摘要
目的 观察复方黄柏液对皮肤破损大鼠创面细胞因子表达、血管生成和创口愈合的影响,并探讨复方黄柏液促进伤口愈合的机制。方法 SD大鼠随机分为对照组、复方黄柏液组、复方黄柏液+GSK组(复方黄柏液+GSK-690693 20 mg/kg),每组12只。构建皮肤缺损大鼠模型后,复方黄柏液组采用复方黄柏液冲洗创面,复方黄柏液+GSK组在用复方黄柏液冲洗创面的同时灌胃GSK-690693,对照组给予等体积无菌生理盐水冲洗创面和灌胃,1次/d,连续干预21 d。在干预后第7、14和21天观察创面恢复情况,计算伤口愈合率;HE染色观察伤口组织病理学变化,并计算再上皮化率;ELISA法检测血清白细胞介素(IL)-1β、肿瘤坏死因子α(TNF-α)和IL-10水平;免疫荧光染色法检测血管生成;实时荧光定量PCR(RT-qPCR)检测伤口组织中血管内皮生长因子(VEGF)、成纤维细胞生长因子(FGF)-2、表皮生长因子(EGF)、Ⅰ型胶原α2(COL1α2)、基质金属蛋白酶2(MMP-2)、MMP-9的mRNA表达;蛋白质印迹(Western blot)检测伤口组织中蛋白激酶B(AKT)、磷酸化AKT(p-AKT)、内皮型一氧化氮合酶(eNOS)和磷酸化eNOS(p-eNOS)蛋白表达。结果 与对照组相比,复方黄柏液组大鼠伤口愈合速度较快,伤口愈合率、再上皮化率、血清IL-10水平、CD31阳性细胞率、伤口组织VEGF、FGF-2、EGF、COL1α2的mRNA水平和p-AKT/AKT、p-eNOS/eNOS比值显著升高,血清IL-1β和TNF-α水平、伤口组织MMP-2和MMP-9的mRNA水平显著降低(P<0.05);使用AKT抑制剂GSK-690693抑制AKT活化,可抑制eNOS磷酸化,并显著减弱复方黄柏液的促血管生成和伤口愈合作用(P<0.05)。结论 复方黄柏液可能通过激活AKT/eNOS通路,促进血管生成,加速皮肤伤口组织修复。
Objective To observe the impacts of compound Huangbai liquid on the expression of cytokines, angiogenesis and wound healing in the damaged skin of rats, and to explore the mechanism further.Methods SD rats were randomly divided into control group, compound Huangbai liquid group, and compound Huangbai liquid + GSK group(compound Huangbai liquid + GSK-690693 20 mg/kg), 12 rats each group. After the skin defect rat model was established, the compound Huangbai liquid group was given compound Huangbai liquid to rinse the wound, the compound Huangbai liquid+GSK group was given GSK-690693 gavage while the compound Huangbai liquid was performed to rinse the wound, and the control group was given an equal volume of sterile normal saline for irrigation of wounds and gavage, once a day, for 21 days. The wound recovery was observed at the 7 th, 14 th and 21 st days after the intervention, and the wound healing and re-epithelialization rates were calculated. HE staining was performed to observe the pathological changes of wound tissue, ELISA was performed to measure the serum levels of interleukin(IL)-1β, tumor necrosis factor-α(TNF-α) and IL-10. Immunofluorescence staining was performed to measure angiogenesis.Real-time quantitative PCR(RT-qPCR) was performed to measure the mRNA expression of vascular endothelial growth factor(VEGF), fibroblast growth factor(FGF)-2, epidermal growth factor(EGF), collagen type Ⅰ α2(COL1α2), matrix metalloproteinase-2(MMP-2), MMP-9 in wound tissue. Western blot was performed to measure the protein expression of protein kinase B(AKT), phosphorylated AKT(p-AKT), endothelial nitric oxide synthase(eNOS) and phosphorylated eNOS(p-eNOS) in wound tissues.Results Compared with the control group, the wound healing and re-epithelialization rates in the compound Huangbai liquid group were faster, serum IL-10 level, CD31 positive cell rate, mRNA levels of VEGF, FGF-2, EGF, COL1α2 in wound tissue and p-AKT/AKT, p-eNOS/eNOS ratio were obviously increased, the serum levels of IL-1β and TNF-α, and mRNA levels of MMP-2 and MMP-9 in wound tissue were obviously decreased(P<0.05). GSK-690693 was able to inhibit eNOS phosphorylation, and obviously attenuate the angiogenesis and wound healing effects of compound Huangbai liquid(P<0.05).Conclusion Compound Huangbai liquid may promote angiogenesis and accelerate skin wound tissue repair by activating the AKT/eNOS pathway.
作者
蒋晓蕾
朱立宏
JIANG Xiaolei;ZHU Lihong(Department of Dermatology,Wuhan Hospital of Traditional Chinese Medicine,Wuhan 430014,China)
出处
《中国皮肤性病学杂志》
CAS
CSCD
北大核心
2022年第12期1390-1397,共8页
The Chinese Journal of Dermatovenereology
基金
武汉市医学科研项目(WZ20C39)
关键词
皮肤伤口愈合
复方黄柏液
血管生成
蛋白激酶B
内皮型一氧化氮合酶
Skin wound healing
Compound Huangbai liquid
Angiogenesis
Protein kinase B
Endothelial nitric oxide synthase
作者简介
通信作者:朱立宏,E-mail:vrb684@163.com
