OSI-027抑制mTOR信号通路减轻高氧诱导的幼鼠肺损伤
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摘要
目的:探讨哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,m TOR)1/2双重抑制剂OSI-027对高体积分数氧(高氧)致sprague-dawley(SD)幼鼠肺损伤及纤维化的抑制作用。方法:72只3周龄SD幼鼠随机分为空气+生理盐水、高氧+生理盐水、高氧+雷帕霉素和高氧+OSI-027组,分别建立动物模型(各组n=18)。高氧干预采用90%氧气持续处理,生理盐水、雷帕霉素、OSI-027干预分别于观察期第1、3、6、8、10、13天经腹腔注射给药,在造模第3、7、14天取各组幼鼠测量体质量变化、肺湿干重比(wet/dry ratio,W/D)、肺组织病理学检查、肺损伤评分、肺泡间隔厚度测定、肺组织免疫组化和蛋白印迹检测m TOR及磷酸化核糖体S6蛋白激酶(pS6K1)蛋白在肺组织的分布和表达。结果:从时间因素看,各组幼鼠体质量(F时间=297.098,P=0.000)、mTOR免疫组化(F时间=379.978,P=0.000)、mTOR(F时间=166.991,P=0.000)和pS6K1(F时间=122.676,P=0.000)蛋白水平都随时间延长而增加。除空气组外,其余各组肺损伤评分(F时间=1410.362,P=0.000)、肺泡间隔厚度(F时间=356.312,P=0.000)、pS6K1免疫组化(F时间=57.992,P=0.000)都随时间延长而升高,肺W/D(F时间=28.915,P=0.000)第3、7天时升高,第14天时下降。从分组因素看,体质量(F分组=176.597,P=0.000)空气组明显高于其他组,肺W/D(F分组=28.484,P=0.000)和肺泡间隔厚度(F分组=296.223,P=0.000)空气组明显低于其他组,除第3天外,mTOR免疫组化(F分组=134.100,P=0.000)高氧组明显高于其他组,PS6K1免疫组化(F分组=234.697,P=0.000)、mTOR(F分组=59.377,P=0.000)和PS6K1(F分组=101.837,P=0.000)蛋白印迹高氧组明显高于其他组,肺损伤评分(F分组=2 420.076,P=0.000)高氧雷帕组明显高于其他组,高氧OSI组明显低于高氧组和高氧雷帕组。结论:高浓度氧可激活肺组织mTOR信号途径;mTOR可能促进了高氧肺损伤纤维化的发生发展,其调控机制可能与抑制mTOR信号通路的活化有关。mTOR复合物1/2(mTORC1/2)双重抑制剂OSI-027能减轻高氧致SD幼鼠肺损伤及纤维化。
Objective:To investigate the inhibitory effect of the mammalian target of rapamycin(mTOR) 1/2 dual inhibitor OSI-027 on hyperoxia-induced lung injury and fibrosis in juvenile Sprague-Dawley(SD) rats. Methods:A total of 72 juvenile SD rats aged 3 weeks were randomly divided into air+normal saline group,hyperoxia+normal saline group,hyperoxia+rapamycin group,and hyperoxia+OSI-027 group,with 18 rats in each group. An animal model was established. Hyperoxia intervention was performed with 90% oxy gen,and normal saline,rapamycin,and OSI-027 interventions were performed via intraperitoneal injection on days 1,3,6,8,10,and13 of observation,respectively. On days 3,7,and 14,the change in body weight,lung wet/dry(W/D) ratio,lung injury scores,and alveolar septal thickness were measured;lung histopathological examination was performed;immunohistochemistry and Western blot were used to evaluate the distribution and expression of mTOR and phosphorylated ribosomal S6 kinase(pS6 K1) in lung tissue. Results:As for the factor of time,there were significant increases over time in body weight(Ftime=297.098,P=0.000),immunohistochemistry of m TOR(Ftime=379.978,P=0.000),m TOR(Ftime=166.991,P=0.000),and pS6 K1(Ftime=122.676,P=0.000). All groups except the air+normal saline group had significant increases in lung injury scores(Ftime=1 410.362,P=0.000),alveolar septum thickness(Ftime=356.312,P=0.000),and pS6 K1 immunohistochemistry(Ftime=57.992,P=0.000) over time,as well as an increase in lung W/D ratio on days 3 and 7(Ftime=28.915,P=0.000) and a reduction in lung W/D ratio on day 14. As for the factor of grouping,the air+normal saline group had a significantly higher body weight(Fgroup=176.597,P=0.000) and significantly lower lung W/D ratio(Fgroup=28.484,P=0.000) and alveolar septum thickness(Fgroup=296.223,P=0.000) than the other groups. At all time points except day 3,the hyperoxia+normal saline group had significantly higher mTOR immunohistochemistry(Fgroup=134.100,P=0.000),pS6 K1 immunohistochemistry(Fgroup=234.697,P=0.000),mTOR(Fgroup=59.377,P=0.000),and p S6 K1(Fgroup=101.837,P=0.000) than the other groups;the