补阳还五汤对肺纤维化小鼠中介导细胞自噬的mTOR蛋白的调控机制探讨
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摘要
目的:观察肺纤维化中介导自噬的哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)的表达、自噬在肺纤维化形成中的作用,探索补阳还五汤对肺纤维化的治疗机制。方法:144只C57BL/6小鼠随机分为6组,分别为假手术组,模型组,强的松组,补阳还五汤高、中、低剂量组,每组24只,假手术组小鼠给予气管注入等量的0. 9%生理盐水,其余各组采用博莱霉素(bleomycin,BLM)气管注入法复制肺纤维化模型,造模后假手术组、模型组给予0. 9%生理盐水(0. 01 g·kg~(-1)·d~(-1)),补阳还五汤高剂量组给予补阳还五汤(28. 08 g·kg~(-1)·d~(-1)),补阳还五汤中剂量组给予补阳还五汤(14. 04 g·kg~(-1)·d~(-1)),补阳还五汤低剂量组给予补阳还五汤(7. 02 g·kg~(-1)·d~(-1)),强的松组给予强的松(0. 455 g·kg~(-1)·d~(-1))灌胃,于造模后第7,14,28天分批提取样本。采用苏木素-伊红(HE)及马松(Masson)染色观察小鼠肺泡炎和纤维化程度;蛋白免疫印迹法(Western blot)检测小鼠肺组织mTOR,核糖体S6,微管相关蛋白1轻链3Ⅱ(microtubule-associate protein 1 light chain 3,MAP1LC3-Ⅱ)的表达;电镜检测观察小鼠肺组织自噬情况。结果:与假手术组比较,模型组小鼠在7,14,28 d肺泡炎及肺纤维化显著(P <0. 01);与模型组比较,给药组各时间点肺泡炎和肺纤维化程度均有不同程度的改善(P <0. 05),补阳还五汤高剂量组下降最明显(P <0. 05)。与假手术组比较,模型组小鼠肺组织mTOR,S6蛋白表达水平显著上调(P <0. 01),与模型组比较,各给药组mTOR,S6蛋白表达下调(P <0. 01);与假手术组比较,模型组小鼠肺组织LC3-Ⅱ表达水平下调(P <0. 01),与模型组比较,各给药组LC3-Ⅱ上调(P <0. 01)。假手术组细胞形态良好,未见自噬。模型组细胞形态最差,存在个别自噬。各给药组细胞形态及自噬数量好于模型组,其中补阳还五汤高、中剂量组自噬数量最多。结论:在BLM所致小鼠肺纤维化形成的过程中,小鼠肺组织中mTOR蛋白被激活,自噬抑制,mTOR蛋白通过抑制自噬参与了肺纤维化的发病;补阳还五汤对BLM所致小鼠肺纤维化有一定的治疗作用,其机制可能与其下调介导细胞自噬的mTOR蛋白表达有关。
Objective: To observe the expression of mammalian target of rapamycin( mTOR) that mediates autophagy in pulmonary fibrosis and the effect of autophagy in the formation of pulmonary fibrosis,in order to explore the treatment mechanism of Buyang Huanwu Tang on pulmonary fibrosis. Method: Totally 144 C57 BL/6 mice were randomly divided into 6 groups: sham operation group,model group,prednisone group,high-dose Buyang Huanwu Tang group,medium-dose Buyang Huanwu Tang group and low-dose Buyang Huanwu Tang group,with 24 mice in each group. The sham operation group was injected with the same amount of 0. 9% saline. The remaining groups were treated with bleomycin tracheal injection to replicate the pulmonary fibrosis model. After modeling,sham operation group and model group were given 0. 9% normal saline( 0. 01 g·kg~(-1)·d~(-1)),group highdose Buyang Huanwu Tang group was given Buyang Huanwu Tang( 28. 08 g·kg~(-1)·d~(-1)),medium-dose Buyang Huanwu Tang group was given Buyang Huanwu Tang( 14. 04 g·kg~(-1)·d~(-1)),low-dose Buyang Huanwu Tang group was given Buyang Huanwu Tang( 7. 02 g·kg~(-1)·d~(-1)),and P group was given prednisone( 0. 455 g·kg~(-1)·d~(-1)) by gavage. The samples were taken in batches on the 7 th,14 thand 28 thdays after modeling; degrees of alveolitis and fibrosis in mice were observed by hematoxylin-eosin( HE) staining and Masson staining. The mTOR protein,ribosomal S6 protein and microtubule associate protein 1 hight chain3-Ⅱ( MAP1 LC3-Ⅱ) of mouse lung tissue were detected by Western blot; electron microscopy was used to observe the autophagy of lung tissue in mice.Result: Compared with the sham-operated group,the degrees of alveolitis and pulmonary fibrosis were significantly severer in the model group on 7 th,14 thand 28 thdays( P < 0. 01); compared with the model group,the degrees of alveolitis and pulmonary fibrosis were alleviated at each observation time in the drug-administered groups( P <0. 05). The decreased scores in high-dose Buyang Huanwu Tang group were the most obvious,with statistically significant differences( P < 0. 05). compared with the sham-operated group,the expressions of mTOR and S6 protein in lung tissue of the model group were significantly up-regulated,and each drug-administered group showed down-regulations. Compared with the sham-operated group,the expression of LC3-Ⅱ protein in lung tissue of the model group was significantly down-regulated,and each drug-administered group showed anup-regulation( P <0. 