回阳生肌汤通过加速树突状细胞胞葬功能促进糖尿病小鼠阴证创面愈合的作用机制研究  

Study on Mechanism of Huiyang Shengji Decoction in Promoting Yin Syndrome Wound Healing in Diabetic Mice by Accelerating Dendritic Cell Efferocytosis

作　　者：蔺莉 徐旭英[1] 于芳宁 唐枭 LIN Li;XU Xuying;YU Fangning;TANG Xiao(Beijing Hospital of Traditional Chinese Medicine Affiliated to Capital Medical University,Beijing 100010,China)

机构地区：[1]首都医科大学附属北京中医医院,北京100010

出　　处：《南京中医药大学学报》2025年第2期203-212,共10页Journal of Nanjing University of Traditional Chinese Medicine

基　　金：国家自然科学基金面上项目(82174388);北京市百千万人才工程培养项目(2019A30);第五批全国中医临床优秀人才研修项目(国中医药办人教函〔2021〕271号);北京市医院管理中心“登峰”人才培养计划(DFL20220801)。

摘　　要：目的探讨回阳生肌汤对小鼠糖尿病阴证创面愈合的作用及机制。方法选用SPF级C57BL/6小鼠,腹腔注射链脲佐菌素和皮肤缺损法构建糖尿病溃疡模型小鼠24只,随机分为模型组,回阳生肌汤低、中、高剂量组,每组6只。另取6只小鼠构建普通创面模型作为空白组。空白组和模型组每日蒸馏水灌胃治疗,回阳生肌汤组每日回阳生肌汤灌胃治疗,每日创面拍照,记录创面愈合率,HE染色观察创面肉芽组织生长情况,Western blot检测创面胞葬相关受体Axl、Tyro3、Mertk蛋白表达,免疫组化检测创面组织CD11c表达,qPCR检测创面TNF-α、IL-1β、IL-10、TGF-β1 mRNA表达,TUNEL染色计算创面凋亡细胞(AC)数目。采用小鼠原代骨髓树突状细胞(BMDC),将细胞重悬于含有10%空白血清或5%、10%、20%的回阳生肌含药血清的1640培养基中,在37℃,5%CO 2细胞培养箱中培养24 h后,Western blot检测创面胞葬相关受体;体外共培养BMDC和AC,流式细胞术检测BMDC吞噬AC的吞噬率。结果回阳生肌汤各剂量组创面愈合显著快于模型组(P<0.05);与模型组比较,回阳生肌汤高剂量组小鼠创面组织中Axl、Tyro3的蛋白表达明显升高(P<0.01,P<0.0001),低、中、高剂量组小鼠创面组织中Mertk蛋白表达差异无统计学意义(P>0.05)。回阳生肌汤显著抑制创面组织IL-1β、TNF-αmRNA的表达(P<0.05),促进TGF-β1、IL-10 mRNA的表达(P<0.05),且回阳生肌汤治疗后创面AC数目少于模型组(P<0.05)。体外实验中,与模型组比较,20%回阳生肌含药血清组BMDC中Axl、Mertk的蛋白表达明显升高(P<0.01,P<0.001),5%、10%、20%回阳生肌含药血清组Tyro3蛋白表达均较模型组更高(P<0.01,P<0.001),回阳生肌汤组BMDC吞噬AC的吞噬率高于模型组(P<0.05)。结论回阳生肌汤通过增强树突状细胞(DC)胞葬功能,减少创面AC聚集,促进创面炎症消退和组织修复,加快皮肤创面愈合。OBJECTIVE To explore the effect and mechanism of Huiyang Shengji Decoction on wound healing in mice with diabetic yin syndrome.METHODS SPF C57BL/6 mice were selected,and 24 diabetic ulcer models were established by intraperitoneal injection of streptozotocin and skin defect method.The mice were randomly divided into model group,low-dose,medium-dose and high-dose Huiyang Shengji Decoction groups,with 6 mice in each group.Six mice were selected to establish a common wound model as the blank group.The blank group and model group were treated with distilled water by intragastric administration every day,and the Huiyang Shengji Decoction groups were treated with Huiyang Shengji Decoction by intragastric administration every day.The wounds were photographed every day,and the wound healing rate was recorded.HE staining was used to observe the growth of granulation tissue in the wounds.Western blot was used to detect the expression of Axl,Tyro3 and Mertk proteins related to wound efferocytosis.Immunohistochemistry was used to detect the expression of CD11c in wound tissue.qPCR was used to detect the mRNA expression of TNF-α,IL-1β,IL-10 and TGF-β1 in the wounds.TUNEL staining was used to calculate the number of apoptotic cells(AC)in the wounds.Mouse primary bone marrow dendritic cells(BMDC)were resuspended in 1640 medium containing 10%blank serum or 5%,10%,and 20%Huiyang Shengji Decoction-containing serum.After culturing in a cell culture incubator at 37℃and 5%CO 2 for 24 h,the wound endocytosis-related receptors were detected by Western blot.BMDC and AC were co-cultured in vitro,and the phagocytic rate of BMDC phagocytosis of AC was detected by flow cytometry.RESULTS The wound healing in each dose group of Huiyang Shengji Decoction was significantly faster than that in the model group(P<0.05).Compared with the model group,the protein expression of Axl and Tyro3 in the wound tissue of mice in the high dose group of Huiyang Shengji Decoction was significantly increased(P<0.01,P<0.0001),and there was no significant dif

关 键 词：回阳生肌汤 糖尿病阴证 创面愈合 树突状细胞 胞葬作用 凋亡细胞 炎症 

分 类 号：R285.5[医药卫生—中药学]