加味芍药甘草汤调控P53-273H对子宫腺肌细胞增殖的影响
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摘要
目的研究加味芍药甘草汤通过调控肿瘤抑制因子的突变型P53-273H对子宫腺肌细胞增殖的影响。方法取人子宫腺肌细胞进行细胞培养,随机分为加味芍药甘草汤组、米非司酮组和空白组3组,分别给予对应的药物处理24 h和48 h后,通过流式细胞术(FCM)检测加味芍药甘草汤对人子宫腺肌病病灶细胞凋亡的影响;反转录实时定量PCR(RT-q PCR)技术检测子宫腺肌细胞P53-273H m RNA、白血病抑制因子受体(LIFR)m RNA表达的影响;蛋白质印迹法(Western blot)检测药物对细胞中P53-273H、白血病抑制因子(LIF)、信号转导与转录激活子3(STAT3)、磷酸化的信号转导与转录激活子3(p-STAT3)蛋白表达的影响。结果 FCM检测显示,加味芍药甘草汤组细胞的凋亡率明显高于空白组(P<0.01),其差异具有统计学意义。RT-q PCR检测显示,加味芍药甘草汤组细胞的P53-273H m RNA、LIFR m RNA的相对表达量较空白组低,与空白组比较P<0.05,其差异有统计学意义。Western blot检测显示,加味芍药甘草汤处理24 h和48 h后,子宫腺肌细胞中的P53-273H、LIF、STAT3、p-STAT3蛋白的表达量均低于米非司酮组和空白组,与米非司酮组比较P<0.05,与空白组比较P<0.05,差异均具有统计学意义。结论加味芍药甘草汤能在一定程度上抑制子宫腺肌细胞的增殖、并促进其凋亡,其作用机制可能是加味芍药甘草汤通过调控P53/LIF/STAT3信号通路而实现的。
Objective To study the influence of Jiawei Shaoyao Gancao Tang(Modified Debark Peony Root and Liquorice Root Decoction,JSGT) on proliferation of adenomyosis(AM) cells through regulating P53-273 H. Methods The human AM cells were collected and cultured,and then divided randomly into JSGT group,mifepristone group and blank group. After treatment with corresponding medicinals for 24 h and 48 h,the influence of JSGT on apoptosis of AM focus cells was detected by using flow cytometry(FCM). The expressions of P53-273 H m RNA and LIFR m RNA of AM cells were detected by using realtime reverse transcription quantitative polymerase chain reaction(RT-PCR). The protein expressions of P53-273 H,LIF,STAT3 and p-STAT3 were detected by using Western blotting assay. Results The results of FCM showed that the apoptosis rate was significantly higher in JSGT group than that in blank group(P < 0. 01). The results of real-time RT-PCR showed that the relative expressions of P53-273 H m RNA and LIFR m RNA were lower in JSGT group than those in blank group(P < 0. 05). The results of Western blotting assay showed that,after intervention with JSGT for 24 h and 48 h,the protein expressions of P53-273 H,LIF,STAT3 and p-STAT3 were lower in JSGT group than those in mifepristone group(P < 0. 05) and blank group(P < 0. 05). Conclusion JSGT can inhibit the proliferation and improve the apoptosis of AM cells,and the mechanism may be related to that JSGT can regulate P53/LIF/STAT3 signal pathway.
Objective To study the influence of Jiawei Shaoyao Gancao Tang(Modified Debark Peony Root and Liquorice Root Decoction,JSGT) on proliferation of adenomyosis(AM) cells through regulating P53-273 H. Methods The human AM cells were collected and cultured,and then divided randomly into JSGT group,mifepristone group and blank group. After treatment with corresponding medicinals for 24 h and 48 h,the influence of JSGT on apoptosis of AM focus cells was detected by using flow cytometry(FCM). The expressions of P53-273 H m RNA and LIFR m RNA of AM cells were detected by using realtime reverse transcription quantitative polymerase chain reaction(RT-PCR). The protein expressions of P53-273 H,LIF,STAT3 and p-STAT3 were detected by using Western blotting assay. Results The results of FCM showed that the apoptosis rate was significantly higher in JSGT group than that in blank group(P < 0. 01). The results of real-time RT-PCR showed that the relative expressions of P53-273 H m RNA and LIFR m RNA were lower in JSGT group than those in blank group(P < 0. 05). The results of Western blotting assay showed that,after intervention with JSGT for 24 h and 48 h,the protein expressions of P53-273 H,LIF,STAT3 and p-STAT3 were lower in JSGT group than those in mifepristone group(P < 0. 05) and blank group(P < 0. 05). Conclusion JSGT can inhibit the proliferation and improve the apoptosis of AM cells,and the mechanism may be related to that JSGT can regulate P53/LIF/STAT3 signal pathway.
