深层海水促进糖尿病模型小鼠创面愈合:激活Wnt/β-catenin通路的机制研究
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摘要
背景:研究证实深层海水可通过激活AMPK通路下调小鼠对糖的摄入,进而有效缓解高脂肪饮食诱导的小鼠糖尿病进程,然而目前有关深层海水在糖尿病创伤难愈合中的作用研究鲜有报道。目的:探讨深层海水对糖尿病小鼠创面的修复作用机制。方法:取健康昆明种雄性小鼠48只(购于昆明医科大学实验动物中心),通过腹腔注射链脲佐菌素建立糖尿病模型;饲养14 d后建立糖尿病小鼠背部创面模型,随机分4组处理,其中3组分别喂食纯净水(对照组)、自来水(自来水组)、深层海水(深层海水组),抑制剂组喂食深层海水的同时股静脉注射Wnt/β-catenin抑制剂XAV-939。创面造模后第3,7,10天,检测对照组、自来水组、深层海水组小鼠血糖和体质量、创面愈合及创面组织中β-catenin、GSK-3β和Rspo-3蛋白表达,观察对照组、深层海水组、抑制剂组创面愈合率、创面组织形态变化及血清中丙二醛、超氧化物歧化酶和谷胱甘肽过氧化物酶水平。实验获得中国科学院昆明动物研究所实验动物伦理委员会批准(批准号:KPRC-2017091)。结果与结论:(1)深层海水对糖尿病小鼠小鼠体质量和血糖值无影响;(2)深层海水组不同时间点的创面愈合率明显高于自来水组和对照组(P均<0.01);(3)Western blotting检测显示与对照组和自来水组比较,深层海水组创面组织中β-catenin、Rspo-3蛋白表达升高,GSK-3β蛋白表达下降;(4)苏木精-伊红染色显示,与对照组比较,深层海水组新生肉芽组织中血管内皮细胞和新生毛细血管增多,炎性细胞浸润较少,成纤维细胞增生明显;抑制剂组与对照组病理表现无明显差异(P> 0.05);深层海水组不同时间点的创面愈合率明显高于自来水组和抑制剂组(P均<0.01);(5)与对照组比较,深层海水组超氧化物歧化酶、谷胱甘肽过氧化物酶活性升高(P <0.01,P <0.001),丙二醛浓度下降(P <0.05,P <0.01);抑制剂组3指标水平与对照组比较无明显差异(P> 0.05);(6)结果表明,深层海水通过激活Wnt/β-catenin通路促进糖尿病小鼠创面的愈合。
BACKGROUND: Deep seawater has been shown to effectively alleviate the process of diabetes in mice induced by high fat diet through activating AMPK pathway, but there are few studies about the role of deep seawater in the wound healing of diabetes mellitus.OBJECTIVE: To explore the mechanism of deep seawater on the cutaneous wound healing in diabetic mice.METHODS: Forty-eight healthy male Kunming mice(provide by Laboratory Animal Center of Kunming Medical University) were selected and were given intraperitoneal injection of streptozotocin to establish diabetic model. After 14 days of incubation, the back would model was established, and the mouse models were randomly divided into four groups: control group(purified water), tap water group, deep seawater group and inhibitor group(deep seawater + venous injection of Wnt/β-catenin inhibitor, XAV-939). Subsequently, the blood glucose and body mass of model mice were observed at 3, 7 and 10 days after wound modeling, and the healing of the wound was observed. The morphological changes of the wound were evaluated. The expression levels of β-catenin, GSK-3β and Rspo-3, malondialdehyde, superoxide dismutase and glutathione peroxidase in the serum were detected. The study was approved by the Laboratory Animal Ethics Committee of Kunming Institute of Zoology, China Academy of Sciences, approval No. KPRC-2017091.RESULTS AND CONCLUSION:(1) Deep seawater showed no significant effect on the body mass and blood glucose.(2) The wound healing rate in the deep seawater group was significantly higher than that in the tap water and control groups at different time points(all P < 0.01).(3)Western blot results showed that the expression levels of β-catenin and Rspo-3 were higher in the deep seawater group than the control and tap water groups, but GSK-3β was down-regulated.(4) Hematoxylin-eosin staining showed that compared with the control group, vascular endothelial cells, new capillaries and fibroblast cells proliferation in the deep seawater group were increased, but the number of inflammatory cells got opposite results. However, there was no significant difference between the deep seawater + XAV-939 group and control group(P >0.05). The healing rate of wound at different time points in the deep seawater group was significantly higher than that in the tap water and control groups(all P < 0.01).(5) Compared with the control group, the expression levels of superoxide dismutase and glutathione peroxidase in serum in the deep seawater group were increased(P < 0.01 and P < 0.001), and malondialdehyde decreased(P < 0.05 and P < 0.01).There was no significant difference between deep seawater + XAV-939 and control groups(P > 0.05).(6) To conclude, deep seawater promotes cutaneous wound healing in diabetic mice via activating Wnt/β-catenin pathway.