hyperoxia+rapamycin group had significantly higher lung injury scores than the other groups(Fgroup=2 420.076,P=0.000),and the hyperoxia+OSI-027 group had significantly lower scores than the hyperoxia+normal saline group and the hyperoxia+rapamycin group. Conclusion:A high concentration of oxygen can activate the mTOR signaling pathway in lung tissue;mTOR may promote the development and progression of hyperoxia-induced pulmonary fibrosis,possibly by inhibiting activation of the mTOR signaling pathway. OSI-027 can alleviate hyperoxia-induced lung injury and fibrosis in juvenile SD rats.
Objective:To investigate the inhibitory effect of the mammalian target of rapamycin(mTOR) 1/2 dual inhibitor OSI-027 on hyperoxia-induced lung injury and fibrosis in juvenile Sprague-Dawley(SD) rats. Methods:A total of 72 juvenile SD rats aged 3 weeks were randomly divided into air+normal saline group,hyperoxia+normal saline group,hyperoxia+rapamycin group,and hyperoxia+OSI-027 group,with 18 rats in each group. An animal model was established. Hyperoxia intervention was performed with 90% oxy gen,and normal saline,rapamycin,and OSI-027 interventions were performed via intraperitoneal injection on days 1,3,6,8,10,and13 of observation,respectively. On days 3,7,and 14,the change in body weight,lung wet/dry(W/D) ratio,lung injury scores,and alveolar septal thickness were measured;lung histopathological examination was performed;immunohistochemistry and Western blot were used to evaluate the distribution and expression of mTOR and phosphorylated ribosomal S6 kinase(pS6 K1) in lung tissue. Results:As for the factor of time,there were significant increases over time in body weight(Ftime=297.098,P=0.000),immunohistochemistry of m TOR(Ftime=379.978,P=0.000),m TOR(Ftime=166.991,P=0.000),and pS6 K1(Ftime=122.676,P=0.000). All groups except the air+normal saline group had significant increases in lung injury scores(Ftime=1 410.362,P=0.000),alveolar septum thickness(Ftime=356.312,P=0.000),and pS6 K1 immunohistochemistry(Ftime=57.992,P=0.000) over time,as well as an increase in lung W/D ratio on days 3 and 7(Ftime=28.915,P=0.000) and a reduction in lung W/D ratio on day 14. As for the factor of grouping,the air+normal saline group had a significantly higher body weight(Fgroup=176.597,P=0.000) and significantly lower lung W/D ratio(Fgroup=28.484,P=0.000) and alveolar septum thickness(Fgroup=296.223,P=0.000) than the other groups. At all time points except day 3,the hyperoxia+normal saline group had significantly higher mTOR immunohistochemistry(Fgroup=134.100,P=0.000),pS6 K1 immunohistochemistry(Fgroup=234.697,P=0.000),mTOR(Fgroup=59.377,P=0.000),and p S6 K1(Fgroup=101.837,P=0.000) than the other groups;the hyperoxia+rapamycin group had significantly higher lung injury scores than the other groups(Fgroup=2 420.076,P=0.000),and the hyperoxia+OSI-027 group had significantly lower scores than the hyperoxia+normal saline group and the hyperoxia+rapamycin group. Conclusion:A high concentration of oxygen can activate the mTOR signaling pathway in lung tissue;mTOR may promote the development and progression of hyperoxia-induced pulmonary fibrosis,possibly by inhibiting activation of the mTOR signaling pathway. OSI-027 can alleviate hyperoxia-induced lung injury and fibrosis in juvenile SD rats.
