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journal papers
Experimental study on ASTAR CREAM for the treatment of chronic diabetic ulcers
Yufeng Jiang 1 Sha Huang2 Jiping Zou2 Zhangrong Xu1 Xiaobing Fu2
1 Chinese PLA Diabetes Mellitus Diagnosis and Treatment Center, Chinese PLA 306th Hospital
2 Wound Repair Key Laboratory, First Hospital Affiliated to the Chinese PLA General Hospital
Corresponding author: Xiaobing Fu, Wound Repair Key Laboratory, First Hospital Affiliated to the Chinese PLA General Hospital
Summary
Objective: To investigate the role and mechanism of ASTAR CREAM, a topical Chinese patent medicine, in the promotion of skin wound healing in diabetic mice. Methods: the same week-old male C57 mice (SPF grade) were used to establish diabetic skin ulcers model. The animals were randomized into three groups, Group A, the wound was treated with topical ASTAR CREAM; Group B, the wound was treated with topical compound sulfadiazine zinc gel as positive control; Group C, the wound was covered with Vaseline gauze without other treatment as blank control, 18 mice per group. After the wound formation, the drugs were directly applied daily according to the grouping design, and the dressing was changed once a day for each group of mice. At Day 3, 7, 10, 14, 17 and 21, the wound size was traced onto the plastic transparent film, and was analyzed by image analysis software MIAS2000 to calculate the average healing rate of the wound. At Day 7 and 17, three C57 mice were randomly selected from each group and sacrificed by cervical dislocation. Full-thickness skin specimen was collected from the wounded area and within 2mm range around the wounded area, the granulation tissue at the junction of wound margin was clipped, and then conventional hematoxylin-eosin staining (HE staining) was performed. 4-5 sections per mouse were used for HE staining. The wound thickness, collagen thickness and arrangement, epithelium formation, blood vessel formation and inflammatory cell infiltration were observed. Results: The results of the healing rate showed that ASTAR CREAM (Drug A) and sulfadiazine (Drug B) were able to promote wound healing; the healing rates between Group A and Group B were not significant different in early stage (0-7 days), but Group A showed a significant advantage in later stage (> 7 days), the overall healing time of which was shorter than that of Group B. The pathological findings for HE staining: At 7d after the injury, more granulation tissue was visible in Group A, and the fibroblasts and capillaries were more than Group B. The thickness of granulation tissue between Group A and Group B was not different (P> 0.05); ASTAR CREAM showed significant healing advantages, including inflammation relief, wound epithelization, well-differentiated epidermal cells and new collagen alignment, the wound tissue structure returned to normal; sulfadiazine silver therapy group also had obvious healing effect. Some stratified epithelium could be seen in most of the wounds, fewer inflammatory cells under epithelium, more new collagen, but more chaotic than Group A; in blank control group, some epithelial tissue formed, and epidermal cells mostly were monolayer and disordered. New collagen arrangement was disordered and more inflammatory cells were still visible in the whole dermis. Conclusion: ASTAR CREAM and compound sulfadiazine zinc gel had the role of promoting the healing of diabetic skin wound. At the beginning of the healing process, the effect of ASTAR CREAM did not differ significantly from that of compound sulfadiazine zinc gel, but in the late showed better healing-promoting effect.
1. Materials and Methods
1.1 Test drugs:
1.1.1 ASTAR CREAM(A):
It was manufactured by Tianjin Darentang Jingwanhong Pharmaceutical Co., Ltd., specification: medicinal ointment, 20 g/branch; Executive Standard WS3-B-3063-98-2008; approval number Z12020440; lot number 211874.
1.1.2 Compound sulfadiazine zinc gel (B):
It was manufactured by Chengdu First Drug Research Institute Co., Ltd., specification: ointment formulation, 30 g/branch; Executive Standard WS-428 (X341) -98 (1); approval number: H19991358; lot number 111203.
1.2 Experimental animals:
The animals used in this study were 54 male C57 miceweight 18 to 22.7g (20.6±1.08g), and age 8 to 9 weeks. This study had been approved by the Institutional Ethics Committee. After fasted 8 hours, the fasting glucose of the mice was measured before establishing the model, and then 60mg/Kg of 1% streptozotocin (STZ, buffer PH = 4.3) was injected intraperitoneally for 3 consecutive days. After 7 days, the fasting blood sugar (FBS) was measured; thereafter the fasting blood glucose was monitored once a week. The mice with the following conditions were used for this experiment: polydipsia, polyuria and FBS≥300mg/dl. The hair on the back of the mice was removed, and the circular full-thickness skin wounds were prepared along the mark, with the diameter of 1cm.
