蠲痛饮对子宫内膜异位症大鼠PI3K/Akt/mTOR信号通路蛋白的影响
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摘要
目的:探讨蠲痛饮对子宫内膜异位症(EMS)大鼠磷脂酰肌醇-3-激酶(PI3K)/蛋白激酶B(Akt)/雷帕霉素靶分子(mTOR)信号通路蛋白的影响。方法:采用自体子宫内膜移植法建立EMS大鼠模型,随机将48只大鼠分为正常组、模型组、蠲痛饮低、中、高剂量组、通路阻滞剂组(LY294002) 6组,分别给予蠲痛饮高、中、低剂量(42. 9,14. 3,4. 8 g·kg~(-1))灌胃,通路阻滞剂组予PI3K通路阻滞剂LY294002(0. 04 g·kg~(-1))腹腔注射,正常组及模型组每天按照10 mL·kg~(-1)给予生理盐水灌胃,各组持续用药28 d。应用透射电镜观察大鼠异位内膜组织超微结构,免疫组化法检测异位内膜组织PI3K,Akt,mTOR蛋白表达,实时荧光定量聚合酶链式反应(Real-time PCR)检测核糖体蛋白S6激酶(p70S6K) mRNA相对含量。结果:与正常组比较,模型组异位内膜PI3K,Akt,mTOR蛋白和p70S6K mRNA的表达都明显升高(P <0. 05);与模型组比较,蠲痛饮中、高剂量、通路阻滞剂组干预后PI3K,Akt,mTOR蛋白和p70S6K mRNA的表达明显降低(P <0. 05)。透射电镜下可见蠲痛饮低、中、高剂量组和通路阻滞剂组均能促进腺上皮细胞萎缩或欠整齐,微绒毛减少或消失,胞核固缩,胞内线粒体肿胀或减少,间质细胞凋亡。结论:PI3K/Akt/mTOR信号通路参与子宫内膜异位症发生;蠲痛饮可能通过下调PI3K/Akt/mTOR通路蛋白的表达及p70S6K mRNA表达,从而抑制上皮间质细胞活性,促进细胞凋亡,治疗子宫内膜异位症。
Objective: To discuss the effect of Juantong decoction on phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin( PI3K/Akt/mTOR) signaling pathway proteins in rats with endometriosis( EMS). Method: The EMS Rat model was established by autologous endometrial transplantation.And 48 rats were randomly divided into 6 group,namely the sham operation group,the model group,the low-dose Juantong decoction group,the middle-dose Juantong decoction group,high-dose Juantong decoction group,and the PI3K pathway blocker group( LY294002). Then,the high-dose Juantong decoction group,the middle-dose Juantong decoction group and the low-dose Juantong decoction group were given different doses( 42. 9,14. 3,4. 8 g·kg~(-1)). The pathway blocker group( LY294002) was given LY294002( 0. 04 g·kg~(-1)) through peritoneal injection weekly. The sham operation group and the model group were given saline irrigation( 10 mL·kg~(-1)). The administration lasted for 28 days. At last,the ectopic endometrial tissues in rats were observed by transmission electron microscope,the proteins of PI3K,Akt and mTOR in the tissue were detected by immunohistochemical method,and the p70 ribosomal S6 kinase( p70 S6 K) mRNA expression of ectopic endometrial tissue was tested by Real-time fluorescence quantitative polymerase chain reaction( Real-time PCR) method. Result: Compared with the normal group,the protein expressions of PI3K,Akt and mTOR and the mRNA expression of p70 S6 K of ectopic endometrium in the model group increased significantly( P < 0. 05). And compared with the model group,the protein expressions of PI3K,Akt and mTOR and the mRNA expression of p70S6K decreased significantly after the intervention with middle and high doses of Juantong decoction and Western medicine Group( P < 0. 05). Under the lens,the glandular epithelial cells in middle and high-dose Juantong decoction and western medicine groups were atrophy or untidy,the microvilli decreased or disappeared,the cell nuclei were shrunk,the mitochondria were visible,and there were apoptotic mesenchymal cells. The glandular epithelial cells in low-dose Juantong decoction group were irregular,the microvilli were decreased,and the mitochondrial swelling was visible in the cytoplasm.Conclusion: The proteins of PI3K/Akt/mTOR signaling pathway participate in the occurrence of endometriosis.Juantong decoction can inhibit the activity of epithelial mesenchymal cells and promote apoptosis by reducing the protein expressions of PI3K,Akt and mTOR and the mRNA expression of P70S6K in endometriotic tissues of model rats,thus inhibiting the PI3K/Akt/mTOR signaling pathway in the treatment of endometriosis.
