暗场)观察细胞状态及荧光细胞数,绿色荧光为成功转入细胞中的带荧光的siRNA;B:U87和U251转染效率比较;C:各组细胞存活率统计直方图;与Control组比较,aP<0.05;与VB组比较,bP<0.05;与si-SHP-1+VB组比较,cP<0.05;SHP-1:酪氨酸磷酸酶1;VB:毛蕊花糖苷;TMZ:替莫唑胺
图3 转染SHP-1 siRNA对VB诱导的U87胶质母细胞瘤细胞凋亡作用的影响A:流式细胞术检测各组U87胶质母细胞瘤细胞凋亡作用;B:各组U87胶质母细胞瘤细胞凋亡率比较;与Control组比较,aP<0.05;与VB组比较,bP<0.05;与si-SHP-1+VB组比较,cP<0.05;VB:毛蕊花糖苷;SHP-1:酪氨酸磷酸酶1;TMZ:替莫唑胺
图4 SHP-1 siRNA转染对VB抑制U87胶质母细胞瘤细胞迁移侵袭作用的影响A:Transwell法检测各组U87胶质母细胞瘤细胞迁移能力;B:各组U87胶质母细胞瘤细胞迁移细胞数比较;C:Transwell法检测各组U87胶质母细胞瘤细胞侵袭能力;D:各组U87胶质母细胞瘤细胞侵袭细胞数比较;与Control组比较,aP<0.05;与VB组比较,bP<0.05;与si-SHP-1+VB组比较,cP<0.05;VB:毛蕊花糖苷;SHP-1:酪氨酸磷酸酶1;TMZ:替莫唑胺
图5 U87细胞SHP-1/STAT3信号通路相关蛋白表达水平A:Western blot蛋白免疫印迹图;B:蛋白相对表达水平比较;与Control组比较,aP<0.05;与VB组比较,bP<0.05;与si-SHP-1+VB组比较,cP<0.05;VB:毛蕊花糖苷;SHP-1:酪氨酸磷酸酶1;TMZ:替莫唑胺;STAT3:信号转导及转录激活子;p-STAT3:磷酸化STAT3
图6 各组裸鼠体质量和移植瘤体积变化情况A:各组裸鼠体质量变化情况;B:各组裸鼠移植瘤体积;与model组比较,aP<0.05;与VB组比较,bP<0.05;与Scramble siRNA+VB组比较,cP<0.05;VB:毛蕊花糖苷;SHP-1:酪氨酸磷酸酶1
图7 各组裸鼠移植瘤中SHP-1/STAT3信号通路相关蛋白表达情况A:Western blot相关蛋白免疫印迹图;B、C:蛋白相对表达水平统计;与model组比较,aP<0.05;与VB组比较,bP<0.05;与si-SHP-1+VB组比较,cP<0.05;VB:毛蕊花糖苷;SHP-1:酪氨酸磷酸酶1;STAT3:信号转导及转录激活子;p-STAT3:磷酸化STAT3;MMP:基质金属蛋白酶;GAPDH:甘油醛-3-磷酸脱氢酶
[1]
Ostrom QT, Gittleman H, Liao P, et al. CBTRUS statistical report: primary brain and central nervous system tumors diagnosed in the United States in 2007-2011[J]. Neuro-oncology, 2014, 16 Suppl 4(suppl 4): iv1-iv63.
[2]
Yan W, Zhang W, You G, et al. Correlation of IDH1 mutation with clinicopathologic factors and prognosis in primary glioblastoma: a report of 118 patients from China[J]. PLoS One, 2012, 7(1): e30339.
[3]
Preusser M, Lim M, Hafler DA, et al. Prospects of immune checkpoint modulators in the treatment of glioblastoma[J]. Nat Rev Neurol, 2015, 11(9): 504-514.
[4]
Kijima N, Hosen N, Kagawa N, et al. Wilms’ tumor 1 is involved in tumorigenicity of glioblastoma by regulating cell proliferation and apoptosis[J]. Anticancer Res, 2014, 34(1): 61-67.
[5]
Prados MD, Byron SA, Tran NL, et al. Toward precision medicine in glioblastoma: the promise and the challenges[J]. Neuro Oncol, 2015, 17(8): 1051-1063.
[6]
Friesen C, Hormann I, Roscher M, et al. Opioid receptor activation triggering downregulation of cAMP improves effectiveness of anti-cancer drugs in treatment of glioblastoma[J]. Cell Cycle, 2014, 13(10): 1560-1570.
