| Objective By establishing a full-thickness skin wound rats model,to investigate the effect of Fucoidan(FUC)dressing on wound healing in rats with skin wound injury.Method 1.Experimental grouping and design: First of all,45 male SD rats were given adaptive feeding for one week and randomly divided into three groups(15 rats/group): Control group,2% fucoidan dressing group(2%FUC)and 5% fucoidan dressing group(5%FUC).Dressings for each group were prepared as follows: 50 μL of normal saline,20mg/m L and 50mg/m L fucoidan solution were dropped onto three alginate dressings(size: 2×2cm square)to make the Control dressing,2%FUC dressing and 5%FUC dressing.After the rats were anesthetized,their backs were shaved and labeled under sterile conditions.The fullthickness skin was removed at the marked position on the back of the rats with sterile surgical scissors to form a rat skin wound model,which was wrapped with the corresponding dressing and fixed with medical tape according to the grouping.Finally,the rats were returned to their cages with free access to water and food,housed in single cages,and the dressing was changed at the same time every day.The day of successful modeling was the 0th day.On the 3rd,7th and 14 th day after modeling,5 rats in each group were randomly selected and sacrificed after anesthesia,and the tissue within 5 mm of the wound and the surrounding tissue were collected.2.Wound observation and recording: The wound healing was observed at the same time every day,and photographs were taken at 0,3,7,and 14 days to calculate the wound healing rate.3.Hematoxylin-Eosin(H&E)staining was used to observe the pathological changes of the wounds of rats in each group on the 3rd,7th,and 14 th days.4.Masson staining was used to observe the distribution and content of collagen in the wounds of rats in each group on the 3rd,7th,and 14 th days.5.Immunohistochemistry(IHC)staining was used to detect the expression levels of interleukin-1β(IL-1β),Tumor necrosis factor-α(TNF-α)and Transforming growth factor-β1(TGF-β1)in wound tissues of rats in each group on the 7th,and 14 th day.6.Western blot(WB)was used to detect the expression levels of α-mooth muscle actin(α-SMA),collagen type I(Col I)and p-Smad/2/3 in wound tissues of rats in each group on days 7 and 14.Result 1.The observation results of wound areas showed that the wound healing of rats in the 5%FUC group was the most obvious.The wound healing rates of the 5%FUC group at 3d and 14 d after injury were 2.4 and 1.3 times higher than those of the Control group,respectively(P < 0.05)After 7 days of injury,the wound healing rate in the 5%FUC group and the 2%FUC group was 1.9 times(P<0.01)and 1.5 times(P<0.05)of that in the Control group,respectively,and the wound healing rate in the 5%FUC group was 1.3 times of that in the 2%FUC group(P<0.05).2.The results of H&E staining showed that compared with the Control group,the 5%FUC group had lighter inflammatory reaction,faster granulation tissue formation,obvious fibroblast proliferation,and a large number of collagen fibers proliferation.However,there was no significant development of skin accessory glands in the 5%FUC group compared with the Control group and the 2%FUC group.3.Masson staining results showed that compared with the Control group,collagen in the dermis of the 2%FUC group and the 5%FUC group was more abundant,more mature,more compact and orderly,and a large number of collagen fiber bundles were formed.After 7 days of injury,collagen content in the 2%FUC group and the 5%FUC group was significantly higher than that in the Control group,which was 1.3 times(P <0.05)and 1.4 times(P<0.01),respectively.The collagen content in the 5%FUC group was 1.2 times higher than that in the Control group 14 days after injury(P<0.05),while there was no significant difference between the 2%FUC group and the Control group(P>0.05).4 The results of IHC experiments showed that compared with the Control group,the mean density of TNF-α and IL-1β in the 5%FUC group was significantly decreased by 30.3% and 8.5%(P < 0.01),respectively,and the mean density of TGF-β1 was significantly increased by 16.0%(P < 0.01)after 7 days of injury.The mean density of TGF-β1 in the 2%FUC group was significantly higher than that in the Control group by 7.7%(P < 0.05).There was no significant difference in the expression of inflammatory factors between the two groups(P > 0.05).After 14 days of injury,compared with the Control group,the mean density of TNF-α and IL-1β in the 5%FUC group decreased by 14.3% and 7.2%,respectively(P < 0.01),while the mean density of TGF-β1 increased by 16.5%(P < 0.01).The mean density of TNF-α in the 2%FUC group was significantly lower than that in the Control group by 6.1%(P < 0.05),and the mean density of TGF-β1 was increased by 10%(P < 0.01).5.The results of Western blot showed that compared with the Control group,the protein expression levels of Col I,α-SMA,and p-Smad/2/3 in the 5%FUC group were significantly increased by 1.16 times,2.16 times,and 3.24 times,respectively(P<0.01)after 7 days of injury.The protein expression levels of Col I and α-SMA in the 2%FUC group were significantly increased by 0.70 times and 1.48 times(P<0.01).After 14 days of injury,compared with the Control group,Col I,α-SMA and p-Smad/2/3 protein expression levels in the 5%FUC group were significantly up-regulated by 0.35 times,0.63 times(P<0.05)and 1.82 times(P <0.01),respectively.The expression of p-Smad/2/3 protein in the 2%FUC group was 2.02 times higher than that in the Control group(P <0.01).Fucoidan dressing promoted TGF-β1/Smad pathway activation,upregulated α-SMA expression,and increased Col I production.Conclusion 1.Fucoidan dressing could promote wound healing in rats,and 5% fucoidan dressing has a better effect but may inhibit the development of skin accessory glands.2.Fucoidan dressing could reduce inflammatory response and promote collagen production after injury in rats,and the mechanism is related to upregulation of TGF-β1/Smad signaling pathway. |
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