BACKGROUND: Deep seawater has been shown to effectively alleviate the process of diabetes in mice induced by high fat diet through activating AMPK pathway, but there are few studies about the role of deep seawater in the wound healing of diabetes mellitus.OBJECTIVE: To explore the mechanism of deep seawater on the cutaneous wound healing in diabetic mice.METHODS: Forty-eight healthy male Kunming mice(provide by Laboratory Animal Center of Kunming Medical University) were selected and were given intraperitoneal injection of streptozotocin to establish diabetic model. After 14 days of incubation, the back would model was established, and the mouse models were randomly divided into four groups: control group(purified water), tap water group, deep seawater group and inhibitor group(deep seawater + venous injection of Wnt/β-catenin inhibitor, XAV-939). Subsequently, the blood glucose and body mass of model mice were observed at 3, 7 and 10 days after wound modeling, and the healing of the wound was observed. The morphological changes of the wound were evaluated. The expression levels of β-catenin, GSK-3β and Rspo-3, malondialdehyde, superoxide dismutase and glutathione peroxidase in the serum were detected. The study was approved by the Laboratory Animal Ethics Committee of Kunming Institute of Zoology, China Academy of Sciences, approval No. KPRC-2017091.RESULTS AND CONCLUSION:(1) Deep seawater showed no significant effect on the body mass and blood glucose.(2) The wound healing rate in the deep seawater group was significantly higher than that in the tap water and control groups at different time points(all P < 0.01).(3)Western blot results showed that the expression levels of β-catenin and Rspo-3 were higher in the deep seawater group than the control and tap water groups, but GSK-3β was down-regulated.(4) Hematoxylin-eosin staining showed that compared with the control group, vascular endothelial cells, new capillaries and fibroblast cells proliferation in the deep seawater group were increased, but the number of inflammatory cells got opposite results. However, there was no significant difference between the deep seawater + XAV-939 group and control group(P >0.05). The healing rate of wound at different time points in the deep seawater group was significantly higher than that in the tap water and control groups(all P < 0.01).(5) Compared with the control group, the expression levels of superoxide dismutase and glutathione peroxidase in serum in the deep seawater group were increased(P < 0.01 and P < 0.001), and malondialdehyde decreased(P < 0.05 and P < 0.01).There was no significant difference between deep seawater + XAV-939 and control groups(P > 0.05).(6) To conclude, deep seawater promotes cutaneous wound healing in diabetic mice via activating Wnt/β-catenin pathway.
引文
[1]Cho H,Blatchley MR,Duh EJ,et al.Acellular and cellular approaches to improve diabetic wound healing.Adv Drug Deliv Rev.2018.pii:S0169-409X(18)30193-5.[Epub ahead of print]
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[3]Ishihara J,Ishihara A,Starke RD,et al.The heparin binding domain of von Willebrand factor binds to growth factors and promotes angiogenesis in wound healing.Blood.2019;pii:blood.2019000510.[Epub ahead of print]
[4]Liao HE,Shibu MA,Kuo WW,et al.Deep sea minerals prolong life span of streptozotocin-induced diabetic rats by compensatory augmentation of the IGF-I-survival signaling and inhibition of apoptosis.Environ Toxicol.2016;31(7):769-781.
[5]Mohd Nani SZ,Majid FA,Jaafar AB,et al.Potential health benefits of deep sea water:a review.Evid Based Complement Alternat Med.2016;2016:6520475.
[6]Schrameyer V,York PH,Chartrand K,et al.Contrasting impacts of light reduction on sediment biogeochemistry in deep-and shallow-water tropical seagrass assemblages(Green Island,Great Barrier Reef).Mar Environ Res.2018;136:38-47.
[7]Hsu CL,Chang YY,Chiu CH,et al.Cardiovascular protection of deep-seawater drinking water in high-fat/cholesterol fed hamsters.Food Chem.2011;127(3):1146-1152.
[8]Hsu TC,Chiu CC,Lin HL,et al.Attenuated effects of deep-sea water on hepatic apoptosis in STZ-induced diabetic rats.Chin J Physiol.2015;58(3):197-205.