[7]
Vertuani S, Beghelli E, Scalambra E, et al. Activity and stability studies of verbascoside, a novel antioxidant, in dermo-cosmetic and pharmaceutical topical formulations[J]. Molecules, 2011, 16(8): 7068-7080.
[8]
Zhou L, Feng Y, Jin Y, et al. Verbascoside promotes apoptosis by regulating HIPK2-p53 signaling in human colorectal cancer[J]. BMC Cancer, 2014, 14: 747.
[9]
Bi L, Yu Z, Wu J, et al. Honokiol inhibits constitutive and inducible STAT3 signaling via PU.1-Induced SHP1 expression in acute myeloid leukemia cells[J]. Tohoku J Exp Med, 2015, 237(3): 163-172.
[10]
Kim DJ, Tremblay ML, Digiovanni J. Protein tyrosine phosphatases, TC-PTP, SHP1, and SHP2, cooperate in rapid dephosphorylation of stat3 in keratinocytes following UVB irradiation[J]. PLoS One, 2010, 5(4): e10290.
[11]
Han Y, Amin HM, Franko B, et al. Loss of SHP1 enhances JAK3/STAT3 signaling and decreases proteosome degradation of JAK3 and NPM-ALK in ALK+ anaplastic large-cell lymphoma[J]. Blood, 2006, 108(8): 2796-2803.
[12]
Pesce M, Franceschelli S, Ferrone A, et al. Verbascoside down-regulates some pro-inflammatory signal transduction pathways by increasing the activity of tyrosine phosphatase SHP-1 in the U937 cell line[J]. J Cell Mol Med, 2015, 19(7): 1548-1556.
[13]
Liu X, Wang X, Du W, et al. Suppressor of fused (Sufu) represses Gli1 transcription and nuclear accumulation, inhibits glioma cell proliferation, invasion and vasculogenic mimicry, improving glioma chemo-sensitivity and prognosis[J]. Oncotarget, 2014, 5(22): 11681-1194.
[14]
Etemad L, Zafari R, Moallem SA, et al. Teratogenic effect of verbascoside, main constituent of lippia citriodora leaves, in mice[J]. Iran J Pharm Res, 2016, 15(2): 521-525.
[15]
Hegazy SA, Wang P, Anand M, et al. The tyrosine 343 residue of nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK) is important for its interaction with SHP1, a cytoplasmic tyrosine phosphatase with tumor suppressor functions[J]. J Biol Chem, 2010, 285(26): 19813-19820.
[16]
Duan S, Yu Y, Lai C, et al. Vincristine-loaded and sgc8-modified liposome as a potential targeted drug delivery system for treating acute lymphoblastic leukemia[J]. J Biomed Nanotechnol, 2018, 14(5): 910-921.
[17]
Hao D, Wen X, Liu L, et al. Sanshool improves UVB-induced skin photodamage by targeting JAK2/STAT3-dependent autophagy[J]. Cell Death Dis, 2019, 10(1): 19.
[18]
Andersson EI, Brück O, Braun T, et al. STAT3 mutation Is associated with STAT3 activation in CD30+ ALK- ALCL[J]. Cancers (Basel), 2020, 12(3): 702.
[19]
Han Y, Amin HM, Frantz C, et al. Restoration of shp1 expression by 5-AZA-2’-deoxycytidine is associated with downregulation of JAK3/STAT3 signaling in ALK-positive anaplastic large cell lymphoma[J]. Leukemia, 2006, 20(9): 1602-1609.
[20]
Xie Z, Koyama T, Suzuki J, et al. Coronary reperfusion following ischemia: different expression of bcl-2 and bax proteins, and cardiomyocyte apoptosis[J]. Jpn Heart J, 2001, 42(6): 759-770.
[21]
Yin Y, Sui C, Meng F, et al. The omega-3 polyunsaturated fatty acid docosahexaenoic acid inhibits proliferation and progression of non-small cell lung cancer cells through the reactive oxygen species-mediated inactivation of the PI3K/Akt pathway[J]. Lipids Health Dis, 2017, 16(1): 87.
[22]
Das T, Bhattacharya S, Biswas A, et al. Inhibition of leukemic U937 cell growth by induction of apoptosis, cell cycle arrest and suppression of VEGF, MMP-2 and MMP-9 activities by cytotoxin protein NN-32 purified from Indian spectacled cobra (Naja naja) venom[J]. Toxicon, 2013, 65: 1-4.
