by The vessel density of untreated diabetic wounds was significantly decreased compared with nondiabetic wounds (P < 0. 05), and the pericyte coverage of new vessels was discrete and incomplete. However , the treatments for it are limited and the cost is high [2]. Therefore , developing effective and economical therapies for correcting impaired healing of diabetic wounds is an urgent clinical demand. Diabetes impairs wound healing through magnifying the inflammatory response, inhibiting angiogenesis, and decreasing extracellular matrix (ECM) deposition [3]. The ideal treatment relies on correcting the multiple deficits simultaneously through highly integrated therapeutic approaches. Traditional Chinese medicine (TCM) is characterized by the use of herbal formulas that are usually grouped by two or more medicinal herbs, which can effectively produce synergetic effects to be greater than the sum of the individual effects and reduce side effects, providing novel therapeutic strategies for diabetic ulcer. Studies showed that combining TCM with conventional treatments in diabetic wound management received better clinical outcome [4]. Tuo-Li-Xiao-Du-San 6H05 (trifluoroacetate salt) (TLXDS) is a refined Chinese 6H05 (trifluoroacetate salt) medicine formula consisting of four herbs: Danggui (Radix Angelica sinensis), Huangqi (Radix Astragali), Baizhi (Angelica dahurica), and Zaojiaoci (thorns of Gleditsia sinensis), in the ratio of 5: 5: 4: 4 (15 g for the former two and 12 g for the latter two). It is derived from orthodox manual of surgery (Wai Ke Zheng Zong in Chinese) formulated by a famous TCM physician Shigong Chen in 1617 AD and has been used for the treatment of various refractory wounds, including pressure ulcer, venous leg ulcer, abscesses, and carbuncle. In Chinese medicine theory, TLXDS includes the therapeutic method of TUO represented byRadix Angelica sinensisandRadix Astragaliand the therapeutic method of TOU represented byAngelica dahuricaand thorns ofGleditsia sinensis. Therapeutic method of TUO means raising Qi (vital energy) and nourishing Blood (body circulation), while therapeutic method of TOU refers to cleansing wound environment and eliminating toxins. For the pharmacological action of Col13a1 each single herb in TLXDS, Radix Astragaliis used as Qi invigorator [5] andAngelica sinensisis prescribed as blood circulation activator [6]; Angelica dahuricais classified as a sweat-inducing drug able to counter harmful external influences on the skin, such as cold, heat, dampness, and dryness [7]; the thorns ofGleditsia sinensishave been used for the treatment of inflammatory diseases including swelling, suppuration, carbuncle, and skin diseases [8]. Hundreds of years of practice has proven the wound healing effect of TLXDS on various refractory wounds [9], and the pharmacological actions of herbs in TLXDS suggest it might be a potential remedy for diabetic wound. However , the effect of TLXDS on healing of diabetic wounds has not been explored before. The purpose of this study is to evaluate the efficacy of TLXDS on diabetic wound by using an excisional cutaneous wound model of streptozotocin-induced diabetic rats and also to clarify its active mechanism by immunohistochemical, qRT-PCR, and western blot analyses. == 2 . Materials and Methods == == 2 . 1 . Preparation of Tuo-Li-Xiao-Du-San Ethanol Extract == Tuo-Li-Xiao-Du-San is comprised of four herbs, Astragalus membranaceus, Angelica sinensis, Angelica dahurica, and thorns ofGleditsia sinensis, in the ratio of 5: 5: 4: 4 (15 g for the former two and 12 g for the latter two). The herbs were obtained from and authenticated by TASLY Pharmaceutical Group Co. Ltd. (Tianjin, China). The 70% ethanol extract of mixture of four herbs was prepared by the department of Pharmaceutical Sciences, Tianjin University of Traditional Chinese Medicine (Tianjin, China) using standardized procedure [10, 11]. Briefly, the crude herbs were powered and then extracted by 70% ethanol for three times. Gather the extracts and filter to remove the solid fragment. The solvents were removed by freeze-drying. The condensate was stored at 20C. The extracts are freshly prepared by dissolving in sterile 6H05 (trifluoroacetate salt) water to an appropriate concentration as herb solution for the later experiment. == 2 . 2 . Animals == Sprague-Dawley male rats (n= 120, 10 weeks old, SPF) weighing 290 10 g.