[9]Ha BG,Shin EJ,Park JE,et al.Anti-diabetic effect of balanced deep-sea water and its mode of action in high-fat diet induced diabetic mice.Mar Drugs.2013;11(11):4193-212.
[10]陈玉姣,白晓苏,曾丽红.补气生肌方促进糖尿病性难愈创面血管新生和创面修复愈合的作用机制[J].实用药物与临床,2017,20(10):1127-1130.
[11]丁寅佳,崔磊,赵旭,等.富血小板纤维蛋白联合脂肪干细胞对糖尿病小鼠皮肤创面愈合的影响研究[J].中国美容医学,2019,28(2):8-13.
[12]Choi SM,Lee KM,Kim HJ,et al.Effects of structurally stabilized EGF and bFGF on wound healing in type I and type II diabetic mice.Acta Biomater.2018;66:325-334.
[13]Milan PB,Lotfibakhshaiesh N,Joghataie MT,et al.Accelerated wound healing in a diabetic rat model using decellularized dermal matrix and human umbilical cord perivascular cells.Acta Biomater.2016;45:234-246.
[14]王志永,和晓培,薜欣,等.难愈性创面的发生机制及人工真皮在其治疗中的应用进展[J].临床合理用药杂志,2017,10(28):180-181.
[15]向鹏君,季晖,顾铭,等.糖尿病创面的炎症机制研究进展[J].药学研究,2017,36(11):667-670.
[16]胡啸明,柳国斌.糖尿病足性溃疡愈合机制的研究进展[J].上海中医药杂志,2017,51(S1):217-219,223.
[17]沈耀明.严重烧伤全身与局部微量元素含量变化特点及对创面修复的机制探讨[J].中国医药指南,2014,12(34):117-118.
[18]张文浩,吴起,马军,等.负压封闭引流联合肝素灌洗治疗兔烫伤并海水浸泡创面[J].南方医科大学学报,2015,35(10):1481-1486.
[19]姚波,刘万宏,傅亚.影响创面愈合的营养因素研究进展[J].基因组学与应用生物学,2012,31(6):640-643.
[20]张海峰,吕游,王洪莎,等.硫酸锌对糖尿病皮肤损伤的修复作用[J].中国老年学杂志,2016,36(23):5777-5780.
[21]李为明,崔进,徐鹏远,等.深层海水对小鼠创面愈合的促进作用[J].重庆医学,2014,43(4):462-464.
[22]韩昌鹏,姜文成,李福伦,等.Wnt信号通路与创面愈合[J].中国美容医学,2012,21(7):1257-1260.
[23]Tang H,Xu Y,Zhang Z,et al.SDF-1/CXCR4 induces epithelialmesenchymal transition through activation of the Wnt/β-catenin signaling pathway in rat chronic allograft nephropathy.Mol Med Rep.2019;19(5):3696-3706.
[24]Xie S,Fu W,Yu G,et al.Discovering small molecules as Wnt inhibitors that promote heart regeneration and injury repair.JMol Cell Biol.2019.pii:mjz023.[Epub ahead of print]
[25]Leavitt T,Hu MS,Marshall CD,et al.Scarless wound healing:finding the right cells and signals.Cell Tissue Res.2016;365(3):483-493.
[26]赵亚男,刘明,张玥,等.Wnt信号通路与皮肤创面愈合的关系[J].现代生物医学进展,2015,15(11):2173-2176,2184.
[27]Yang HL,Tsai YC,Korivi M,et al.Lucidone promotes the cutaneous wound healing process via activation of the PI3K/AKT,Wnt/β-catenin and NF-κB signaling pathways.Biochim Biophys Acta Mol Cell Res.2017;1864(1):151-168.
[28]Qi W,Yang C,Dai Z,et al.High levels of pigment epithelium-derived factor in diabetes impair wound healing through suppression of Wnt signaling.Diabetes.2015;64(4):1407-1419.
[29]Pizzute T,Li J,Zhang Y,et al.Fibroblast growth factor ligand dependent proliferation and chondrogenic differentiation of synovium-derived stem cells and concomitant adaptation of Wnt/mitogen-activated protein kinase signals.Tissue Eng Part A.2016;22(15-16):1036-1046.
[30]Hu F,Yan Y,Wang CW,et al.Article effect and mechanism of ganoderma lucidum polysaccharides on human fibroblasts and skin wound healing in mice.Chin J Integr Med.2019;25(3):203-209.
[31]Ma T,Fu B,Yang X,et al.Adipose mesenchymal stem cell-derived exosomes promote cell proliferation,migration,and inhibit cell apoptosis via Wnt/β-catenin signaling in cutaneous wound healing.J Cell Biochem.2019;120(6):10847-10854.
