龟板软膏调控毛囊干细胞中的Wnt/β-catenin信号通路修复大鼠急性皮肤创面
|
摘要
背景:龟板散由龟板、黄连和冰片组成,是治疗皮肤疮疡的有效方剂。中药单体机制明确,龟板的有效单体十四酸甾醇酯可以激活并调控毛囊干细胞中的Wnt/β-catenin信号通路。目的:探讨龟板软膏对大鼠急性皮肤创面的修复效果,以及其作用是否通过调控毛囊干细胞的Wnt/β-catenin信号通路实现。方法:取龟板散各组分中的有效单体十四酸甾醇酯(S8)、盐酸小檗碱及右旋龙脑制备龟板软膏。构建SD大鼠急性皮肤创面模型并随机分为3个药物组:龟板软膏组、S8组和空白对照组,连续给药10d。造模后连续3 d注射BrdU(50 mg/kg)标记增殖细胞。分别于修复第3,5,7及10天计算创面愈合率,苏木精-伊红染色和Masson染色观察创面病理学变化,Western blot法和免疫荧光检测Wnt/β-catenin信号通路蛋白β-catenin、LEF1、C-myc及毛囊干细胞的表面标志物CD34和Integrinβ1的表达情况。实验方案经广州中医药大学动物实验伦理委员会批准,批准号为20190218001。结果与结论:①修复第5,7,10天,龟板软膏组的创面愈合率均高于其他组(P <0.05);②龟板软膏组新生表皮与真皮连接紧密,新生皮肤附属器较S8组和空白对照组多,胶原纤维致密,修复效果优于其他组;③龟板软膏组和S8组Wnt/β-catenin信号通路蛋白β-catenin、LEF1、C-myc及毛囊干细胞的表面标志物CD34、Integrinβ1的阳性表达量均高于空白对照组,β-catenin、CD34和Integrinβ1的阳性表达均上调,LEF1和C-myc的表达呈先上调后下降变化,差异有统计学意义(P <0.05);④龟板软膏组中BrdU标记的增殖细胞数量较其他组多,且同时表达CD34阳性和LEF1阳性;⑤结果提示:龟板软膏可能通过调控毛囊干细胞中的Wnt/β-catenin信号通路,加快大鼠急性皮肤创面的修复,龟板软膏由S8、盐酸小檗碱及右旋龙脑3种有效单体组成,具有稳定性和安全性,其对急性皮肤创面的促愈合效果优于单一有效单体。
BACKGROUND: Traditional prescription Guiban San composed of tortoise shell, coptis chinensis and borneol is used for treating skin injury.As per the clear mechanism of traditional Chinese medicine monomer, the effective monomer cholesterol myristate of tortoise shell can activate and regulate the Wnt/β-catenin signal pathway in hair follicle stem cells.OBJECTIVE: To explore the effect of tortoise shell ointment on acute skin wound in rats and the mechanism whether is by regulating Wnt/β-catenin signal pathway in hair follicle stem cells.METHODS: Tortoise shell ointment was prepared with cholesterol myristate, berberine and borneol--the effective monomers from components of Guiban San. Acute skin wound model was made in Sprague-Dawley rats and rat models were then randomly divided into three groups: tortoise shell ointment group, cholesterol myristate(S8) group and control group. Medication in each group was administered continuously for 10 days. BrdU(50 mg/kg) for proliferative cell labeling was administered for 3 consecutive days. The healing rate of the wound was analyzed in different groups at 3, 5, 7, and 10 days after repair. Hematoxylin-eosin staining and Masson staining were conducted for histopathological observations. The expression of β-catenin, LEF1, C-myc in Wnt/β-catenin signal pathway and CD34, Integrin β1 in hair follicle stem cells were detected by western blot and immunofluorescence. The study protocol was approved by the Animal Ethics Committee of Guangzhou University of Chinese Medicine with an approval No. 20190218001.RESULTS AND CONCLUSION:(1) At days 5, 7, and 10 after repair, the wound healing rate in the tortoise shell ointment group was significantly higher than that in the other groups(P < 0.05).(2) Compared with the S8 and control groups, tortoise shell ointment group showed better repair effect with more neoformative hair follicles and denser collagen fibers.(3) The expressions of β-catenin, LEF1, C-myc,CD34, Integrin β1 in the tortoise shell ointment and S8 group were higher than those in the control group. The positive expression of β-catenin,CD34 and Integrin β1 were increased during wound healing. LEF1 and C-myc were increased during the prophase and metaphase of wound healing but decreased during the later stage. The difference was statistically significant among three groups(P < 0.05). The number of BrdU-labeled proliferative cells in the tortoise shell ointment group was higher than that in the S8 and control groups, with concurrent positive expressions of CD34 and LEF1. To conclude, tortoise shell ointment may stably and safely promote the repair of acute skin wound in rats via regulating Wnt/β-catenin signal pathway in hair follicle stem cells, and the healing effect is superior to any single effective monomer.