引文
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[12] Kallet RH,Matthay MA. Hyperoxic acute lung injury[J]. Respir Care,2013,58(1):123-141.
[13] Tee AR,Blenis J. mTOR,translational control and human disease[J]. Semin Cell Dev Biol,2005,16(1):29-37.
[14] Yu KR,Park SB,Jung JW,et al. HMGA2 regulates the in vitro aging and proliferation of human umbilical cord blood-derived stromal cells through the mTOR/p70S6K signaling pathway[J]. Stem Cell Res,2013,10(2):156-165.
[15] Parrales A,López E,Lee-Rivera I,et al. ERK1/2-dependent activation of m TOR/mTORC1/p70S6K regulates thrombin-induced RPE cell proliferation[J]. Cell Signal,2013,25(4):829-838.
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[17] Dowling RJ,Topisirovic I,Fonseca BD,et al. Dissecting the role of mTOR:lessons from m TOR inhibitors[J]. Biochim Biophys Acta,2010,1804(3):433-439.
[18] Yap TA,Garrett MD,Walton MI,et al. Targeting the PI3K-AKTmTOR pathway:progress,pitfalls,and promises[J]. Curr Opin Pharmacol,2008,8(4):393-412.
[19] Sun Y,Zhao S,Li X,et al. Local application of rapamycin reduces epidural fibrosis after laminectomy via inhibiting fibroblast proliferation and prompting apoptosis[J]. J Orthop Surg Res,2016,11(1):58.
[20] Yoshizaki A,Yanaba K,Yoshizaki A,et al. Treatment with rapamycin prevents fibrosis in tight-skin and bleomycin-induced mouse models of systemic sclerosis[J]. Arthritis Rheum,2010,62(8):2476-2487.
[21] Wu H,Chen J,Xu J,et al. Blocking rpS6 phosphorylation exacerbates Tsc1 deletion-induced kidney growth[J]. J Am Soc Nephrol,2016,27(4):1145-1158.
[22] Sarbassov DD,Ali SM,Sengupta S,et al. Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB[J]. Mol Cell,2006,22(2):159-168.
[23] Gentzler RD,Altman JK,Platanias LC. An overview of the mTOR pathway as a target in cancer therapy[J]. Expert Opin Ther Targets,2012,16(5):481-489.
[24] Mitra A,Luna JI,Marusina AI,et al. Dual mTOR inhibition is required to prevent TGF-beta-mediated fibrosis:implications for scleroderma[J]. J Invest Dermatol,2015,135(11):2873-2876.
[2] Helmerhorst HJ,Roos-Blom MJ,van Westerloo DJ,et al. Association between arterial hyperoxia and outcome in subsets of critical illness:a systematic review,meta-analysis,and meta-regression of cohort studies[J]. Crit Care Med,2015,43(7):1508-1519.
[3] Higgins RD,Jobe AH,Koso-Thomas M,et al. Bronchopulmonary dysplasia:executive summary of a workshop[J]. J Pediatr,2018,197:300-308.
[4] Thiyagarajan V,Lee KW,Leong MK,et al. Potential natural mTOR inhibitors screened by in silico approach and suppress hepatic stellate cells activation[J]. J Biomol Struct Dyn,2017[Epub ahead of print]. DOI:10.1080/07391102.2017.1411295.
[5] Lawrence J,Nho R. The role of the mammalian target of rapamycin(mTOR)in pulmonary fibrosis[J]. Int J Mol Sci,2018,19(3):E778.
