| Objective: To explore the effect of timolol maleate on hypertrophic scar and its possible mechanism.Methods:1.In vitro experiments:Human umbilical vein endothelial cells(HUVEC)were extracted and cultured.Four experimental groups were set up: control group,low concentration group,medium concentration group,and high concentration group.The cells were treated with different concentrations of Timolol Maleate(TM)and observed changes.CCK-8 was used to detect the effect of TM on cell proliferation,and a scratch test was used to detect the effect of TM on cell migration.Annexin V-FITC /PI double staining method was used to detect the effect of TM on apoptosis.Western blot was used to detect the expression of VEGF and CD34 in HUVEC under the action of drugs,and the expression levels of key proteins PI3 K,AKT,m TOR,and their phosphorylated proteins in the PI3K/AKT/m TOR pathway.Statistical data were analyzed using SPSS 24.0 software.Values are expressed as mean standard deviation.The differences among multiple groups were calculated by one-way ANOVA,and the pairwise comparison between groups was conducted by independent sample t-test,and the difference was statistically significant if P < 0.05.2.In vivo experiment:Twenty healthy Japanese white rabbits were selected as experimental animals and randomly divided into four groups: model group(blank control group),normal saline group(negative control group),Triamcinolone Acetonide Acetate,triamcinolone acetate)administration group(positive control group),and TM administration group.One hundred and sixty hypertrophic scar models with a diameter of 1cm were established in the abdomen of both ears,four on each side.The rats in the normal saline group and the medication group were given the scar model at the same concentration and the same frequency three weeks after the modeling,i.e.,21 days from now.The model group was not treated.The level of scar appearance was observed by the naked eye,and the changes in the rabbit ear scar model were measured,photographed,and recorded.The cicatrix hyperplasia index and VSS value were calculated to evaluate the cicatrix level.HE staining was used to observe the histopathological changes among different groups.The expression levels of VEGF and CD34 were determined by immunohistochemistry and western blot.The statistical method is the same as the cell experiment.Results:1.In vitro experiments:CCK-8 experiment: The results showed that TM could reduce the vitality of HUVEC cells and inhibit their proliferation,and with the increase of administration concentration,the inhibition degree of TM on cells became more and more obvious(P<0.05);Scratch test: compared with the control group,TM can inhibit the migration of HUVEC(P<0.05);Annexin V-FITC /PI double staining method: The data showed that TM could promote HUVEC apoptosis in a concentration-dependent manner(P<0.05);The results of western blot showed that the expression levels of VEGF and CD34 in HUVEC decreased in a concentration-dependent manner under the action of TM,and the difference was statistically significant compared with the control group(P< 0.05).Under the action of TM,the protein expression levels of key proteins PI3 K,AKT,m TOR,and their phosphorylated products p-PI3 K,p-AKT,and p-m TOR in HUVEC decreased gradually with the increase of drug concentration.Compared with the control group,the difference was statistically significant(P < 0.05).2.In vivo experiment:Macroscopic observation: The wounds were epithelialized 21 days after the operation.The scars of each group showed different degrees of hyperplasia along with the time.The scar hyperplasia in the model group and the negative control group(normal saline group)was significant,but no significant difference was noticed between the two groups with the naked eye.In the TM group,the scar was lighter in color,slightly higher than the surface of surrounding normal skin.The surface was flat,and the scar was soft.The degree of central hyperplasia of the wound surface had been significantly reduced.The scar thickness in the TAC group was decreased as compared with that in the TM group,but it was redder than that in the TM group with obvious congestion.HI: After the establishment of models,the HI gradually increased with time and stabilized at 28 days,and began to decline.There was no significant difference in HI results between the model group and the negative control group(normal saline group)(P > 0.05).Compared with the model group,HI values in the TAC group and TM group were decreased,and the differences were statistically significant(P < 0.05).VSS score:there was no significant difference among groups before treatment and after treatment,the score of the TM group in the TAC group was lower than that of the model group(P< 0.05).the VSS results of the TAC group and tm group were similar.the VSS score of the TAC group was slightly lower than that of the TM group and the difference was not statistically significant(P < 0.05).HE staining results: Typical hypertrophic scar changes were observed in the model group and the negative control group(normal saline group): obvious thick and dense fibroblast/myofibroblast accumulation,and a large amount of collagen deposition,which was arranged disorderly and in a vortex shape.Besides,there were obvious neovascularization and obvious infiltration of inflammatory cells.In contrast,the proliferation of dermis was decreased in TAC and TM groups,and the dense collagen fibers had been significantly reduced,with a regular arrangement.The neovascularization and inflammatory cell infiltration were milder,and the extracellular matrix precipitation was also reduced.The expressions of VEGF and CD34 in the hypertrophic scar tissue of rabbit ears detected by immunohistochemistry showed that after staining with VEGF antibodies,the stained area in the TM group was smaller than that in the model group and the normal saline group,with a lighter color,and the expression of VEGF was decreased(P < 0.05).The expression levels of VEGF in the TAC group and TM group were similar,but TAC group was slightly lower than TM group without statistical difference(P > 0.05).After staining with CD34 antibody,the number and density of microvessels in the TM group were significantly reduced compared with those in the model group,and the MVD value in the TM group was reduced compared with those in the model group and the normal saline group(P < 0.05).The MVD values in the TAC group were similar to those in the TM group,but were slightly lower in the TAC group than in the TM group with no statistical difference(P > 0.05).The expressions of VEGF and CD34 in hypertrophic scar tissue of rabbit ears were detected by Western Blot.Compared with the control group,the expression levels of VEGF and VEGF protein in the negative control group(normal saline group)were not significantly changed,and the protein expression levels in the TM group and the positive control group(TAC group)were decreased(P < 0.05).Compared with the control group,the expression levels of CD34 protein in the negative control group(normal saline group)were not significantly different,and the protein expression levels of the TM group and the positive control group(TAC group)were decreased to different degrees(P < 0.05).The above results indicated that TM could inhibit the expression levels of VEGF and CD34 in hypertrophic scars of rabbit ears.Conclusion:1.Timolol maleate inhibits the proliferation and migration of HUVEC,promotes their apoptosis,and affects the expression levels of VEGF and CD34 proteins.2.The pharmacological effects of timolol maleate on HUVEC may be mediated through the PI3K/AKT/m TOR pathway.3Timolol maleate has a good effect on the treatment of hypertrophic scars in rabbit ears.The mechanism may be to treat hypertrophic scar by reducing the secretion of vascular endothelial growth factor and reducing the number of microvessels,thereby inhibiting the hyperproliferation of blood vessels by HUVEC in scar tissue. |
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