外用溃疡散对皮肤溃疡大鼠创面愈合的作用机制及安全性研究

Mechanism and Safety Study of External Use of Kuiyangsan on Wound Healing for Skin Ulcer in Rats

目的 探讨外用溃疡散对大鼠足部皮肤溃疡创面愈合的作用机制及其安全性。方法 健康雄性SD大鼠110只,以随机数字表法分为2组,20只用于安全性实验,分为完整皮肤组、破损皮肤组,其余90只再以随机数字表法分为对照组(15只)、模型组(15只)、生长因子组(30只)、外用溃疡散组(30只)。采用高脂高糖饮食结合腹腔注射链脲佐菌素的方法联合足背部制造溃疡创面的方法构建大鼠皮肤溃疡模型。生长因子组给予外用重组人碱性成纤维细胞生长因子(rh-bFGF)150 IU/cm^(2)、外用溃疡散组给予外用溃疡散外敷创面1 g/cm^(2)。观察创面愈合情况,计算创面愈合率。分别在治疗前、治疗后第7、14天时采集创面肉芽组织制作苏木精-伊红(HE)染色切片,进行组织病理学观察;聚合酶链式反应(PCR)检测创面肉芽组织中转化生长因子β1(TGF-β1)、大鼠信号转导分子3(Smad3)的表达;免疫组织化学法检测含半胱氨酸的天冬氨酸蛋白水解酶-3(cleaved-caspase-3)的表达;原位末端凋亡法(TUNEL)对组织细胞凋亡情况进行检测。将20只健康雄性SD大鼠,背部脊柱两侧剃毛,破损皮肤组用细砂纸摩擦造成剃毛区局部擦伤,于剃毛区左右两侧分别涂外用溃疡散与凡士林自身对照,进行给药后皮肤刺激性观察、尿液检测及β2微球蛋白(β2-MG)检测。结果 与对照组比较,给药第7、14天,模型组创面愈合率明显降低(P<0.01),可见炎症细胞浸润,有少量新生毛细血管和少量胶原纤维。与模型组比较,外用溃疡散组与生长因子组创面愈合率提高(P<0.01),创面组织有较多成纤维细胞,炎症细胞浸润较少修复情况较好;创面中TGF-β1、Smad3 mRNA表达水平显著升高(P<0.01),创面cleaved-caspase-3的蛋白表达量明显降低(P<0.01),细胞凋亡量均明显降低(P<0.01)。安全性实验中,两组在对皮肤刺激程度、24 h尿蛋白(albumin,ALB)含量及β2-MG检测比较,差异无统计学意义(P>0.05)。结论 外用溃疡散可以通过TGF-β1/Smad3信号通路,加速大鼠足部创面新生血管生成,胶原纤维沉积,减少细胞凋亡,促进创面愈合,且具有安全性。

Objective To study the mechanism and safety of external use of Kuiyangsan(KYs)on wound healing of skin ulcer in rats.Methods A total of 110 SPF Sprague-Dawley rats were divided into 2 groups by random number table.Twenty rats used for safety experiment were randomly divided into the complete skin group(n=10)and the damaged skin group(n=10),and the other 90 were divided into the control group(n=15),the model group(n=15),the rh-bFGF group(n=30),and the KYS group(n=30)by random number table.An skin ulcer wound was established on the back of rats'feet by a high-fat high-sugar diet combined with intraperitoneal injection of streptozotocin(STz).Recombinant human-basic fibroblast growth factor(rhbFGF,150 IU/cm^(2))was externally administered to rats in the rh-bFGF group.KYS(1 g/cm^(2))was externally applied to rats in the KYS group.Wound healing was observed to calculate the wound healing rate.Sections of the wound granulation tissue were collected for HE staining and histopathological observation before treatment,and at day 7 and 14 after treatment.The gene expressions of tumor growth factorβ1(TGF-β1)and Smad3 in the wound granulation tissue were detected by PCR.The expression of cleaved caspase-3 was determined by immunohistochemistry.The TUNEL Apoptosis Detection Kit was used to detect the apoptosis of tissue cells.In the safety experiments 20 healthy male Sprague-Dawley rats were shaved on both sides of the back spine,and the damaged skin group was rubbed with fine sandpaper to cause local abrasion in the shaved area.The left and right sides of the shaved area were coated with KYS and Vaseline respectively.Skin irritation was observed,urine test andβ2-MG level test were performed.Results Compared with the control group,the wound healing rate significantly decreased in the model group at day 7 and 14 after administration(P<0.01).Inflammatory cell infiltration and few new capillaries and few collagen fibers were observed.Compared with the model group,the wound healing rate of the KYS group and the rh-bFGF group significantly increased(P<0.01).There were more fibroblasts and less infiltration of inflammatory cells in the wound tissue,indicating good repair.mRNA expression levels of TGF-β1 and Smad3 significantly increased(P<0.01).The cleaved caspase-3 protein expression level significantly decreased(P<0.01).The degree of apoptosis in the wound surface significantly decreased(P<0.01).In the safety experiments there were no significant differences in the degree of skin irritation,24 h albumin(ALB)levels,orβ2-MG levels between the two groups(P>0.05).Conclusion KYS accelerated neovasculogenesis and collagen fiber deposition,reduced apoptosis,and promoted wound healing in rats by TGF-β1/Smad3 signaling pathway,and it had a remarkable safety profile.