1.3 Grouping and administration:
STZ-induced diabetic C57 mice were randomized into three groups: Group A, the wound was treated with topical ASTAR CREAM; Group B, the wound was treated with topical compound sulfadiazine zinc gel as positive control; Group C, the wound was covered with Vaseline gauze without other treatment as blank control, 18 mice per group. On the day of preparation, the wounds were fixed with sterile gauze bandage. From Day 2 after wound formation, the drugs were applied directly according to the grouping design. For Group A and B, the drugs were directly applied to the whole wound and covered with the sterile gauze, the gauze corners was fixed by suturing for one needle, Group C was only covered with the gauze. For each group of mice, the dressing was changed once a day.
1.4 Observations and test methods:
At Day 3, 7, 10, 14, 17 and 21, the wound size was traced onto the plastic transparent film, and was analyzed by image analysis software MIAS2000 to calculate the average healing rate of the wound. At Day 7 and 17, three C57 mice were randomly selected from each group and sacrificed by cervical dislocation. Full-thickness skin specimen was collected from the wounded area and within 2 mm. range around the wounded area, the granulation tissue at the junction of wound margin was clipped, and then conventional hematoxylin-eosin staining (HE staining) was performed. The observed contents for HE staining: wound thickness, collagen thickness and arrangement, epithelium formation, blood vessel formation and inflammatory cell infiltration. 4-5 sections per mouse were used for HE staining.
1.5 Statistical analysis
The experimental results were expressed as X ± SD. Multiple F-test was used, and there was significant difference if P<0.05.
2. Results:
2.1 Average healing rate (%) of different time points
| Time points | ASTAR CREAM | Silver sulfadiazine | Blank control |
| 0 | 0 | 0 | 0 |
| 3 | 20±0.21 | 19.8±0.16 | 19.5±0.45 |
| 7 | 65±0.13 | 61± 1.14 | 55±0.79 |
| 10 | 85 ± 1.93 * | 81 ± 0.85 * | 77 ± 1.35 * |
| 17 | 100 * | 95 ± 0.29 * | 87 ± 1.17 * |
| 21 | 100 * | 97 ± 0.37 * | 90 ± 0.96 * |
2.2 Substantially observed results for wound surface (Fig.1)
Macroscopic semi-paste yellow cover was formed in the wound treated with ASTAR CREAM (Group A), covering the wound, and was easy to be removed. The ruddy wound edge was unapparent relative to Group B. The dry and white film-like cover was formed in the wound treated with the sulfadiazine therapy (Group B), and was hard to be uncovered. The uncovered part had a clear ruddy edge.
The results of the healing rate showed that ASTAR CREAM (Drug A) and sulfadiazine (Drug B) were able to promote wound healing; the healing rates between Group A and Group B were not significant different in early stage (0-7 days), it could not determined if Drug A showed better efficacy in promoting wound healing than Drug B, but Group A showed a significant advantage in later stage (>7 days), the overall healing time of which was shorter than that of Group B.
2.3 Results of HE staining (Figure 2, 3, 4)
Group A: ASTAR CREAM treatment group; Group B: sulfadiazine silver treatment group; Group C: blank control group
After injured 7d, the inflammatory cells were more for each group, but the inflammation of the Group C is the most obvious. The more granulation tissue was visible in Group A, and the fibroblasts and capillaries were more than Group B. The thickness of granulation tissue of Group A and B was more thick than Group C (P<0.01), there was no difference between Group A and B (P> 0.05).
After injured 17d, ASTAR CREAM treatment group showed significant healing advantages, including inflammation relief, wound epithelization, well-differentiated epidermal cells and new collagen alignment, the wound tissue structure returned to normal; sulfadiazine silver therapy group also had obvious healing effect. Some stratified epithelium could be seen in most of the wounds, fewer inflammatory cells under epithelium, more new collagen, but more chaotic than Group A; in blank control group, some epithelial tissue formed, and epidermal cells mostly were monolayer and disordered. New collagen arrangement was disordered and more inflammatory cells were still visible in the whole dermis.
3. Discussions：
Oakley first proposed the concept of "diabetic foot" (DF) in 1956. The international working group of diabetic foot disease (IWGDF) defines it as the wound which involves the full-thickness skin under the ankle of the diabetics and has nothing to do with the course of the diabetes, which is generally accepted [3]. The overseas literature reported that about 15% of patients with diabetes experienced foot ulcers, foot ulcers were an important cause of death in patients with diabetes [4]. In China, in chronic wounds of body surface, diabetic ulcers rose from 4.9% [5] 10 years ago to 35.3% [6]. In Inpatient diabetes in large cities of China, about 2% to 4% of patients with diabetic foot hospitalize, 4% to 10% of hospitalized diabetic patients are accompanied by diabetic foot ulcers [7]. With the increase of the aging population in China and the change of the spectrum of disease, the number of diabetic patients in China is now nearly 100 million [8], the resulting problems will bring enormous health economic and social burden to the country, and will also be attracted more and more attention.