[6] Xu Y,Tai W,Qu X,et al. Rapamycin protects against paraquatinduced pulmonary fibrosis:activation of Nrf2 signaling pathway[J].Biochem Biophys Res Commun,2017,490(2):535-540.
[7] Dang H,Wang S,Yang L,et al. Upregulation of Shh and Ptc1 in hyperoxiainduced acute lung injury in neonatal rats[J]. Mol Med Rep,2012,6(2):297-302.
[8] Zhang J,Hu X,Wang S,et al. Protective effects of low-dose rapamycin combined with valsartan on podocytes of diabetic rats[J]. Int J Clin Exp Med,2015,8(8):13275-13281.
[9] Mateo J,Olmos D,Dumez H,et al. A first in man,dose-finding study of the m TORC1/mTORC2 inhibitor OSI-027 in patients with advanced solid malignancies[J]. Br J Cancer,2016,114(8):889-896.
[10] Matute-Bello G,Downey G,Moore BB,et al. An official American Thoracic Society workshop report:features and measurements of experimental acute lung injury in animals[J]. Am J Respir Cell Mol Biol,2011,44(5):725-738.
[11] Carnesecchi S,Deffert C,Pagano A,et al. NADPH oxidase-1 plays a crucial role in hyperoxia-induced acute lung injury in mice[J]. Am J Respir Crit Care Med,2009,180(10):972-981.
[12] Kallet RH,Matthay MA. Hyperoxic acute lung injury[J]. Respir Care,2013,58(1):123-141.
[13] Tee AR,Blenis J. mTOR,translational control and human disease[J]. Semin Cell Dev Biol,2005,16(1):29-37.
[14] Yu KR,Park SB,Jung JW,et al. HMGA2 regulates the in vitro aging and proliferation of human umbilical cord blood-derived stromal cells through the mTOR/p70S6K signaling pathway[J]. Stem Cell Res,2013,10(2):156-165.
[15] Parrales A,López E,Lee-Rivera I,et al. ERK1/2-dependent activation of m TOR/mTORC1/p70S6K regulates thrombin-induced RPE cell proliferation[J]. Cell Signal,2013,25(4):829-838.
[16] Thoreen CC,Sabatini DM. Rapamycin inhibits mTORC1,but not completely[J]. Autophagy,2009,5(5):725-726.
[17] Dowling RJ,Topisirovic I,Fonseca BD,et al. Dissecting the role of mTOR:lessons from m TOR inhibitors[J]. Biochim Biophys Acta,2010,1804(3):433-439.
[18] Yap TA,Garrett MD,Walton MI,et al. Targeting the PI3K-AKTmTOR pathway:progress,pitfalls,and promises[J]. Curr Opin Pharmacol,2008,8(4):393-412.
[19] Sun Y,Zhao S,Li X,et al. Local application of rapamycin reduces epidural fibrosis after laminectomy via inhibiting fibroblast proliferation and prompting apoptosis[J]. J Orthop Surg Res,2016,11(1):58.
[20] Yoshizaki A,Yanaba K,Yoshizaki A,et al. Treatment with rapamycin prevents fibrosis in tight-skin and bleomycin-induced mouse models of systemic sclerosis[J]. Arthritis Rheum,2010,62(8):2476-2487.
[21] Wu H,Chen J,Xu J,et al. Blocking rpS6 phosphorylation exacerbates Tsc1 deletion-induced kidney growth[J]. J Am Soc Nephrol,2016,27(4):1145-1158.
[22] Sarbassov DD,Ali SM,Sengupta S,et al. Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB[J]. Mol Cell,2006,22(2):159-168.
[23] Gentzler RD,Altman JK,Platanias LC. An overview of the mTOR pathway as a target in cancer therapy[J]. Expert Opin Ther Targets,2012,16(5):481-489.
[24] Mitra A,Luna JI,Marusina AI,et al. Dual mTOR inhibition is required to prevent TGF-beta-mediated fibrosis:implications for scleroderma[J]. J Invest Dermatol,2015,135(11):2873-2876.