| Objective: The rabbit ear scar model was established to explore the effect and mechanism of vincristine(VCR)on inhibiting hypertrophic scar;fibroblasts were cultured in vitro to explore the effect and mechanism of various concentrations of VCR on inducing fibroblast apoptosis and reducing extracellular matrix production;which could provide new targets and directions for the prevention and treatment of hypertrophic scar.Methods: New Zealand white rabbits were anesthetized to make the ventral wound of ear to establish the rabbit ear scar model,The rabbits were randomly divided into three groups and the wounds of ear were soaked with various solution: group A was treated with saline gauze;group B was treated with 0.5 μg/ml VCR;group C was treated with 1 μg/ml VCR.After the animals were killed,the full-thickness scar skin samples were taken,fixed by formaldehyde,embedded in paraffin and sectioned.HE staining was used to detect the scar index and the number of fibroblasts in the scar tissue.Masson staining was used to detect the collagen content in scar tissue.Sirius red staining was used to detect the type and content of collagen in scar tissue.Immunohistochemical staining was used to detect the expression of CHOP.TUNEL staining was used to observe cell apoptosis.Fibroblasts were extracted from human skin scar tissue in vitro and treated with different concentrations of VCR.CCK-8 was used to detect the survival rate of fibroblasts after treated with different concentrations of VCR.The expression of collagen I and collagen III as well as the expression of α-smooth muscle actin(α-SMA)before and after VCR treatment were detected by immunofluorescence staining and Western blot detection.The apoptosis rate of fibroblasts was detected by flow cytometry and TUNEL staining after treated with different concentrations of VCR.The expression levels of apoptosis related proteins(cleaved PARP and Bax),anti-apoptotic protein(Bcl-2)and endoplasmic reticulum stress related proteins(GRP78,p-PERK,CHOP)were detected by Western blot after the fibroblasts were treated with different concentrations of VCR.The expressions of endoplasmic reticulum stress related proteins(GRP78,CHOP)and apoptosis related proteins(Bax,Bcl-2)were detected by Western blot after fibroblasts treated with VCR and/or endoplasmic reticulum channel inhibitor 4-phenylbutyric acid(4-PBA).Results: The results of HE staining,Masson staining and Sirius red staining showed that VCR could reduce the scar index,the number of fibroblasts and the content of collagen in the scar tissue of rabbit ears,and there was significant difference compared with the control group(P<0.05).TUNEL staining showed that the apoptosis rate of fibroblasts in scar tissue of VCR treatment group was increased,and the content of CHOP in scar tissue of rabbit ear increased with the increase of VCR concentration.The result of CCK-8 showed that the survival rate of fibroblasts decreased with the increase of VCR concentration(P< 0.05).The expressions of collagen and α-smooth muscle actin in extracellular matrix were significantly decreased by immunofluorescence staining.The results of flow cytometry and TUNEL staining showed that VCR could increase fibroblasts apoptosis rate(P<0.05).Western blot analysis showed that the expressions of cleaved-PARP and Bax increased,the expression of anti-apoptotic protein Bcl-2 decreased,and the expressions of endoplasmic reticulum stress-related proteins(GRP78,p-PERK and CHOP)increased with the increase of VCR concentration.Western blot analysis showed that the expressions of endoplasmic reticulum stress-related proteins(GRP78,CHOP)and apoptosis related proteins(Bax,Bcl-2)after treated with 0.5μg/ml VCR,were partially reversed by endoplasmic reticulum channel inhibitor 4-PBA.Conclusion: VCR can activate endoplasmic reticulum stress pathway,induce apoptosis of human fibroblasts and reduce the production of extracellular matrix collagen;Local application of VCR can inhibit the formation of hypertrophic scar in rabbit ears. |
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