Objective: To discuss the effect of Juantong decoction on phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin( PI3K/Akt/mTOR) signaling pathway proteins in rats with endometriosis( EMS). Method: The EMS Rat model was established by autologous endometrial transplantation.And 48 rats were randomly divided into 6 group,namely the sham operation group,the model group,the low-dose Juantong decoction group,the middle-dose Juantong decoction group,high-dose Juantong decoction group,and the PI3K pathway blocker group( LY294002). Then,the high-dose Juantong decoction group,the middle-dose Juantong decoction group and the low-dose Juantong decoction group were given different doses( 42. 9,14. 3,4. 8 g·kg~(-1)). The pathway blocker group( LY294002) was given LY294002( 0. 04 g·kg~(-1)) through peritoneal injection weekly. The sham operation group and the model group were given saline irrigation( 10 mL·kg~(-1)). The administration lasted for 28 days. At last,the ectopic endometrial tissues in rats were observed by transmission electron microscope,the proteins of PI3K,Akt and mTOR in the tissue were detected by immunohistochemical method,and the p70 ribosomal S6 kinase( p70 S6 K) mRNA expression of ectopic endometrial tissue was tested by Real-time fluorescence quantitative polymerase chain reaction( Real-time PCR) method. Result: Compared with the normal group,the protein expressions of PI3K,Akt and mTOR and the mRNA expression of p70 S6 K of ectopic endometrium in the model group increased significantly( P < 0. 05). And compared with the model group,the protein expressions of PI3K,Akt and mTOR and the mRNA expression of p70S6K decreased significantly after the intervention with middle and high doses of Juantong decoction and Western medicine Group( P < 0. 05). Under the lens,the glandular epithelial cells in middle and high-dose Juantong decoction and western medicine groups were atrophy or untidy,the microvilli decreased or disappeared,the cell nuclei were shrunk,the mitochondria were visible,and there were apoptotic mesenchymal cells. The glandular epithelial cells in low-dose Juantong decoction group were irregular,the microvilli were decreased,and the mitochondrial swelling was visible in the cytoplasm.Conclusion: The proteins of PI3K/Akt/mTOR signaling pathway participate in the occurrence of endometriosis.Juantong decoction can inhibit the activity of epithelial mesenchymal cells and promote apoptosis by reducing the protein expressions of PI3K,Akt and mTOR and the mRNA expression of P70S6K in endometriotic tissues of model rats,thus inhibiting the PI3K/Akt/mTOR signaling pathway in the treatment of endometriosis.
引文
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[2] Saba L,Sulcis R,Melis G B,et al. Endometriosis:the role of magnetic resonance imaging[J]. Acta Radiol,2015,56(3):355-367.
[3] Vercellini P, Vigano P, Somigliana E, et al.Endometriosis:pathogenesis and treatment[J]. Nat Rev Endocrinol,2014,10(5):261-275.
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[5] GUAN Y T, HUANG Y Q, WU J B, et al.Overexpression of chloride channel-3 is associated with the increased migration and invasion ability of ectopic endometrial cells from patients with endometriosis[J].Hum Reprod,2016,31(5):986-998.
[6] Nissim H,Nahum S. Upstream and downstream of mTOR[J]. Genes Dev,2004,18(16):1926-1945
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[8] Mahaut L,Carole N,Charlotte N,et al. The mTOR/Akt inhibitor t EMSirolimus prevents deep infiltrating endometriosis in mice[J]. Am J Pathol,2011,179(2):880-889.
[9]李卫红,李莉,李卫民,等.蠲痛饮治疗子宫内膜异位症的临床观察[J].四川中医,2009,27(10):79-80.
[10]李卫红,谭文举,李莉.蠲痛饮对大鼠子宫内膜异位症TNF-α、IL-1β和ICAM-1影响[J].中国妇幼保健,2013,28(24):4029-4032.
[11]徐叔云.药理实验方法学[M].北京:人民卫生出版社,2002:203-204.
[12]李卫红,谭文举,李莉.蠲痛饮对大鼠子宫内膜异位症TNF-α、IL-1β和ICAM-1影响[J].中国妇幼保健,2013,28(24):4029-4032.
[13]李卫红,李莉,宋小燕.蠲痛饮对子宫内膜异位症大鼠血管生长因子的影响[J].中国中医药信息杂志,2010,17(1):34-35.
[14]李卫红,李莉,谭文举.蠲痛饮对大鼠子宫内膜异位症微血管密度的影响[J].中国实验方剂学杂志,2011,17(22):181-183.
[15]刘洁云,郭洁,吴雅俊,等,琥珀散加减辨治子宫内膜异位症疼痛(血瘀证)的疗效作用机制[J].中国实验方剂学杂志,2017,23(17):205-230.
[16]周艳,刘明珠,宫瘤消胶囊对子宫内膜异位症气滞血瘀证血管生成机制的影响[J].中国实验方剂学杂志,2017,23(21):200-205.
[17] XU G,ZHANG W,Bertram P,et al. Pharmacogenomic profiling of the PI3K/PTEN-Akt-mTOR pathway in common human tumors[J]. Int Joncol,2004,24(4):893-900.
[18] Pene F,Claessens Y E,Muller O,et al. Role of the phosphatidylinositol 3-kinase/Akt and mTOR/P70S6-kinase pathways in the proliferation and apoptosis in multiple myeloma[J]. Oncogene, 2002,21(43):6587-6597.
