湿润暴露疗法/湿润烧伤膏干预PI3K-Akt-mTOR信号通路促进体表慢性难愈合创面修复的实验研究
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摘要
目的探讨湿润暴露疗法/湿润烧伤膏(moist exposed burn therapy/moist exposed burn ointment, MEBT/MEBO)干预磷脂酰肌醇3-激酶(PI3K)-蛋白激酶B (Akt)-哺乳动物雷帕霉素靶蛋白(mTOR)信号通路促进体表慢性难愈合创面的修复机制。方法将160只雄性SD大鼠随机分为空白组24只、对照组(急性全层皮肤缺损组)34只和慢性难愈合创面组102只,慢性难愈合创面组(全层皮肤缺损+注射醋酸氢化可的松)造模成功后再随机分为模型组34只、贝复新组34只和MEBT/MEBO组34只。各组分别干预治疗后观察:(1)创面愈合情况,记录统计创面愈合时间;于第3、7、14天固定焦距拍照创面计算愈合率;(2)并同时于第3、7、14天取创面组织,石蜡切片HE常规染色观察第3、14天病理改变;(3)提取第3、7、14天创面组织蛋白,Western Blot法检测PI3K、p-Akt (S473)、p-mTOR (Ser2448)、p-p70 S6K (Thr389)、p-4E BP1 (Thr37/46)蛋白表达水平。结果 (1)与模型组比较,贝复新组及MEBT/MEBO组显著缩短大鼠创面愈合时间和提高创面愈合率(P<0.05)。(2)造模及干预治疗14天后,贝复新组及MEBT/MEBO组大鼠创面组织病理学形态学改善明显优于模型组。(3)与模型组比较,造模及干预治疗7天后贝复新组及MEBT/MEBO组大鼠创面组织PI3K和磷酸化Akt蛋白高表达(P<0.05),MEBT/MEBO组mTOR及其下游效应分子p70 S6K和4E BP1磷酸化水平显著升高(P<0.05)。结论 MEBT/MEBO可促进体表慢性难愈合性创面的愈合,较好改善其组织病理形态学改变,其作用机制可能与激活PI3K-Akt-mTOR信号通路,促进大量与愈合相关的蛋白质合成有关。
Objective To observe the molecular mechanisms underlying therapeutic benefits of MEBT/MEBO in chronic non-healing cutaneous wound by focusing on the PI3K-Akt-mTOR signaling pathway. Methods Totally 160 male SD rats were randomly divided into normal group(n=24), acute wound group(n=34), and chronic wound group(n=102). After successfully establishing chronic non-healing cutaneous would model, 102 rats were randomly divided into the model group(n=34), rb-FGF group(n=34), and MEBT/MEBO group(n=34).(1) After initiation of treatment with saline, rb-FGF, or MEBO, healing time and rates of healing were recorded and calculated in each group. The healing rate of the wound was calculated after fixing focal length on day 3, 7, and 14.(2) On day 3, 7, and 14, tissues were collected from each group and examined histopathologically by HE staining.(3) Proteins were extracted from tissue samples to detect expression levels of PI3K, p-Akt(S473), p-mTOR(Ser2448), p-p70 S6K(Thr389), and p-4E BP1(Thr37/46) by Western Blot. Results(1) Compared with the model group, the healing time was significantly shortened and the healing rate improved in the rb-FGF group and MEBT/MEBO group(P<0.05).(2) After 14-day modeling and intervention, histopathological changes were more markedly improved in the rb-FGF group and MEBT/MEBO group than in the model group.(3) Compared with the model group, after 14-day modeling and intervention, expressions of PI3 K and phosphor-Akt were higher in the rb-FGF group and MEBT/MEBO group than in the model group(P<0.05). Phosphorylation levels of mTOR and its downstream effector p70 S6K and 4E BP1 were significantly elevated more in the MEBT/MEBO group than in the model group(P<0.05). Conclusions MEBT/MEBO significantly promoted the healing of chronic non-healing cutaneous wound, and improved histopathological changes. Its mechanism might be associated with activating PI3K-Akt-mTOR signaling pathway, promoting proteins syntheses associated with healing.