[23]
Yin S, Wu H, Lv J, et al. SHP-1 arrests mouse early embryo development through downregulation of Nanog by dephosphorylation of STAT3[J]. PLoS One, 2014, 9(1): e86330.
[1] 李康, 冀亮, 赵维, 林乐岷. 自噬在乳腺癌生物学进展中的双重作用[J]. 中华乳腺病杂志(电子版), 2023, 17(04): 195-202.
[2] 孔莹莹, 谢璐涛, 卢晓驰, 徐杰丰, 周光居, 张茂. 丁酸钠对猪心脏骤停复苏后心脑损伤的保护作用及机制研究[J]. 中华危重症医学杂志(电子版), 2023, 16(05): 355-362.
[3] 张晓燕, 肖东琼, 高沪, 陈琳, 唐发娟, 李熙鸿. 转录因子12过表达对脓毒症相关性脑病大鼠大脑皮质的保护作用及其机制[J]. 中华妇幼临床医学杂志(电子版), 2023, 19(05): 540-549.
[4] 刘星辰, 刘娟, 魏宝宝, 刘洁, 刘辉. XIAP与XAF1异常表达与卵巢癌的相关性分析[J]. 中华妇幼临床医学杂志(电子版), 2023, 19(04): 419-427.
[5] 黄瑞娟, 德奇, 巴特, 周彪. 对人脐带间充质干细胞外泌体影响热损伤人皮肤成纤维细胞迁移的分析[J]. 中华损伤与修复杂志(电子版), 2023, 18(03): 229-234.
[6] 张生军, 赵阿静, 李守博, 郝祥宏, 刘敏丽. 高糖通过HGF/c-met通路促进结直肠癌侵袭和迁移的实验研究[J]. 中华普外科手术学杂志(电子版), 2024, 18(01): 21-24.
[7] 刘硕儒, 王功炜, 张斌, 李书豪, 胡成. 新型溶瘤病毒M1激活内质网应激致前列腺癌细胞凋亡的机制[J]. 中华腔镜泌尿外科杂志(电子版), 2023, 17(04): 388-393.
[8] 刘雪峰, 韩海峰, 杨硕, 逯景辉. 腹腔镜腹壁侵袭性纤维瘤病切除联合腹壁重建:单中心经验总结[J]. 中华疝和腹壁外科杂志(电子版), 2023, 17(04): 374-379.
[9] 芦丹, 杨硕, 刘旭. VEGF、HMGB1、hs-CRP/Alb在AECOPD伴呼吸衰竭中的变化及预后分析[J]. 中华肺部疾病杂志(电子版), 2023, 16(04): 532-534.
[10] 孟原竹, 蒋国路, 陈小兵, 蒋莉. 肺结核合并侵袭性肺曲霉感染临床特征及危险因素分析[J]. 中华肺部疾病杂志(电子版), 2023, 16(04): 541-543.
[11] 于迪, 于海波, 吴焕成, 李玉明, 苏彬, 陈馨. 发状分裂相关增强子1差异表达对胆固醇刺激下血管内皮细胞的影响[J]. 中华脑科疾病与康复杂志(电子版), 2023, 13(05): 264-270.
[12] 邓世栋, 刘凌志, 郭大勇, 王超, 黄忠欣, 张晖辉. 沉默SNHG1基因对膀胱癌细胞增殖、凋亡、迁移和铁死亡的影响[J]. 中华临床医师杂志(电子版), 2023, 17(07): 804-811.
[13] 张敏洁, 张小杉, 段莎莎, 施依璐, 赵捷, 白天昊, 王雅晳. 氢气治疗心肌缺血再灌注损伤的作用机制及展望[J]. 中华临床医师杂志(电子版), 2023, 17(06): 744-748.
[14] 郭如烨, 孟黎明, 陈楠, 宋玉莹, 尹海燕, 郭岩. Apelin/APJ系统对帕金森病模型的神经保护作用及机制研究进展[J]. 中华诊断学电子杂志, 2023, 11(04): 276-282.
[15] 邱甜, 杨苗娟, 胡波, 郭毅, 何奕涛. 亚低温治疗脑梗死机制的研究进展[J]. 中华脑血管病杂志(电子版), 2023, 17(05): 518-521.
阅读次数
全文


摘要