[32]Zhang G,Song K,Yan H.MicroRNA-124 represses wound healing by targeting SERP1 and inhibiting the Wnt/β-catenin pathway.Adv Clin Exp Med.2019.[Epub ahead of print]
[33]Liu D,He S,Chen S,et al.Different effects of Wnt/β-catenin activation and Parathyroid hormone on diaphyseal and metaphyseal in the early phase of femur bone healing of mice.Clin Exp Pharmacol Physiol.2019.[Epub ahead of print]
[34]Bao Q,Chen S,Qin H,et al.An appropriate Wnt/β-catenin expression level during the remodeling phase is required for improved bone fracture healing in mice.Sci Rep.2017;7(1):2695.
[35]Rognoni E,Gomez C,Pisco AO,et al.Inhibition ofβ-catenin signalling in dermal fibroblasts enhances hair follicle regeneration during wound healing.Development.2016;143(14):2522-2535.
[36]Liu J,Wang Y,Pan Q,et al.Wnt/beta-catenin pathway forms a negative feedback loop during TGF-beta1 induced human normal skin fibroblast-to-myofibroblast transition.J Dermatol Sci.2012;65(1):38-49.
[37]Schmid A,Sailland J,Novak L,et al.Modulation of Wnt signaling is essential for the differentiation of ciliated epithelial cells in human airways.FEBS Lett.2017;591(21):3493-3506.
[2]Kulprachakarn K,Ounjaijean S,Wungrath J,et al.Micronutrients and natural compounds status and their effects on wound healing in the diabetic foot ulcer.Int J Low Extrem Wounds.2017;16(4):244-250.
[3]Ishihara J,Ishihara A,Starke RD,et al.The heparin binding domain of von Willebrand factor binds to growth factors and promotes angiogenesis in wound healing.Blood.2019;pii:blood.2019000510.[Epub ahead of print]
[4]Liao HE,Shibu MA,Kuo WW,et al.Deep sea minerals prolong life span of streptozotocin-induced diabetic rats by compensatory augmentation of the IGF-I-survival signaling and inhibition of apoptosis.Environ Toxicol.2016;31(7):769-781.
[5]Mohd Nani SZ,Majid FA,Jaafar AB,et al.Potential health benefits of deep sea water:a review.Evid Based Complement Alternat Med.2016;2016:6520475.
[6]Schrameyer V,York PH,Chartrand K,et al.Contrasting impacts of light reduction on sediment biogeochemistry in deep-and shallow-water tropical seagrass assemblages(Green Island,Great Barrier Reef).Mar Environ Res.2018;136:38-47.
[7]Hsu CL,Chang YY,Chiu CH,et al.Cardiovascular protection of deep-seawater drinking water in high-fat/cholesterol fed hamsters.Food Chem.2011;127(3):1146-1152.
[8]Hsu TC,Chiu CC,Lin HL,et al.Attenuated effects of deep-sea water on hepatic apoptosis in STZ-induced diabetic rats.Chin J Physiol.2015;58(3):197-205.
[9]Ha BG,Shin EJ,Park JE,et al.Anti-diabetic effect of balanced deep-sea water and its mode of action in high-fat diet induced diabetic mice.Mar Drugs.2013;11(11):4193-212.
[10]陈玉姣,白晓苏,曾丽红.补气生肌方促进糖尿病性难愈创面血管新生和创面修复愈合的作用机制[J].实用药物与临床,2017,20(10):1127-1130.
[11]丁寅佳,崔磊,赵旭,等.富血小板纤维蛋白联合脂肪干细胞对糖尿病小鼠皮肤创面愈合的影响研究[J].中国美容医学,2019,28(2):8-13.
[12]Choi SM,Lee KM,Kim HJ,et al.Effects of structurally stabilized EGF and bFGF on wound healing in type I and type II diabetic mice.Acta Biomater.2018;66:325-334.
[13]Milan PB,Lotfibakhshaiesh N,Joghataie MT,et al.Accelerated wound healing in a diabetic rat model using decellularized dermal matrix and human umbilical cord perivascular cells.Acta Biomater.2016;45:234-246.
[14]王志永,和晓培,薜欣,等.难愈性创面的发生机制及人工真皮在其治疗中的应用进展[J].临床合理用药杂志,2017,10(28):180-181.
[15]向鹏君,季晖,顾铭,等.糖尿病创面的炎症机制研究进展[J].药学研究,2017,36(11):667-670.
[16]胡啸明,柳国斌.糖尿病足性溃疡愈合机制的研究进展[J].上海中医药杂志,2017,51(S1):217-219,223.
