基于Akt/mTOR自噬通路介导NLRP3炎症小体失活探讨冲和膏促进糖尿病大鼠溃疡创面愈合的作用机制  

Mechanism Study of Chonghe Soft Extract on Promoting Ulcer Wound Healing in Diabetes Rats Based on Akt-mTOR Autophagy-mediated NLRP3 Inflammasome Inactivation

作　　者：党赢 师建平[1] 张锁[1] 刘钰 海日[1] 吴昊[1] 焦雨琦 王璐[1] 白洁[1] 陈旭 DANG Ying;SHI Jianping;ZHANG Suo;LIU Yu;HAI Ri;WU Hao;JIAO Yuqi;WANG Lu;BAI Jie;CHEN Xu(Inner Mongolia Medical College,Hohhot 010110 Inner Mongolia,China)

机构地区：[1]内蒙古医科大学,内蒙古呼和浩特010110

出　　处：《中药新药与临床药理》2024年第6期841-849,共9页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：国家自然青年科学基金项目(82205127);内蒙古自治区高等教育科学研究项目(NJZY23131);内蒙古自治区人才开发基金项目(内人社办发〔2021〕171号);内蒙古自治区草原英才团队滚动支持项目(CYYCTD2024017);内蒙古医科大学实验室开放基金(2023ZN25)。

摘　　要：目的探讨冲和膏通过蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)自噬通路介导核苷酸结合寡聚酸结构域样受体蛋白3(NLRP3)炎症小体失活对糖尿病大鼠溃疡创面愈合的作用机制。方法采用高糖高脂饲料喂养、腹腔注射链脲佐菌素(Streptozocin,STZ)联合皮肤缺损方法构建糖尿病溃疡SD大鼠创面模型36只,将大鼠随机分为模型组、冲和膏组和生长因子组,每组12只。另取12只SD大鼠构建普通创面模型作为空白组。空白组和模型组不进行药物干预,冲和膏组和生长因子组分别外敷冲和膏、生长因子凝胶,观察记录各组大鼠创面愈合情况。给药第7天和第14天分别取材,HE染色观察创面组织病理学改变,并进行成纤维细胞计数;ELISA法检测血清中白细胞介素(IL)-1β、IL-18、肿瘤坏死因子(TNF)-α水平;免疫组化检测创面肉芽组织自噬相关蛋白Beclin-1、LC3-Ⅱ表达水平;Western Blot法检测创面肉芽组织炎症小体相关蛋白NLRP3、含Caspase募集结构域的凋亡相关斑点样蛋白(ASC)、Caspase-1、Pro-Caspase-1及Akt/mTOR自噬通路相关蛋白Akt、p-Akt、mTOR、p-mTOR的表达情况。结果与空白组比较,模型组在第7天和第14天创面病理损伤修复延缓,单位面积成纤维细胞数量较少(P<0.01),炎症因子IL-1β、IL-18、TNF-α水平升高(P<0.01),创面组织中ASC、Pro-Caspase-1、Caspase-1、NLRP3表达水平升高(P<0.01),Beclin-1、LC3-Ⅱ以及mTOR、p-mTOR、Akt、p-Akt表达水平降低(P<0.01);与模型组比,第7天和第14天冲和膏组和生长因子组病理损伤明显改善,单位面积成纤维细胞数量明显增加(P<0.01),炎症因子IL-1β、IL-18、TNF-α水平明显降低(P<0.01),创面组织中ASC、Pro-Caspase-1、Caspase-1、NLRP3表达水平降低(P<0.01),Beclin-1、LC3-Ⅱ以及mTOR、p-mTOR、Akt、p-Akt表达水平升高(P<0.01,P<0.05)。结论冲和膏可降低炎症反应,促进成纤维细胞生成,调控Akt/mTOR通路介导NLRP3炎症小体失活,�Objective To investigate the mechanism of Chonghe soft extract on ulcer wound healing in diabetic rats through protein kinase B(Akt)/mammalian Sirolimus target protein(mTOR)-mediated nucleotides binding oligomeric acid domain-like receptor protein 3(NLRP3)inflammasome inactivation.Methods Thirty six SD rats with diabetic ulcer,which were established by feeding with high glucose and high fat diet and injecting intraperitoneally with streptozocin(STZ)combined with skin defect,were randomly divided into model group,Chonghe soft extract group and growth factor group,with twelve rats in each group.Another twelve SD rats were injected an equal dose of citric acid-sodium citrate buffer solution and used as blank group.The blank group and the model group were not received drug intervention,but the Chonghe soft extract group and the growth factor group were externally applied Chonghe soft extract and growth factor gel,respectively.The wound healing of each group was observed and recorded.After 7 days and 14 days of treatment,the histopathology of wound were observed by HE staining and the number of fibroblasts were counted.The levels of IL-1β,IL-18 and TNF-αin serum were detected by ELISA.The expression of autophagy-related protein Beclin-1 and LC3II in granulation tissue was detected by immunohistochemistry.The expression of NLRP3,apoptosis-associated speck-like protein containing a caspase recruitment domain(ASC),Caspase1,Pro-Caspase1 and Akt/mTOR autophagy pathway-related proteins Akt,p-Akt,mTOR and p-mTOR were detected by Western Blot.Results Compared with the blank control group,the pathological wound repair of the model group was delayed on the 7th day and 14th day,the number of fibroblasts per unit area was decreased(P<0.01).The levels of IL-1β,IL-18 and TNF-αwere increased(P<0.01).The expression levels of ASC,Pro-Caspase1,Caspase1,and NLRP3 were increased in the wound tissues(P<0.01),while the expression levels of Beclin-1,LC3-II,mTOR,p-mTOR,Akt and p-Akt were decreased in the wound tissues(P<0.01).Compared wi

关 键 词：冲和膏 AKT/MTOR NLRP3 糖尿病 溃疡 创面愈合 自噬 大鼠 

分 类 号：R285.5[医药卫生—中药学]