引文
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[15]Levine AJ,Tomasini R,Mckeon FD,et al.The p53 family:guardians of maternal reproduction[J].Nature Reviews Molecular Cell Biology,2017,12(4):259-265.
[16]Feng Z,Zhang C,Kang HJ,et al.Regulation of female reproduction by p53 and its family members[J].Faseb Journal Official Publication of the Federation of American Societies for Experimental Biology,2011,25(7):2464-2468.
[17]Subramani E,Madogwe E,Bordignon V,et al.Dysregulated LIF and its receptor regulated STAT3 pathway:a possible cause for repeated implantation failure in women with dormant genital tuberculosis?[J].Fertility&Sterility,2016,105(4):1076-1084.
[2]Struble J,Reid S,Bedaiwy MA.Adenomyosis;A Clinical Review of a Challenging Gynecologic Condition[J].Journal of Minimally Invasive Gynecology,2015,23(2):164-185.
[3]Gordts S,Campo R,Brosens I.Hysteroscopic diagnosis and excision of myometrial cystic adenomyosis[J].Gynecological Surgery,2014,11(4):273-278.
[4]Schindler AE.Non-contraceptive benefits of oral hormonal contraceptives[J].Int J Endocrinol Metab,2013,11(1):41-47.
[5]王帅.加味芍药甘草汤对子宫腺肌病MAPK/ERK信号通路干预机制[D].广州:广州中医药大学,2015.Wang S.Effect of Modified Shaoyaogancao Decoction on adenomyosis via MAPK/ERK signal pathway[D].Guangzhou:Guangzhou University of Chinese Medicine,2015.
[6]曾玉燕,李坤寅,关永格.加味芍药甘草汤及含药血清对子宫腺肌病E2、ER及芳香化酶P450的影响[J].北京中医药大学学报,2017,40(9):722-728.Zeng YY,Li KY,Guan YG.Influence of Jiawei Shaoyao Gancao Tang and its medicated serum on estrogen,estrogen receptor and aromatase P450 in treatment of adenomyosis[J].Journal of Beijing University of Traditional Chinese Medicine,2017,40(9):722-728.
[7]Garcia L,Isaacson K.Adenomyosis:review of the literature[J].Journal of Minimally Invasive Gynecology,2011,18(4):428-437.
[8]Gordts S,Campo R,Brosens I.Hysteroscopic diagnosis and excision of myometrial cystic adenomyosis[J].Gynecological Surgery,2014,11(4):273-278.
[9]Akira S,Mine K,Kuwabara Y,et al.Efficacy of longterm,low-dose gonadotropin-releasing hormone agonist therapy(draw-back therapy)for adenomyosis[J/OL].Medical Science Monitor,2009,15(1):CR1-4[2009-01-02].http://www.medscimonit.com/fulltxt.php?ICID=869510.
[10]Liu X,Wang W,Wang Y,et al.Clinical Predictors of Long-term Success in Ultrasound-guided High-intensity Focused Ultrasound Ablation Treatment for Adenomyosis:A Retrospective Study[J/OL].Medicine,2016,95(3):e2443[2016-01-22].http://210.38.102.135:8000/rwt/32/http/MSYXTLUQPJUB/10.1097/MD.0000000000002443.DOI:10.1097/MD.0000000000002443.
[11]Stoner CS,Pearson GD,Ko9 A,et al.Effect of thioredoxin deletion and p53 cysteine replacement on human p53 activity in wild-type and thioredoxin reductase null yeast[J].Biochemistry,2009,48(38):9156-9169.
[12]Munksgaard PS,Blaakaer J.The association between endometriosis and ovarian cancer:a review of histological,genetic and molecular alterations[J].Gynecologic Oncology,2012,124(1):164-169.
[13]Bischoff FZ,Heard M,Simpson JL.Somatic DNA alterations in endometriosis:high frequency of chromosome 17and p53 loss in late-stage endometriosis[J].Journal of Reproductive Immunology,2002,55(1/2):49-64.
[14]Xu W,Harrison SC,Eck MJ.Three-dimensional structure of the tyrosine kinase c-Src[J].Nature,1997,385(6617):595-602.
[15]Levine AJ,Tomasini R,Mckeon FD,et al.The p53 family:guardians of maternal reproduction[J].Nature Reviews Molecular Cell Biology,2017,12(4):259-265.
[16]Feng Z,Zhang C,Kang HJ,et al.Regulation of female reproduction by p53 and its family members[J].Faseb Journal Official Publication of the Federation of American Societies for Experimental Biology,2011,25(7):2464-2468.
[17]Subramani E,Madogwe E,Bordignon V,et al.Dysregulated LIF and its receptor regulated STAT3 pathway:a possible cause for repeated implantation failure in women with dormant genital tuberculosis?[J].Fertility&Sterility,2016,105(4):1076-1084.