BACKGROUND: Traditional prescription Guiban San composed of tortoise shell, coptis chinensis and borneol is used for treating skin injury.As per the clear mechanism of traditional Chinese medicine monomer, the effective monomer cholesterol myristate of tortoise shell can activate and regulate the Wnt/β-catenin signal pathway in hair follicle stem cells.OBJECTIVE: To explore the effect of tortoise shell ointment on acute skin wound in rats and the mechanism whether is by regulating Wnt/β-catenin signal pathway in hair follicle stem cells.METHODS: Tortoise shell ointment was prepared with cholesterol myristate, berberine and borneol--the effective monomers from components of Guiban San. Acute skin wound model was made in Sprague-Dawley rats and rat models were then randomly divided into three groups: tortoise shell ointment group, cholesterol myristate(S8) group and control group. Medication in each group was administered continuously for 10 days. BrdU(50 mg/kg) for proliferative cell labeling was administered for 3 consecutive days. The healing rate of the wound was analyzed in different groups at 3, 5, 7, and 10 days after repair. Hematoxylin-eosin staining and Masson staining were conducted for histopathological observations. The expression of β-catenin, LEF1, C-myc in Wnt/β-catenin signal pathway and CD34, Integrin β1 in hair follicle stem cells were detected by western blot and immunofluorescence. The study protocol was approved by the Animal Ethics Committee of Guangzhou University of Chinese Medicine with an approval No. 20190218001.RESULTS AND CONCLUSION:(1) At days 5, 7, and 10 after repair, the wound healing rate in the tortoise shell ointment group was significantly higher than that in the other groups(P < 0.05).(2) Compared with the S8 and control groups, tortoise shell ointment group showed better repair effect with more neoformative hair follicles and denser collagen fibers.(3) The expressions of β-catenin, LEF1, C-myc,CD34, Integrin β1 in the tortoise shell ointment and S8 group were higher than those in the control group. The positive expression of β-catenin,CD34 and Integrin β1 were increased during wound healing. LEF1 and C-myc were increased during the prophase and metaphase of wound healing but decreased during the later stage. The difference was statistically significant among three groups(P < 0.05). The number of BrdU-labeled proliferative cells in the tortoise shell ointment group was higher than that in the S8 and control groups, with concurrent positive expressions of CD34 and LEF1. To conclude, tortoise shell ointment may stably and safely promote the repair of acute skin wound in rats via regulating Wnt/β-catenin signal pathway in hair follicle stem cells, and the healing effect is superior to any single effective monomer.
引文
[1]Li J,Zheng H,Wang J,et al.Expression of Kruppel-like factor KLF4 in mousehair follicle stem cells contributes to cutaneous wound healing.PLoS One.2012;7(6):e39663.
[2]Pineda CM,Park S,Mesa KR,et al.Intravital imaging of hair follicle regeneration in the mouse.Nat Protoc.2015;10(7):1116-1130.