[17]沈耀明.严重烧伤全身与局部微量元素含量变化特点及对创面修复的机制探讨[J].中国医药指南,2014,12(34):117-118.
[18]张文浩,吴起,马军,等.负压封闭引流联合肝素灌洗治疗兔烫伤并海水浸泡创面[J].南方医科大学学报,2015,35(10):1481-1486.
[19]姚波,刘万宏,傅亚.影响创面愈合的营养因素研究进展[J].基因组学与应用生物学,2012,31(6):640-643.
[20]张海峰,吕游,王洪莎,等.硫酸锌对糖尿病皮肤损伤的修复作用[J].中国老年学杂志,2016,36(23):5777-5780.
[21]李为明,崔进,徐鹏远,等.深层海水对小鼠创面愈合的促进作用[J].重庆医学,2014,43(4):462-464.
[22]韩昌鹏,姜文成,李福伦,等.Wnt信号通路与创面愈合[J].中国美容医学,2012,21(7):1257-1260.
[23]Tang H,Xu Y,Zhang Z,et al.SDF-1/CXCR4 induces epithelialmesenchymal transition through activation of the Wnt/β-catenin signaling pathway in rat chronic allograft nephropathy.Mol Med Rep.2019;19(5):3696-3706.
[24]Xie S,Fu W,Yu G,et al.Discovering small molecules as Wnt inhibitors that promote heart regeneration and injury repair.JMol Cell Biol.2019.pii:mjz023.[Epub ahead of print]
[25]Leavitt T,Hu MS,Marshall CD,et al.Scarless wound healing:finding the right cells and signals.Cell Tissue Res.2016;365(3):483-493.
[26]赵亚男,刘明,张玥,等.Wnt信号通路与皮肤创面愈合的关系[J].现代生物医学进展,2015,15(11):2173-2176,2184.
[27]Yang HL,Tsai YC,Korivi M,et al.Lucidone promotes the cutaneous wound healing process via activation of the PI3K/AKT,Wnt/β-catenin and NF-κB signaling pathways.Biochim Biophys Acta Mol Cell Res.2017;1864(1):151-168.
[28]Qi W,Yang C,Dai Z,et al.High levels of pigment epithelium-derived factor in diabetes impair wound healing through suppression of Wnt signaling.Diabetes.2015;64(4):1407-1419.
[29]Pizzute T,Li J,Zhang Y,et al.Fibroblast growth factor ligand dependent proliferation and chondrogenic differentiation of synovium-derived stem cells and concomitant adaptation of Wnt/mitogen-activated protein kinase signals.Tissue Eng Part A.2016;22(15-16):1036-1046.
[30]Hu F,Yan Y,Wang CW,et al.Article effect and mechanism of ganoderma lucidum polysaccharides on human fibroblasts and skin wound healing in mice.Chin J Integr Med.2019;25(3):203-209.
[31]Ma T,Fu B,Yang X,et al.Adipose mesenchymal stem cell-derived exosomes promote cell proliferation,migration,and inhibit cell apoptosis via Wnt/β-catenin signaling in cutaneous wound healing.J Cell Biochem.2019;120(6):10847-10854.
[32]Zhang G,Song K,Yan H.MicroRNA-124 represses wound healing by targeting SERP1 and inhibiting the Wnt/β-catenin pathway.Adv Clin Exp Med.2019.[Epub ahead of print]
[33]Liu D,He S,Chen S,et al.Different effects of Wnt/β-catenin activation and Parathyroid hormone on diaphyseal and metaphyseal in the early phase of femur bone healing of mice.Clin Exp Pharmacol Physiol.2019.[Epub ahead of print]
[34]Bao Q,Chen S,Qin H,et al.An appropriate Wnt/β-catenin expression level during the remodeling phase is required for improved bone fracture healing in mice.Sci Rep.2017;7(1):2695.
[35]Rognoni E,Gomez C,Pisco AO,et al.Inhibition ofβ-catenin signalling in dermal fibroblasts enhances hair follicle regeneration during wound healing.Development.2016;143(14):2522-2535.
[36]Liu J,Wang Y,Pan Q,et al.Wnt/beta-catenin pathway forms a negative feedback loop during TGF-beta1 induced human normal skin fibroblast-to-myofibroblast transition.J Dermatol Sci.2012;65(1):38-49.
[37]Schmid A,Sailland J,Novak L,et al.Modulation of Wnt signaling is essential for the differentiation of ciliated epithelial cells in human airways.FEBS Lett.2017;591(21):3493-3506.