A variety of abnormal physiological factors are known to lead to poor healing of diabetic ulcers, including growth factor production, function damage, neovascularization obstacles, macrophage dysfunction, collagen aggregation, epithelial barrier function, an abnormal number of granulation tissue and the imbalance between extracellular matrix composition and metalloproteinase remodeling. Because traditional Chinese medicines play multi-target, multi-link and multi-level comprehensive regulatory and synergic role against the major influencing factors for wound healing [9], "Medicine Origin" described "the external therapy was the most important in the surgical methods", the role of traditional Chinese medicine for the treatment of diabetic ulcers is now more and more concerned. ASTAR ……..CREAM was developed initially in the 1960s for the treatment of burns and other refractory wounds caused by bullets and artillery fire, based on the folk remedies, such as "Huanglianjiedu ointment", "Shengji yuhong plaster"and others. The silver zinc preparations have been used clinically in the standard treatment of wounds for a long time, and are considered to have good efficacy to promote wound healing. This study compared the promoting effect of ASTAR CREAM and compound sulfadiazine zinc gel on the healing of diabetic foot ulcers, and found: The results of the healing rate showed that ASTAR CREAM (Drug A) and sulfadiazine (Drug B) were able to promote wound healing; the healing rates between Group A and Group B were not significant different in early stage (0-7 days), but Group A showed a significant advantage in later stage (>7 days), the overall healing time of which was shorter than that of Group B. The pathological findings for HE staining: At 7d after the injury, the inflammatory cells were more in each group, but the inflammation of the Group C is the most obvious. The more granulation tissue was visible in Group A, and the fibroblasts and capillaries were more than Group B. The thickness of granulation tissue of Group A and B was more thick than Group C (P<0.01), there was no difference between Group A and B (P> 0.05); At 17d after the injury, ASTAR CREAM showed significant healing advantages, including inflammation relief, wound epithelization, well-differentiated epidermal cells and new collagen alignment, the wound tissue structure returned to normal; sulfadiazine silver therapy group also had obvious healing effect. Some stratified epithelium could be seen in most of the wounds, fewer inflammatory cells under epithelium, more new collagen, but more chaotic than Group A; in blank control group, some epithelial tissue formed, and epidermal cells mostly were monolayer and disordered. New collagen arrangement was disordered and more inflammatory cells were still visible in the whole dermis.
Most of the herbs selected for ASTAR CREAM are the goods that have the bitter, cold, salty and acid properties, in which Garden Burnet can purge intense heat and detoxicate detoxification, astringe to arrest bleeding, inhibit the protease activity of the fiber, and shorten the bleeding and clotting time, protect the wound, and is an important drug in the treatment of the wound; Rhubarb is an main drug for removing blood stasis, can promote blood circulation, and increase the local blood supply and oxygenation of the ulcers. Modern research shows that rhubarb can enhance the phagocytotic ability of leukocytes, reduce local capillary permeability and relieve swelling, thus achieving anti-bacterial, anti-inflammatory and analgesic effect and improving stress resistance capacity against the local damage; Gardenia has bitter taste and cold nature, can prevent fungal infection of the wound, and reduce the local inflammation; "Bencao Jingshu" recorded that Sangusis Draconis was "an important drug for decreasing blood stasis and producing new blood". Modern pharmacological studies confirm that it has inhibitory effect on a variety of fungal skin and can promote wound healing; Frankincense and Myrrh can invigorate the blood circulation and relieve pain, relieve the swelling and promote tissue regeneration; Lithospermum has the effect of cooling blood and promoting blood circulation, the experiments show that it plays a role in promoting the proliferation of granulation tissue, and can inhibit a variety of bacterial growth; Angelica can enrich the blood and promote the healing of chronic wounds, and reduce swelling and pus; Borneol not only has anti-inflammatory analgesic effect, also has good transdermal absorption function and promote the absorption of other drugs; Sesame oil and Beeswax are the adjuvant drugs, can adjust the wound pH, moisturize the skin, and are considered as the pharmaceutical excipients. The combination of various drugs can promote blood circulation and detoxication, reduce swelling and ease pain, as well as remove the necrotic tissue and promote tissue regeneration. The effects of relieving the swelling, promoting blood circulation, removing the necrotic tissue, detoxification and promoting tissue regeneration were not found in the compound sulfamethoxazole pyrimidine zinc gel, which may be the main reason for its efficacy advantage. The healing mechanism of the refractory wounds is not completely clear, the slower division and proliferation of the fibroblasts and epidermal cells in the local wound, the decrease of extracellular matrix synthesis and the accelerated decomposition are considered as the pathological basis for the prolonged unhealed wounds. The above experimental study shows that ASTAR CREAM can play a role against the main factors for wound healing, and play its multi-target, multi-link and multi-level regulatory role in promoting cell division and proliferation through the regulation of the synthesis and secretion of growth factors, stimulating wound neovascularization, improving and regulating wound matrix repair formation as well as nourishing the wound, thus ultimately to promote wound healing. Due to the complex wound healing process, a more in-depth research on specific mechanism of ASTAR CREAM for promoting the diabetic ulcers from the molecular and cellular level is also needed in order to explain it more clearly.
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