[3]Si Y,Bai J,Wu J,et al.LncRNA PlncRNA 1 regulates proliferation and differentiation of hair follicle stem cells through TGFβ1 mediated Wnt/βcatenin signal pathway.Mol Med Rep.2018;17(1):1191-1197.
[4]Du KT,Deng JQ,He XG,et al.MiR-214 Regulates the Human Hair Follicle Stem Cell Proliferation and Differentiation by Targeting EZH2 and Wnt/β-Catenin Signaling Way In Vitro.Tissue Eng Regen Med.2018;15(3):341-350.
[5]Lim X,Nusse R.Wnt signaling in skin development,homeostasis,and disease.Cold Spring Harb Perspect Biol.2013;5(2).pii:a008029.doi:10.1101/cshperspect.a008029.
[6]Nowak JA,Polak L,Pasolli HA,et al.Hair Follicle Stem Cells Are Specified and Function in Early Skin Morphogenesis.Cell Stem Cell.2008;3(1):33.
[7]Chen DF,Du SH,Zhang HL,et al.Autocrine BMP4 signaling involves effect of cholesterol myristate on proliferation of mesenchymal stem cells.Steroids.2009;74(13-14):1066-1072.
[8]李瑜,魏刚,唐铱,等.十四酸甾醇酯调控Wnt/β-catenin信号通路促皮肤创面修复的研究[J].中药新药与临床药理,2018,29(3):302-306.
[9]Wang K,Feng X,Chai L,et al.The metabolism of berberine and its contribution to the pharmacological effects.Drug Metab Rev.2017;49(2):139-157.
[10]Chen J,Su JY,Li L,et al.A new source of natural d-borneol and its characteristic.J Med Plants Res.2011;5(15):3440-3447.
[11]唐铱,李春,黎湘君,等.龟板提取物激活Wnt/β-catenin通路促大鼠触须部皮肤创面修复[J].中药新药与临床药理,2017,28(3):298-303.
[12]Ramírez E,Mendieta L,Flores G,et al.Neurogenesis and morphological-neural alterations closely related to amyloidβ-peptide(25-35)-induced memory impairment in male rats.Neuropeptides.2018;67:9-19.
[13]J Martin Wojtowicz,Nohjin Kee.BrdU assay for neurogenesis in rodents.Nat Protoc.2006;1(3):1399-1405.
[14]杨鸣华,刘祎,孔令义.基于中药有效单体成分的新药研究[J].世界科学技术-中医药现代化,2016,18(3):329-336.
[15]Das U,Behera SS,Pramanik K.Ethno-Herbal-Medico in Wound Repair:An Incisive Review.Phytother Res.2017;31(4):579-590.
[16]Chen G,Xu Y,Jing J,et al.The anti-sepsis activity of the components of Huanglian Jiedu Decoction with high lipid A-binding affinity.Int Immunopharmacol.2017;46:87-96.
[17]Ruan H,Zhan YY,Hou J,et al.Berberine binds RXRαto suppressβ-catenin signaling in colon cancer cells.Oncogene.2017;36(50):6906-6918.
[18]Lin YH,Lin JH,Chou SC,et al.Berberine-loaded targeted nanoparticles as specific Helicobacter pylori eradication therapy:in vitro and in vivo study.Nanomedicine(Lond).2015;10:57-71.
[19]Pietra D,Borghini A,Bianucci AM.In vitro studies of antifibrotic and cytoprotective effects elicited by proto-berberine alkaloids in human dermal fibroblasts.Pharmacol Rep.2015;67(6):1081-1089.
[20]Bao J,Yang B,Sun Y,et al.A berberine-loaded electrospun poly-(epsilon-caprolactone)nanofibrous membrane with hemostatic potential and antimicrobial property for wound dressing.J Biomed Nanotechnol.2013;9(7):1173-1180.