01). The cells in the sham-operated group were well-formed,and no autophagy was observed. The model group had the worst cell morphology,with individual autophagy. The cell morphology and autophagy in each drugadministered group were better than those in the model group. The high-dose Buyang Huanwu Tang group and the medium-dose Buyang Huanwu Tang group had the highest number of autophagosome. Conclusion: The mTOR protein is activated in mice lung tissue,autophagy is inhibited,mTOR protein participates in the pathogenesis of pulmonary fibrosis by inhibiting autophagy; Buyang Huanwu Tang has a certain therapeutic effect on BLM-induced pulmonary fibrosis in mice,and its mechanism may be related to the down-regulation of mTOR protein expression that mediates autophagy.
Objective: To observe the expression of mammalian target of rapamycin( mTOR) that mediates autophagy in pulmonary fibrosis and the effect of autophagy in the formation of pulmonary fibrosis,in order to explore the treatment mechanism of Buyang Huanwu Tang on pulmonary fibrosis. Method: Totally 144 C57 BL/6 mice were randomly divided into 6 groups: sham operation group,model group,prednisone group,high-dose Buyang Huanwu Tang group,medium-dose Buyang Huanwu Tang group and low-dose Buyang Huanwu Tang group,with 24 mice in each group. The sham operation group was injected with the same amount of 0. 9% saline. The remaining groups were treated with bleomycin tracheal injection to replicate the pulmonary fibrosis model. After modeling,sham operation group and model group were given 0. 9% normal saline( 0. 01 g·kg~(-1)·d~(-1)),group highdose Buyang Huanwu Tang group was given Buyang Huanwu Tang( 28. 08 g·kg~(-1)·d~(-1)),medium-dose Buyang Huanwu Tang group was given Buyang Huanwu Tang( 14. 04 g·kg~(-1)·d~(-1)),low-dose Buyang Huanwu Tang group was given Buyang Huanwu Tang( 7. 02 g·kg~(-1)·d~(-1)),and P group was given prednisone( 0. 455 g·kg~(-1)·d~(-1)) by gavage. The samples were taken in batches on the 7 th,14 thand 28 thdays after modeling; degrees of alveolitis and fibrosis in mice were observed by hematoxylin-eosin( HE) staining and Masson staining. The mTOR protein,ribosomal S6 protein and microtubule associate protein 1 hight chain3-Ⅱ( MAP1 LC3-Ⅱ) of mouse lung tissue were detected by Western blot; electron microscopy was used to observe the autophagy of lung tissue in mice.Result: Compared with the sham-operated group,the degrees of alveolitis and pulmonary fibrosis were significantly severer in the model group on 7 th,14 thand 28 thdays( P < 0. 01); compared with the model group,the degrees of alveolitis and pulmonary fibrosis were alleviated at each observation time in the drug-administered groups( P <0. 05). The decreased scores in high-dose Buyang Huanwu Tang group were the most obvious,with statistically significant differences( P < 0. 05). compared with the sham-operated group,the expressions of mTOR and S6 protein in lung tissue of the model group were significantly up-regulated,and each drug-administered group showed down-regulations. Compared with the sham-operated group,the expression of LC3-Ⅱ protein in lung tissue of the model group was significantly down-regulated,and each drug-administered group showed anup-regulation( P <0. 01). The cells in the sham-operated group were well-formed,and no autophagy was observed. The model group had the worst cell morphology,with individual autophagy. The cell morphology and autophagy in each drugadministered group were better than those in the model group. The high-dose Buyang Huanwu Tang group and the medium-dose Buyang Huanwu Tang group had the highest number of autophagosome. Conclusion: The mTOR protein is activated in mice lung tissue,autophagy is inhibited,mTOR protein participates in the pathogenesis of pulmonary fibrosis by inhibiting autophagy; Buyang Huanwu Tang has a certain therapeutic effect on BLM-induced pulmonary fibrosis in mice,and its mechanism may be related to the down-regulation of mTOR protein expression that mediates autophagy.
引文
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[25]Araya J,Kojima J,Takasaka N,et al.Insufficient autophagyin idiopathic pulmonary fibrosis[J].Am JPhysiol Lung Cell Mol Physiol,2013,304(1):56-69.
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[28]PEI J J,Hugon J.m TOR-dependent signaling in Alzheimer's disease[J].J Cell Mol Med,2008,12(6B):2525-2532.
[29]SUI L,REN J.Role of Mtor in memory[J].Chin Pharmacol Bull,2011,27:748-751.
[30]Kamada Y,Funakoshi T,Shintani T,et al.Tor mediated inductionof autophagy viaan Apg1 protein kinase complex[J].J Cell Biol,2000,150(6):1507-1513.
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[33]肖美凤,刘金玲,杨岩涛,等.补阳还五汤的研究现状及其新药创制关键技术[J].中草药,2018,49(7):1688-1694.
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[2]Katzenstein A L,Zisman D A,Litzky L A,et al.Usualinterstitial pneumonia:histologic study of biopsy and explant specimens[J].Am J Surg Pahol,2002,26(12):1567-1577.
[3]Chiarini F,Evangelisti C,Mc Cubrey J A,et al.Current treatment strategies for inhibiting m TOR in cancer[J].Trends Pharmacol Sci,2015,36(2):124-135.
[4]HUANG K,Fingar D C.Growing knowledge of the m TOR signaling network[J].Semin Cell Dev Biol,2014,36:79-90.
[5]MA X M,Blenis J.Molecular mechanisms of m TOR-mediated transla-tional control[J].Nat Revi Mol Cell Biol,2009,10(5):307-318.
[6]彭敏,阳学风.m TOR信号通路与肝星状细胞功能的关系[J].世界华人消化杂志,2017,25(35):3141-3148.
[7]Kamada Y,Funakoshi T,Shintani T,et al.Tor mediated induction of autophagy via an Apg1 protein kinase complex[J].J Cell Biol,2000,150(6):1507-1513.
[8]Noda T,Ohsumi Y.TOR,a phosphatidylinositol kinase homologue,controls autophagy in yeast[J].J Biol Chem,1998,273(7):3963-3966.
[9]董洪亮,刘乃国,苗双,等.1,25-(OH)-2D-3对肺纤维化大鼠中PI3K、AKT、m TOR表达的影响及其机制研究[J].中国现代医学杂志,2018,28(19):1-6.
[10]林艺凯,马爱平,周伟跃,等.m TOR信号通路在博来霉素诱导小鼠肺纤维化中的作用机制研究[J].中国呼吸与危重监护杂志,2018,17(2):178-182.
[11]Chitra P,Saiprasad G,Manikandan R,et al.Berberine inhibits Smad and non-Smad signaling cascades and enhances autophagy against pulmonary fibrosis[J].JMol Med,2015,93(9):1015-1031.
[12]Patel A S,LIN L,Geyer A,et al.Autophagy in idiopathic pulmonary fibrosis[J].PLo S One,2012,7(7):e41394.
[13]GUI X H,CHEN H W,CAI H R,et al.Leptin promotes pulmonary fibrosis development by inhibiting autophagy via PI3K/Akt/m TOR pathway[J].Biochem Biophys Res Commun,2018,498(3):660-666.
[14]Jessica L,Richard N.The role of the mammalian target of rapamycin(m TOR)in pulmonary fibrosis[J].Int JMol Sci,2018,19(3):E778.
[15]杨昆,李勇华,王飞,等.补阳还五汤防治特发性肺纤维化的作用及机制探讨[J].北京中医药大学学报,2017,40(7):550-557.
[16]王振兴,孙中莉,王明杰,等.补阳还五汤对肺纤维化中HMGB1-RAGE信号通路的调控作用[J].中国实验方剂学杂志,2017,23(13):138-144.
[17]杨晗,王飞,杨昆.补阳还五汤对博莱霉素所致肺纤维化大鼠肺组织ERK表达的影响[J].中华中医药杂志,2017,32(4):1727-1730.
[18]郝丽君.补阳还五汤大鼠含药血清对HMGB1诱导的HFL-1细胞相关炎症因子的影响[D].成都:成都中医药大学,2016.
[19]李水芹,李学君,王飞,等.补阳还五汤大鼠含药血清对肺成纤维细胞TGF-β1/Smad/ERK信号通路的影响[J].辽宁中医杂志,2015,42(12):2436-2439.
[20]杨晗,王飞,王全林,等.补阳还五汤对肺纤维化大鼠肺组织TGF-β1/Smad3表达的影响[J].中国实验方剂学杂志,2013,19(24):240-244.
[21]Limjunyawong N,Mitzner W,Horton M R.A mouse model of chronic idiopathic pulmonary fibrosis[J].Physiol Rep,2014,2(2):e00249.
[22]Demedts M,Behr J,Buhl R,et al.High-dose acetylcysteine in idiopathic pulmonary fibrosis[J].NEngl J Med,2005,353(21):2229-2242.
[23]Szapiel S V,Elson Na,Fulmer J D,et al.Bleomycininduced interstitial pulmonary disease in the nude athymic mouse[J].Am Rev Respir Dis,1979,120(9):893-899.
[24]陶金,孙钰,王静成,等.细胞自噬与纤维化疾病的研究进展[J].江苏医药,2018,44(4):432-437.
[25]Araya J,Kojima J,Takasaka N,et al.Insufficient autophagyin idiopathic pulmonary fibrosis[J].Am JPhysiol Lung Cell Mol Physiol,2013,304(1):56-69.
[26]Cabrera S,Maciel M,Herrera I,et al.Essential role for the ATG4B protease and autophagy in bleomycininduced pulmonary fibrosis[J].Autophagy,2015,11(4):670-684.
[27]Ricci A,Cherubini E,Scozzi D,et al.Decreased expression of autophagic beclin 1 protenin in idiopathic pulmonary fibrosis fibroblasts[J].J Cell Physiol,2013,228(7):1516-1524.
[28]PEI J J,Hugon J.m TOR-dependent signaling in Alzheimer's disease[J].J Cell Mol Med,2008,12(6B):2525-2532.
[29]SUI L,REN J.Role of Mtor in memory[J].Chin Pharmacol Bull,2011,27:748-751.
[30]Kamada Y,Funakoshi T,Shintani T,et al.Tor mediated inductionof autophagy viaan Apg1 protein kinase complex[J].J Cell Biol,2000,150(6):1507-1513.
[31]Ravikumar B,Sarkar S,Davies J E,et al.Regulation of mammalian autophagy in physiology and patho physiology[J].Physiol Rev,2010,90(4):1383-1435.
[32]Cordani M,Butera G,Pacchiana R,et al.Molecular interplay between mutant p53 proteins and autophagy in cancer cells[J].BBA Rev Canc,2017,1867(1):19-28.
[33]肖美凤,刘金玲,杨岩涛,等.补阳还五汤的研究现状及其新药创制关键技术[J].中草药,2018,49(7):1688-1694.
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