[19] Otto C, Wessler S, Fritzemeier K H. Exploiting nongenomic estrogen receptor-mediated signaling for the development of pathway-selective estrogen receptor ligands[J]. Ernst Schering Found Symp Proc,2006(1):163-181.
[20] HUANG S,Houghton P J. Targeting mTOR signaling for cancer therapy[J]. Curr Opin Pharmacol,2003,3(4):371-377.
[21] ZHOU X,TAN M,Hawthorne V S,et al. Activation of the Akt/mammalian target of rapamycin/4E-BP1pathway by Erb B2 overexpression predicts tumor progression in breast cancer[J]. Clin Cancer Res,2004,10(20):6779-6788.
[22] Jung C H,Ro S H,CAO J,et al. mTOR regulation of autophagy[J]. Febs Lett,2010,584(7):1287-1295.
[2] Saba L,Sulcis R,Melis G B,et al. Endometriosis:the role of magnetic resonance imaging[J]. Acta Radiol,2015,56(3):355-367.
[3] Vercellini P, Vigano P, Somigliana E, et al.Endometriosis:pathogenesis and treatment[J]. Nat Rev Endocrinol,2014,10(5):261-275.
[4] Burney R O, Giudice L C. Pathogenesis and pathophysiology of endometriosis[J]. Fertil Steril,2012,98(3):511-519.
[5] GUAN Y T, HUANG Y Q, WU J B, et al.Overexpression of chloride channel-3 is associated with the increased migration and invasion ability of ectopic endometrial cells from patients with endometriosis[J].Hum Reprod,2016,31(5):986-998.
[6] Nissim H,Nahum S. Upstream and downstream of mTOR[J]. Genes Dev,2004,18(16):1926-1945
[7]万鹏云,傅芬,PI3K/Akt/mTOR信号通路对子宫内膜异位症的调节作用[J].中国妇幼保健,2013,28(33):5577-5579.
[8] Mahaut L,Carole N,Charlotte N,et al. The mTOR/Akt inhibitor t EMSirolimus prevents deep infiltrating endometriosis in mice[J]. Am J Pathol,2011,179(2):880-889.
[9]李卫红,李莉,李卫民,等.蠲痛饮治疗子宫内膜异位症的临床观察[J].四川中医,2009,27(10):79-80.
[10]李卫红,谭文举,李莉.蠲痛饮对大鼠子宫内膜异位症TNF-α、IL-1β和ICAM-1影响[J].中国妇幼保健,2013,28(24):4029-4032.
[11]徐叔云.药理实验方法学[M].北京:人民卫生出版社,2002:203-204.
[12]李卫红,谭文举,李莉.蠲痛饮对大鼠子宫内膜异位症TNF-α、IL-1β和ICAM-1影响[J].中国妇幼保健,2013,28(24):4029-4032.
[13]李卫红,李莉,宋小燕.蠲痛饮对子宫内膜异位症大鼠血管生长因子的影响[J].中国中医药信息杂志,2010,17(1):34-35.
[14]李卫红,李莉,谭文举.蠲痛饮对大鼠子宫内膜异位症微血管密度的影响[J].中国实验方剂学杂志,2011,17(22):181-183.
[15]刘洁云,郭洁,吴雅俊,等,琥珀散加减辨治子宫内膜异位症疼痛(血瘀证)的疗效作用机制[J].中国实验方剂学杂志,2017,23(17):205-230.
[16]周艳,刘明珠,宫瘤消胶囊对子宫内膜异位症气滞血瘀证血管生成机制的影响[J].中国实验方剂学杂志,2017,23(21):200-205.
[17] XU G,ZHANG W,Bertram P,et al. Pharmacogenomic profiling of the PI3K/PTEN-Akt-mTOR pathway in common human tumors[J]. Int Joncol,2004,24(4):893-900.
[18] Pene F,Claessens Y E,Muller O,et al. Role of the phosphatidylinositol 3-kinase/Akt and mTOR/P70S6-kinase pathways in the proliferation and apoptosis in multiple myeloma[J]. Oncogene, 2002,21(43):6587-6597.
[19] Otto C, Wessler S, Fritzemeier K H. Exploiting nongenomic estrogen receptor-mediated signaling for the development of pathway-selective estrogen receptor ligands[J]. Ernst Schering Found Symp Proc,2006(1):163-181.
[20] HUANG S,Houghton P J. Targeting mTOR signaling for cancer therapy[J]. Curr Opin Pharmacol,2003,3(4):371-377.
[21] ZHOU X,TAN M,Hawthorne V S,et al. Activation of the Akt/mammalian target of rapamycin/4E-BP1pathway by Erb B2 overexpression predicts tumor progression in breast cancer[J]. Clin Cancer Res,2004,10(20):6779-6788.
[22] Jung C H,Ro S H,CAO J,et al. mTOR regulation of autophagy[J]. Febs Lett,2010,584(7):1287-1295.