Objective To observe the molecular mechanisms underlying therapeutic benefits of MEBT/MEBO in chronic non-healing cutaneous wound by focusing on the PI3K-Akt-mTOR signaling pathway. Methods Totally 160 male SD rats were randomly divided into normal group(n=24), acute wound group(n=34), and chronic wound group(n=102). After successfully establishing chronic non-healing cutaneous would model, 102 rats were randomly divided into the model group(n=34), rb-FGF group(n=34), and MEBT/MEBO group(n=34).(1) After initiation of treatment with saline, rb-FGF, or MEBO, healing time and rates of healing were recorded and calculated in each group. The healing rate of the wound was calculated after fixing focal length on day 3, 7, and 14.(2) On day 3, 7, and 14, tissues were collected from each group and examined histopathologically by HE staining.(3) Proteins were extracted from tissue samples to detect expression levels of PI3K, p-Akt(S473), p-mTOR(Ser2448), p-p70 S6K(Thr389), and p-4E BP1(Thr37/46) by Western Blot. Results(1) Compared with the model group, the healing time was significantly shortened and the healing rate improved in the rb-FGF group and MEBT/MEBO group(P<0.05).(2) After 14-day modeling and intervention, histopathological changes were more markedly improved in the rb-FGF group and MEBT/MEBO group than in the model group.(3) Compared with the model group, after 14-day modeling and intervention, expressions of PI3 K and phosphor-Akt were higher in the rb-FGF group and MEBT/MEBO group than in the model group(P<0.05). Phosphorylation levels of mTOR and its downstream effector p70 S6K and 4E BP1 were significantly elevated more in the MEBT/MEBO group than in the model group(P<0.05). Conclusions MEBT/MEBO significantly promoted the healing of chronic non-healing cutaneous wound, and improved histopathological changes. Its mechanism might be associated with activating PI3K-Akt-mTOR signaling pathway, promoting proteins syntheses associated with healing.
引文
[1] 姜玉峰,付小兵.体表慢性难愈合创面的研究进展[J].感染·炎症·修复,2011,12(1):59-61.
[2] 曾鸿孟,唐乾利.体表慢性难愈合创面的研究进展[J].中国烧伤创疡杂志,2016,28(5):340-344.
[3] Gurtner GC,Werner S,Barrandon Y,et al.Wound repair and regeneration[J].Nature,2008,453(7193):314-321.
[4] Eming SA,Martin P,Tomic-Canic M.Wound repair and regeneration:mechanisms,signaling,and translation[J].Sci Transl Med,2014,6(265):265sr6.
[5] Sen CK,Gordillo GM,Roy S,et al.Human skin wounds:a major and snowballing threat to public health and the economy[J].Wound Repair Regen,2009,17(6):763-771.
[6] Jiang Y,Huang S,Fu X,et al.Epidemiology of chronic cutaneous wounds in China[J].Wound Repair Regen,2011,19(2):181-188.
[7] Han G,Ceilley R.Chronic wound healing:A review of current management and treatments[J].Adv Ther,2017,34(3):599-610.
[8] 卢栋,郭璐,唐乾利,等.MEBT/MEBO治疗慢性难愈合创面60例临床疗效观察[J].中国烧伤创疡杂志,2010,22(5):346-350.
[9] 陈方凯,李晓辉,龚海粟,等.MEBT/MEBO治疗慢性难愈性创面的临床疗效分析[J].中国烧伤创疡杂志,2014,26(2):119-124.
[10] 李杰辉,黄欣,唐乾利,等.MEBT/MEBO治疗糖尿病足溃疡的临床疗效观察[J].中国烧伤创疡杂志,2016,28(1):22-25.
[11] 吴标良,唐乾利,冯烈,等.烧伤皮肤再生医疗技术治疗糖尿病足部溃疡疗效的系统评价[J].中国全科医学,2014,17(32):3851-3854.
[12] 唐乾利,黄欣,王宇,等.湿润暴露疗法/湿润烧伤膏治疗慢性难愈合创面的超微病理及丝裂原活化蛋白激酶激酶和c-myc mRNA表达的机制研究[J].中国全科医学,2015,18(3):294-299.
[13] 唐乾利,付军,韩珊珊,等.MEBT/MEBO对大鼠慢性难愈性皮肤溃疡创面肉芽组织EGF、EGFR表达的影响[J].中国烧伤创疡杂志,2012,24(6):470-480.
[14] 唐乾利,韩珊珊,付军,等.MEBT/MEBO对皮肤创面愈合过程中VEGF、bFGF、EGF mRNA表达影响的研究[J].右江民族医学院学报,2012,34(5):597-601.