[21]Wang S,Zhang D,Hu JS,et al.A clinical and mechanistic study of topical borneol-induced analgesia.EMBO Molecular Medicine.2017;9(6):802-815.
[22]Zhou Y,Li W,Chen L,et al.Enhancement of intestinal absorption of akebia saponind by borneol andprobenecid in situ and in vitro.Environ.Toxicol.harmacol.2010;29(3):229-234.
[23]Zhang QL,Fu BM,Zhang ZJ.Borneol,a novel agent that improves central nervous system drug delivery by enhancing blood-brain barrier permeability.Drug Deliv.2017;24(1):1037-1044.
[24]Mikesh LM,Aramadhaka LR,Moskaluk C,et al.Proteomic anatomy of human skin.J Proteomics.2013;84:190-200.
[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]Gao C,Xiao G,Hu J.Regulation of Wnt/β-catenin signaling by posttranslational modifications.Cell Biosci.2014;4(1):13.
[27]Wang HD,Yang L,Yu XJ,et al.Immunolocalization ofβ-catenin and Lef-1 during postnatal hair follicle development in mice.Acta Histochem.2012;114(8):773-778.
[28]Wang N,Yang T,Li J,et al.The expression and role of c-Myc in mouse hair follicle morphogenesis and cycling.Acta Histochem.2012;114(3):199-206.
[29]Shen Q,Yu W,Fang Y,et al.Beta-catenin can induce hair follicle stem cell differentiation into transit-amplifying cells through c-myc activation.Tissue Cell.2017;49(1):28-34.
[30]Ojeh N,Pastar I,Tomic-Canic M,et al.Stem Cells in Skin Regeneration,Wound Healing,and Their Clinical Applications.Int J Mol Sci.2015;16(10):25476-25501.
[31]李佳,安恒庆,王峰,等.组织块法与酶消化法培养大鼠毛囊干细胞的比较[J].中国组织工程研究,2015,19(01):91-95.
[2]Pineda CM,Park S,Mesa KR,et al.Intravital imaging of hair follicle regeneration in the mouse.Nat Protoc.2015;10(7):1116-1130.
[3]Si Y,Bai J,Wu J,et al.LncRNA PlncRNA 1 regulates proliferation and differentiation of hair follicle stem cells through TGFβ1 mediated Wnt/βcatenin signal pathway.Mol Med Rep.2018;17(1):1191-1197.
[4]Du KT,Deng JQ,He XG,et al.MiR-214 Regulates the Human Hair Follicle Stem Cell Proliferation and Differentiation by Targeting EZH2 and Wnt/β-Catenin Signaling Way In Vitro.Tissue Eng Regen Med.2018;15(3):341-350.
[5]Lim X,Nusse R.Wnt signaling in skin development,homeostasis,and disease.Cold Spring Harb Perspect Biol.2013;5(2).pii:a008029.doi:10.1101/cshperspect.a008029.
[6]Nowak JA,Polak L,Pasolli HA,et al.Hair Follicle Stem Cells Are Specified and Function in Early Skin Morphogenesis.Cell Stem Cell.2008;3(1):33.
[7]Chen DF,Du SH,Zhang HL,et al.Autocrine BMP4 signaling involves effect of cholesterol myristate on proliferation of mesenchymal stem cells.Steroids.2009;74(13-14):1066-1072.
[8]李瑜,魏刚,唐铱,等.十四酸甾醇酯调控Wnt/β-catenin信号通路促皮肤创面修复的研究[J].中药新药与临床药理,2018,29(3):302-306.
[9]Wang K,Feng X,Chai L,et al.The metabolism of berberine and its contribution to the pharmacological effects.Drug Metab Rev.2017;49(2):139-157.
[10]Chen J,Su JY,Li L,et al.A new source of natural d-borneol and its characteristic.J Med Plants Res.2011;5(15):3440-3447.
[11]唐铱,李春,黎湘君,等.龟板提取物激活Wnt/β-catenin通路促大鼠触须部皮肤创面修复[J].中药新药与临床药理,2017,28(3):298-303.
