The Experimental Study On The Treatment Of Hypertrophic Scar In C57 Mice Induced By Bleomycin

Hyperplastic scar is a kind of pathological scar,which is the inevitable result of abnormal wound repair.In the course of its development,a variety of cells,mainly fibroblasts,are abnormal enhanced,and cell hyperplasia,which eventually lead to dermal hyperplasia resulting in lesions.In recent years,many scholars at home and abroad have paid attention to the study of hypertrophic scar.However,due to its complicated pathogenesis and lack of good animal models,the prevention of hypertrophic scar is extremely difficult,and there is no effective treatment so far.A large number of studies have confirmed that CO₂ laser has a good therapeutic effect on hypertrophic scars,but the specific mechanism is still unclear,which limits the development and further application of this technology.It has been reported that the expression of GLI-1 protein increases during scar formation,indicating that Hedgehog signaling pathway is activated.After specific blockade of Hedgehog signaling pathway,the expression of vascular endothelial growth factor?VEGF?,alpha-SMA and collagen type I proteins in hypertrophic scars decrease,and scar proliferation is inhibited.This suggests that blocking Hedgehog signaling pathway may prevent the occurrence and development of hyperplastic scar.For a long time,C57BL/6J mice have been widely used in the establishment of HS models.Experiments show that a certain dose of Bleomycin?BLM?can induce the skin of mice to produce scar-like pathological characteristics.In this study,BLM was used to induce hypertrophic scar in C57BL/6J mice,and some important factors in the process of hypertrophic scar formation were detected.The hypertrophic scar was treated with CO₂ laser,thus explaining the treatment of hypertrophic scar by CO₂laser.It is of great significance and valuable to explore the mechanism of hypertrophic scar.ObjectiveTo observe the expression of Hedgehog signaling pathway in hypertrophic scar of C57BL/6Jmale mice induced by bleomycin,To explore the effect of CO₂ laser on Hedgehog signaling pathway in hypertrophic scar.Materials and Methods1.Eighty-four?84?6-week-old C57BL/6Jmale mice were randomly divided into four groups:group A was a blank control group,subcutaneously injected with 100?l PBS once a day;and group B,C and D were treated with 0.5 mg/mL,1 mg/mL and 3mg/mL BLM?bleomycin?,100?l per subcutaneous injection,once a day.2.The corresponding concentration of BLM was injected subcutaneously each time,draw materials at week3,week4 and week5.After HE and Masson's staining,the distribution of blood vessels,the morphology and arrangement of collagen fibers in skin tissue were observed under microscope,the degree of skin fibrosis was analyzed,and the pathological changes of skin scar tissue in rats were observed.3.The expression of GLI-1 and alpha-SMA protein in skin tissue was detected by immunofluorescence staining.The dermal thickness of skin samples was measured by ImageJ software.The effect of different doses of BLM on scar induction and the effect of BLM on Hedgehog signaling pathway were investigated.4.Fourty-nine?49?C57BL/6J male mice were subcutaneously injected with 1 mg/mL BLM to make scar models.Four?4?weeks later,seven mice were randomly executed.Histological staining and immunofluorescence staining were performed to detect the success of the scar models.The remaining mice were randomly divided into three groups:A,B and C.Group A was not treated and continued to feed.Group B was treated with 10 mJ energy laser.Group C was treated with 20 mJ energy CO2 laser.At the sixth weeks and ninth weeks,seven mouse were killed randomly.Histological staining was used to observe the pathological changes of skin scar tissue in mice,and immunofluorescence staining was used to observe the expression of alpha-SMA and GL1 in skin scar tissue of mice.5.To analyze the therapeutic effect of CO2 laser with different energy on HS model of mice established by BLM,and to explore the mechanism of CO2 laser in HS treatment.Results1.Histological staining results showed that the collagen fibers in the control group were similar to those in the normal skin.The collagen fibers in the control group were arranged regularly without obvious coarsening,fibroblasts arranged orderly,and inflammatory cells infiltrated.At the 3rd week,the mice in the experimental group showed pathological characteristics similar to scar tissue.At the 4th week,the scar was even worse,but there was no obvious change after the 5th week.The specific manifestations were:fibroblasts were obviously disordered,whirlpool or nodular,collagen deposition,collagen bundle thickening,disordered arrangement,visible inflammatory cell infiltration and capillary hyperplasia,subcutaneous adipose tissue was knotted,subcutaneous adipose tissue is replaced by connective tissue.The fibrosis induced by 0.5 mg/mL BLM in group B was less obvious than that in other two groups.The fibrosis induced by 3 mg/mL BLM in group D was obvious and a large number of inflammatory cells infiltrated.However,the increase of collagen fibers was not uniform,even fibrotic dermal papillae appeared in adipose and muscular layers.The fibrous rings in group C were also obvious,and the thickness of fibrosis was uniform compared with that in group D.2.Immunofluorescence showed that the expression of GLI-1 protein increased gradually with the increase of BLM dosage and time.The thickness of dermis in group A was similar to that of normal skin at the third,fourth and fifth weeks,but increased slowly in group B.The thickness of dermis in group D increased significantly but not uniformly,while that in group C increased significantly and uniformly.At the 4th and 5th weeks,the thickness was about 2 times of that of group A.3.In the CO₂ laser treatment group,the results of group A?model control group?and group B?10mJ CO₂ laser treatment group?were similar,while those of group C?20mJ CO₂ laser treatment group?were reduced in skin scar,fibrosis and dermal collagen thickness,and the effect was obvious.In addition,immunofluorescence results showed that the expression of alpha-SMA and GLI-1 in skin scar tissue of group C was significantly decreased after CO2 laser treatment.Conclusion1.After 4 weeks,the collagen fibers of mice in 1 mg/mL dosage groups increased obviously and uniformly,with a large number of inflammatory cells infiltrating and capillaries increasing.The thickness of dermis was about 2 times of that of normal skin,and the repeatability was high.It could be used as a reliable method to make HS model.2.The low expression of GLI-1 in normal mice indicates that the Hedgehog signaling pathway is not activated.The high expression of GLI-1 in mice stimulated by BLM indicates that the Hedgehog signaling pathway is activated.3.20 mJ CO2 laser can inhibit the scar induced by BLM.The possible reason is that this energy can promote the apoptosis of fibroblasts and inhibit Hedgehog signaling pathway.10 mJ CO2 lattice laser cannot inhibit the scar induced by BLM,and even promote it to a certain extent.This may be the low energy CO2 laser damage on skin is not enough to initiate self-repair mechanism.