[15] Laplante M,Sabatini DM.mTOR signaling in growth control and disease[J].Cell,2012,149(2):274-293.
[16] Dibble CC,Manning BD.Signal integration by mTORC1 coordinates nutrient input with biosynthetic output[J].Nat Cell Biol,2013,15(6):555-564.
[17] Saxton RA,Sabatini DM.mTOR signaling in growth,metabolism,and disease[J].Cell,2017,168(6):960-976.
[18] 赵京禹,付小兵,雷永红,等.大鼠小面积全层皮肤缺损创面模型的制备[J].感染·炎症·修复,2008,9(1):64-64.
[19] 沈道修,顾月芳,任晓英.一种研究中西药抗炎作用的塑料环肉芽肿定量法[J].中西医结合杂志,1983,3(1):49-50,67,5.
[20] 张云杰.中医药治疗慢性皮肤溃疡研究概况[J].山东中医杂志,2014,33(12):1040-1042.
[21] Huang YY,Jiang M,Zhang C,et al.Benefits of Chinese medicine among patients with diabetic foot:An expert review from clinical studies[J].Curr Vasc Pharmacol,2015,13(4):520-525.
[22] Tang QL,Han SS,Feng J,et al.Moist exposed burn ointment promotes cutaneous excisional wound healing in rats involving VEGF and bFGF[J].Mol Med Rep,2014,9(4):1277-1282.
[23] 唐乾利,李利青,李辉,等.MEBT/MEBO对大鼠糖尿病足创面组织TGF-β1、Smad3、P-smad3表达及形态学结构的影响[J].重庆医科大学学报,2017,(3):283-288.
[24] 唐乾利,曾鸿孟,王澍,等.皮肤再生医疗技术对大鼠慢性难愈合创面不同时段PTEN表达的影响[J].中华中医药学刊,2017,(9):2218-2221.
[25] 唐乾利,王权胜,尚新志.MEBT/MEBO对慢性难愈合皮肤创面基础研究现状与展望[J].中国烧伤创疡杂志,2009,21(2):95-99.
[26] 徐荣祥,萧摩.烧伤皮肤再生疗法与创面愈合的机制[J].中国烧伤创疡杂志,2003,15(4):253-261.
[27] 李利青,何晓微,黄欣,等.烧伤皮肤再生医疗技术治疗糖尿病皮肤溃疡机理的总结探析[J].中国烧伤创疡杂志,2014,26(1):38-44,46.
[28] 崔文慧.mTOR信号通路与创伤愈合的相关性研究[D].重庆:重庆医科大学,2012:8-12.
[29] Cantley LC.The role of phosphoinositide 3-kinase in human disease[J].Harvey Lect,2004,100:103-122.
[30] Dibble CC,Cantley LC.Regulation of mTORC1 by PI3K signaling[J].Trends Cell Biol,2015,25(9):545-555.
[31] Mills RE,Taylor KR,Podshivalova K,et al.Defects in skin gamma delta T cell function contribute to delayed wound repair in rapamycin-treated mice[J].J Immunol,2008,181(6):3974-3983.
[2] 曾鸿孟,唐乾利.体表慢性难愈合创面的研究进展[J].中国烧伤创疡杂志,2016,28(5):340-344.
[3] Gurtner GC,Werner S,Barrandon Y,et al.Wound repair and regeneration[J].Nature,2008,453(7193):314-321.
[4] Eming SA,Martin P,Tomic-Canic M.Wound repair and regeneration:mechanisms,signaling,and translation[J].Sci Transl Med,2014,6(265):265sr6.
[5] Sen CK,Gordillo GM,Roy S,et al.Human skin wounds:a major and snowballing threat to public health and the economy[J].Wound Repair Regen,2009,17(6):763-771.
[6] Jiang Y,Huang S,Fu X,et al.Epidemiology of chronic cutaneous wounds in China[J].Wound Repair Regen,2011,19(2):181-188.
[7] Han G,Ceilley R.Chronic wound healing:A review of current management and treatments[J].Adv Ther,2017,34(3):599-610.
[8] 卢栋,郭璐,唐乾利,等.MEBT/MEBO治疗慢性难愈合创面60例临床疗效观察[J].中国烧伤创疡杂志,2010,22(5):346-350.