[12]Ramírez E,Mendieta L,Flores G,et al.Neurogenesis and morphological-neural alterations closely related to amyloidβ-peptide(25-35)-induced memory impairment in male rats.Neuropeptides.2018;67:9-19.
[13]J Martin Wojtowicz,Nohjin Kee.BrdU assay for neurogenesis in rodents.Nat Protoc.2006;1(3):1399-1405.
[14]杨鸣华,刘祎,孔令义.基于中药有效单体成分的新药研究[J].世界科学技术-中医药现代化,2016,18(3):329-336.
[15]Das U,Behera SS,Pramanik K.Ethno-Herbal-Medico in Wound Repair:An Incisive Review.Phytother Res.2017;31(4):579-590.
[16]Chen G,Xu Y,Jing J,et al.The anti-sepsis activity of the components of Huanglian Jiedu Decoction with high lipid A-binding affinity.Int Immunopharmacol.2017;46:87-96.
[17]Ruan H,Zhan YY,Hou J,et al.Berberine binds RXRαto suppressβ-catenin signaling in colon cancer cells.Oncogene.2017;36(50):6906-6918.
[18]Lin YH,Lin JH,Chou SC,et al.Berberine-loaded targeted nanoparticles as specific Helicobacter pylori eradication therapy:in vitro and in vivo study.Nanomedicine(Lond).2015;10:57-71.
[19]Pietra D,Borghini A,Bianucci AM.In vitro studies of antifibrotic and cytoprotective effects elicited by proto-berberine alkaloids in human dermal fibroblasts.Pharmacol Rep.2015;67(6):1081-1089.
[20]Bao J,Yang B,Sun Y,et al.A berberine-loaded electrospun poly-(epsilon-caprolactone)nanofibrous membrane with hemostatic potential and antimicrobial property for wound dressing.J Biomed Nanotechnol.2013;9(7):1173-1180.
[21]Wang S,Zhang D,Hu JS,et al.A clinical and mechanistic study of topical borneol-induced analgesia.EMBO Molecular Medicine.2017;9(6):802-815.
[22]Zhou Y,Li W,Chen L,et al.Enhancement of intestinal absorption of akebia saponind by borneol andprobenecid in situ and in vitro.Environ.Toxicol.harmacol.2010;29(3):229-234.
[23]Zhang QL,Fu BM,Zhang ZJ.Borneol,a novel agent that improves central nervous system drug delivery by enhancing blood-brain barrier permeability.Drug Deliv.2017;24(1):1037-1044.
[24]Mikesh LM,Aramadhaka LR,Moskaluk C,et al.Proteomic anatomy of human skin.J Proteomics.2013;84:190-200.
[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]Gao C,Xiao G,Hu J.Regulation of Wnt/β-catenin signaling by posttranslational modifications.Cell Biosci.2014;4(1):13.
[27]Wang HD,Yang L,Yu XJ,et al.Immunolocalization ofβ-catenin and Lef-1 during postnatal hair follicle development in mice.Acta Histochem.2012;114(8):773-778.
[28]Wang N,Yang T,Li J,et al.The expression and role of c-Myc in mouse hair follicle morphogenesis and cycling.Acta Histochem.2012;114(3):199-206.
[29]Shen Q,Yu W,Fang Y,et al.Beta-catenin can induce hair follicle stem cell differentiation into transit-amplifying cells through c-myc activation.Tissue Cell.2017;49(1):28-34.
[30]Ojeh N,Pastar I,Tomic-Canic M,et al.Stem Cells in Skin Regeneration,Wound Healing,and Their Clinical Applications.Int J Mol Sci.2015;16(10):25476-25501.
[31]李佳,安恒庆,王峰,等.组织块法与酶消化法培养大鼠毛囊干细胞的比较[J].中国组织工程研究,2015,19(01):91-95.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |