| Objectives To explore the skin fibroblasts(SF)cultured in vitro from the back dermal tissues of neonatal pups in SD rats,induced by angiotensin ?(angiotensin ?,Ang ?)into skin myofibroblasts(SMF))In the process of Hedgehog signaling in the development and regulation of skin myofibroblasts,and the study of N-acetyl-seryl-aspartyl-lysylproline(N-acetyl-seryl-aspartyl-lysyl-proline(Ac-SDKP)inhibits angiotensin II-induced differentiation of skin fibroblasts into myofibroblasts by regulating Hedgehog(HH)signaling,thereby exerting its anti-skin fibrosis effect Function,providing a more scientific theoretical basis for future clinical applications.Methods SF was isolated from the back dermis of newborn SD neonatal rats in primary culture and cultured to the second generation of cells,induced by Ang?to differentiate into myofibroblasts,and blocked with Ac-SDKP,SMO blocker(GDC-0449),GLI Retardant(GANT61)pretreatment.The cell migration ability was detected by scratch test.The location and expression of ?-smooth muscle actin(?-SMA)were detected by immunohistochemical staining.Western-blotting technique was used to detect collagen type ?(Col?),transgenic factor proteins GLI(GLI1,GLI2,GLI3),transmembrane Patched protein(PTC),smoothened proto-oncogene(SMO),SHH,The level of ?-SMA.The statistical method is to sort out the original data obtained in the experiment by Excel 2013,and then build a database to perform statistical analysis through the SPSS22.0 software package.The data are expressed as the mean ± standard deviation(sx±).The t test was used between groups,and single factor analysis of variance was used among multiple groups.P<0.05 was considered statistically significant.Results 1 Cell scratch test showed that Ac-SDKP induced skin myofibroblast scratch test at 0 hours compared with 48 hours.The results showed that the migration of skin myofibroblasts induced by Ang ?was significantly enhanced compared to normal myofibroblasts.The combination of Ang ?and Ac-SDKP administered showed that the combination of Ang ?and Ac-SDKP was significantly better than Ac-SDKP-induced migration of skin myofibroblasts.2 The results of immunocytochemistry showed that the positive expression of ?-SMA in the cells of Ang?-induced group was stronger than that of the blank control group;while the Ac-SDKP pretreatment group could significantly inhibit the positive expression of ?-SMA.3 Immunoblotting results showed that SMO receptor blocker GDC-0449 was used to induce skin myofibroblasts.Compared with the control group,Col I,?-SMA,SHH,SMO,GLI1 and GLI2 were positive in the Ang?induced group The expressions were up-regulated by 1.9 times,4.3 times,3.5 times,2.4 times,1.7 times,and 3 times,PTC and GLI3 positive expressions were down-regulated by 86% and 80%,respectively.Compared with the Ang?-induced group,the Ang and ?GDC-0449 blockers The positive expressions of Col I,?-SMA,SHH,SMO,GLI1,and GLI2 were down-regulated by 50%,55%,49%,52%,43%,and 45%,and the positive expressions of PTC and GLI3 were up-regulated by 1.9 times and 2.9-fold,respectively.(P<0.05).The GLI61 blocker GANT61 was used to induce skin myofibroblasts.Compared with the control group,the positive expressions of Col I,?-SMA,SHH,SMO,GLI1,and GLI2 were up-regulated by 2.9 times,5.1 times,and 4.9 times,3.2 times,5 times,1.7 times,PTC and GLI3 positive expression were down-regulated by 82% and 69%(P<0.05),compared with Ang induced group,Col I,?? in Ang and GANT61 blocker group ?-The positive expressions of SMA,SHH,SMO,GLI1,and GLI2 were down-regulated by 32%,37%,20%,51%,48%,and 49%,respectively.The positive expressions of PTC and GLI3 were up-regulated by 5.4-fold and 1.4-fold,respectively(P<0.05).Ac-SDKP was used to induce skin myofibroblasts.Compared with the control group,the positive expressions of Col I,?-SMA,SHH,SMO,GLI1,and GLI2 were up-regulated by 2.9 times,1.4 times,1.6 times,and 4.8 in the Ang?-induced group.Times,2 times,1.8 times,PTC,GLI3 positive expression were down-regulated by 85% and 63%(P<0.05),compared with Ang?induced group,Col I,?-SMA,SHH,The positive expressions of SMO,GLI1,and GLI2 were down-regulated by 56%,66%,73%,50%,41%,and 51%,respectively,and the positive expressions of PTC and GLI3 were up-regulated by 2.7-fold and 5.6-fold,respectively(P<0.05).Conclusions Ac-SDKP may antagonize Ang?-mediated differentiation of skin myofibroblasts by inhibiting Hedgehog signaling and exert its anti-fibrotic effect.Figure 11;Table 8;Reference 61... |
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