[9] 陈方凯,李晓辉,龚海粟,等.MEBT/MEBO治疗慢性难愈性创面的临床疗效分析[J].中国烧伤创疡杂志,2014,26(2):119-124.
[10] 李杰辉,黄欣,唐乾利,等.MEBT/MEBO治疗糖尿病足溃疡的临床疗效观察[J].中国烧伤创疡杂志,2016,28(1):22-25.
[11] 吴标良,唐乾利,冯烈,等.烧伤皮肤再生医疗技术治疗糖尿病足部溃疡疗效的系统评价[J].中国全科医学,2014,17(32):3851-3854.
[12] 唐乾利,黄欣,王宇,等.湿润暴露疗法/湿润烧伤膏治疗慢性难愈合创面的超微病理及丝裂原活化蛋白激酶激酶和c-myc mRNA表达的机制研究[J].中国全科医学,2015,18(3):294-299.
[13] 唐乾利,付军,韩珊珊,等.MEBT/MEBO对大鼠慢性难愈性皮肤溃疡创面肉芽组织EGF、EGFR表达的影响[J].中国烧伤创疡杂志,2012,24(6):470-480.
[14] 唐乾利,韩珊珊,付军,等.MEBT/MEBO对皮肤创面愈合过程中VEGF、bFGF、EGF mRNA表达影响的研究[J].右江民族医学院学报,2012,34(5):597-601.
[15] Laplante M,Sabatini DM.mTOR signaling in growth control and disease[J].Cell,2012,149(2):274-293.
[16] Dibble CC,Manning BD.Signal integration by mTORC1 coordinates nutrient input with biosynthetic output[J].Nat Cell Biol,2013,15(6):555-564.
[17] Saxton RA,Sabatini DM.mTOR signaling in growth,metabolism,and disease[J].Cell,2017,168(6):960-976.
[18] 赵京禹,付小兵,雷永红,等.大鼠小面积全层皮肤缺损创面模型的制备[J].感染·炎症·修复,2008,9(1):64-64.
[19] 沈道修,顾月芳,任晓英.一种研究中西药抗炎作用的塑料环肉芽肿定量法[J].中西医结合杂志,1983,3(1):49-50,67,5.
[20] 张云杰.中医药治疗慢性皮肤溃疡研究概况[J].山东中医杂志,2014,33(12):1040-1042.
[21] Huang YY,Jiang M,Zhang C,et al.Benefits of Chinese medicine among patients with diabetic foot:An expert review from clinical studies[J].Curr Vasc Pharmacol,2015,13(4):520-525.
[22] Tang QL,Han SS,Feng J,et al.Moist exposed burn ointment promotes cutaneous excisional wound healing in rats involving VEGF and bFGF[J].Mol Med Rep,2014,9(4):1277-1282.
[23] 唐乾利,李利青,李辉,等.MEBT/MEBO对大鼠糖尿病足创面组织TGF-β1、Smad3、P-smad3表达及形态学结构的影响[J].重庆医科大学学报,2017,(3):283-288.
[24] 唐乾利,曾鸿孟,王澍,等.皮肤再生医疗技术对大鼠慢性难愈合创面不同时段PTEN表达的影响[J].中华中医药学刊,2017,(9):2218-2221.
[25] 唐乾利,王权胜,尚新志.MEBT/MEBO对慢性难愈合皮肤创面基础研究现状与展望[J].中国烧伤创疡杂志,2009,21(2):95-99.
[26] 徐荣祥,萧摩.烧伤皮肤再生疗法与创面愈合的机制[J].中国烧伤创疡杂志,2003,15(4):253-261.
[27] 李利青,何晓微,黄欣,等.烧伤皮肤再生医疗技术治疗糖尿病皮肤溃疡机理的总结探析[J].中国烧伤创疡杂志,2014,26(1):38-44,46.
[28] 崔文慧.mTOR信号通路与创伤愈合的相关性研究[D].重庆:重庆医科大学,2012:8-12.
[29] Cantley LC.The role of phosphoinositide 3-kinase in human disease[J].Harvey Lect,2004,100:103-122.
[30] Dibble CC,Cantley LC.Regulation of mTORC1 by PI3K signaling[J].Trends Cell Biol,2015,25(9):545-555.
[31] Mills RE,Taylor KR,Podshivalova K,et al.Defects in skin gamma delta T cell function contribute to delayed wound repair in rapamycin-treated mice[J].J Immunol,2008,181(6):3974-3983.
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