石香膏干预糖尿病溃疡创面模型大鼠创面组织晚期糖基化终产物及其受体与内皮型一氧化氮合成酶表达的变化

Shixiang Paste interferes with the expression of advanced glycation endproducts and its receptors and endothelial nitric oxide synthase in wound tissue of diabetic ulcer rats

背景:糖尿病溃疡作为糖尿病的常见并发症之一,其低愈合率和高截肢率已严重危害人类健康,尽管目前多种中医外用膏药已证实能促进创面愈合,但其发挥作用的分子机制尚未明确。目的:探讨石香膏治疗糖尿病溃疡创面的可能作用机制。方法:将40只清洁级雄性SD大鼠随机分正常对照组、糖尿病溃疡组、细胞生长因子组、石香膏组,每组10只。除正常对照组外,其余3组均制备糖尿病大鼠模型;1周后4组均采用全层皮肤缺损法制备溃疡创面模型。造模成功1周后,石香膏组、细胞生长因子组分别给予外用细胞生长因子及外敷石香膏干预,糖尿病溃疡组及正常对照组仅使用双层消毒干纱布覆盖固定。分别在干预后第7,14天取各组大鼠背部创面肉芽组织标本,Western-blot法检测大鼠背部创面肉芽组织中晚期糖基化终产物受体、内皮型一氧化氮合成酶蛋白表达水平;苏木精-伊红染色检测大鼠背部创面样本病理变化;免疫组化法检测大鼠背部创面肉芽组织中晚期糖基化终产物表达水平。结果与结论:①苏木精-伊红染色结果显示,干预第7,14天正常对照组与糖尿病溃疡组镜下均可见大片未修复组织,而石香膏与细胞生长因子组在干预后第7,14天后创面修复良好;②Western-blot法检测结果显示,与正常对照组对比,糖尿病溃疡组的晚期糖基化终产物受体表达量上升,内皮型一氧化氮合成酶表达量下降(P<0.05);石香膏组和细胞生长因子组中晚期糖基化终产物受体相对表达量相对下降,内皮型一氧化氮合成酶相对表达量相对上升(P<0.05);与糖尿病溃疡组对比,石香膏组和细胞生长因子组中晚期糖基化终产物受体表达量相对下降,内皮型一氧化氮合成酶表达量相对上升(P<0.05);与细胞生长因子组相比,石香膏组晚期糖基化终产物受体表达量相对下降,内皮型一氧化氮合成酶表达量相对上升(P<0.05);③免疫组化法检测结果显示,与正常对照组对比,糖尿病溃疡组、细胞生长因子组晚期糖基化终产物表达水平上升,石香膏组的晚期糖基化终产物的表达水平下降(P<0.05);与糖尿病溃疡组比较,各给药组晚期糖基化终产物的表达水平上升(P<0.05);与石香膏组比较,细胞生长因子组的晚期糖基化终产物表达水平下降(P<0.05);④提示石香膏可以促进糖尿病溃疡创面修复,抑制创面中晚期糖基化终产物、晚期糖基化终产物受体表达,促进内皮型一氧化氮合成酶表达可能是其作用机制之一。

BACKGROUND:Diabetic ulcer,as one of the common complications of diabetes,has caused serious harm to human health due to its low healing rate and high amputation rate.Although traditional Chinese medicine has been proved to improve wound healing,the molecular mechanism of this effect has not been clarified.OBJECTIVE:To investigate the possible mechanism of Shixiang Paste in treating diabetic ulcers METHODS:Forty clean male Sprague-Dawley rats were randomly divided into four groups(n=10 per group):a normal control group,a diabetic ulcer group,a cell growth factor group and a Shixiang Paste group.Except for the normal control group,a diabetic rat model was established in the other three groups.After 1 week,an ulcer wound model was established in four groups by using the full-thickness skin defect method.One week after successful modeling,rats in the Shixiang Paste and cell growth factor groups were treated with external Shixiang Paste and cell growth factor,respectively.Wounds in the diabetic ulcer and normal control groups were only covered and fixed with double sterile dry gauze.The granulation tissue samples were taken from the wound on the back of rats on the 7th and 14th days after intervention.Western blot was used to detect the protein expression of receptors of advanced glycation endproducts and endothelial nitric oxide synthase in the granulation tissue from the back wound of rats.Hematoxylin-eosin staining was used to detect pathological changes in rat back wound samples.Immunohistochemical staining was used to detect the expression of advanced glycation endproducts in the granulation tissue from the wound on the back of rats.RESULTS AND CONCLUSION:The results of hematoxylin-eosin staining showed that large areas of unrepaired tissue were seen under the microscope in the normal control group and the diabetic ulcer group on the 7th and 14th days after intervention,while in the Shixiang Paste and cell growth factor groups,the wounds healed well on the 7th and 14th days after intervention.Western blot results showed that,compared with the normal control group,the expression of receptors of advanced glycation endproducts was decreased in the diabetic ulcer group was increased,and the expression of endothelial nitric oxide synthase(P<0.05).In addition,the expression of receptors of advanced glycation endproducts was relatively decreased,and the expression of endothelial nitric oxide synthase was relatively increased in the Shixiang Paste group and cell growth factor group(P<0.05).Compared with the diabetic ulcer group,the expression of receptors of advanced glycation endproducts was relatively decreased,and the expression of endothelial nitric oxide synthase was relatively increased in the Shixiang Paste group and the cell growth factor group(P<0.05).Compared with the cell growth factor group,the expression of receptors of advanced glycation endproducts was relatively decreased,and the expression of endothelial nitric oxide synthase was relatively increased in the Shixiang Paste group(P<0.05).The results of immunohistochemical staining showed that,compared with the normal control group,the expression of advanced glycation endproducts was increased in the diabetic ulcer group and the cell growth factor group,and the expression of advanced glycation endproducts was decreased in the Shixiang Paste group(P<0.05).Compared with the diabetic ulcer group,the expression of advanced glycation endproducts was increased in the other groups(P<0.05).Compared with the Shixiang Paste group,the expression level of advanced glycation endproducts was decreased in the cell growth factor group(P<0.05).To conclude,Shixiang Paste can promote the repair of diabetic ulcer wound.Inhibiting the expression of advanced glycation endproducts and receptors and enhancing the expression of endothelial nitric oxide synthase in wound tissue may be one of the mechanisms.