Kuai Le, Zhang Jing-Ting, Deng Yu, Xu Shun, Xu Xun-Zhe, Wu Min-Feng, Guo Dong-Jie, Chen Yu, Wu Ren-Jie, Zhao Xing-Qiang, Nian Hua, Li Bin, Li Fu-Lun
Department of Dermatology of Yueyang Hospital, Shanghai University of TCM, Shanghai, 200437, China.
Pharmaceutical Center of Yueyang Hospital, Shanghai University of TCM, Shanghai, 200437, China.
BMC Complement Altern Med. 2018 Jan 29;18(1):32. doi: 10.1186/s12906-017-2074-8.
Sheng-ji Hua-yu(SJHY) formula is one of the most useful Traditional Chinese medicine (TCM) in the treatment of the delayed diabetic wound. However, elucidating the related molecular biological mechanism of how the SJHY Formula affects excessive inflammation in the process of re-epithelialization of diabetic wound healing is a task urgently needed to be fulfilled. The objectives of this study is to evaluate the effect of antagonisic expression of pro-/anti-inflammatory factors on transforming growth factor-β(TGF-β) superfamily (activin and follistatin) in the process of re-epithelialization of diabetic wound healing in vivo, and to characterize the involvement of the activin/follistatin protein expression regulation, phospho-Smad (pSmad2), and Nuclear factor kappa B p50 (NF-kB) p50 in the diabetic wound healing effects of SJHY formula.
SJHY Formula was prepared by pharmaceutical preparation room of Yueyang Hospital of Integrated Traditional Chinese and Western Medicine. Diabetic wound healing activity was evaluated by circular excision wound models. Wound healing activity was examined by macroscopic evaluation. Activin/follistatin expression regulation, protein expression of pSmad2 and NF-kB p50 in skin tissue of wounds were analyzed by Real Time PCR, Western blot, immunohistochemistry and hematoxylin and eosin (H&E) staining.
Macroscopic evaluation analysis showed that wound healing of diabetic mice was delayed, and SJHY Formula accelerated wound healing time of diabetic mice. Real Time PCR analysis showed higher mRNA expression of activin/follistatin in diabetic delayed wound versus the wound in normal mice. Western Blot immunoassay analysis showed reduction of activin/follistatin proteins levels by SJHY Formula treatment 15 days after injury. Immunohistochemistry investigated the reduction of pSmad2 and NF-kB p50 nuclear staining in the epidermis of diabetic SJHY versus diabetic control mice on day 15 after wounding. H&E staining revealed that SJHY Formula accelerated re-epithelialization of diabetic wound healing.
The present study found that diabetic delayed wound healing time is closely related to the high expression level of activin/follistatin, which leads to excessive inflammation in the process of re-epithelization. SJHY Formula accelerates re-epithelialization and healing time of diabetic wounds through decreasing the high expression of activin/follistatin.
生肌化瘀（SJHY）方是治疗糖尿病延迟伤口最有效的中药之一。然而，阐明SJHY方在糖尿病伤口愈合再上皮化过程中影响过度炎症的相关分子生物学机制是一项亟待完成的任务。本研究的目的是评估促炎/抗炎因子的拮抗表达对糖尿病伤口愈合再上皮化过程中转化生长因子-β（TGF-β）超家族（激活素和卵泡抑素）的影响，并确定激活素/卵泡抑素蛋白表达调控、磷酸化Smad（pSmad2）和核因子κB p50（NF-κB p50）在SJHY方对糖尿病伤口愈合作用中的参与情况。
SJHY方由上海中医药大学附属岳阳中西医结合医院药剂室制备。通过圆形切除伤口模型评估糖尿病伤口愈合活性。通过宏观评估检查伤口愈合活性。通过实时定量聚合酶链反应（Real Time PCR）、蛋白质免疫印迹法（Western blot）、免疫组织化学和苏木精-伊红（H&E）染色分析伤口皮肤组织中激活素/卵泡抑素表达调控、pSmad2和NF-κB p50的蛋白表达。
宏观评估分析表明糖尿病小鼠伤口愈合延迟，而SJHY方加速了糖尿病小鼠的伤口愈合时间。实时定量聚合酶链反应分析显示糖尿病延迟伤口中激活素/卵泡抑素的mRNA表达高于正常小鼠伤口。蛋白质免疫印迹法分析显示，损伤后15天，SJHY方治疗可降低激活素/卵泡抑素蛋白水平。免疫组织化学研究发现，在受伤后第15天，糖尿病SJHY组小鼠表皮中pSmad2和NF-κB p50核染色较糖尿病对照组小鼠减少。H&E染色显示，SJHY方加速了糖尿病伤口愈合的再上皮化。
本研究发现糖尿病延迟伤口愈合时间与激活素/卵泡抑素的高表达水平密切相关，这导致再上皮化过程中出现过度炎症。SJHY方通过降低激活素/卵泡抑素的高表达加速糖尿病伤口的再上皮化和愈合时间。
