| Objectives To study the effect and mechanism of Ac-SDKP on the activation and migration of skin fibroblasts mediated by TGF-β1 through Hedgehog signaling pathway.Methods Rat neonatal skin fibroblasts were cultured,and second-generation fibroblasts were used in the experiment.TGF-β1 was used alone to act on skin fibroblasts.The experiment was divided into:(1)control group,(2)TGF-β1 group.TGF-β1 and SMO receptor blocker(GDC-0449)were applied to skin fibroblasts.The experimental groups were:(1)control group,(2)TGF-β1 group,(3)GDC-0449,(4)TGF-β1 and GDC-0449 group.TGF-β1 and GLI transcription factor blocker(GANT61)were used to treat skin fibroblasts.The experimental groups were:(1)control group,(2)TGF-β1 group,(3)GANT61 group,(4)TGF-β1 and GANT61 group.TGF-β1 and GLI transcription factor blocker(GANT61)were used to treat skin fibroblasts.The experimental groups were:(1)control group,(2)TGF-β1 group,(3)GANT61 group,(4)TGF-β1 and GANT61 group.Using TGF-β1 and Ac-SDKP to act on skin fibroblasts,the experiment was divided into:(1)control group,(2)TGF-β1 group,(3)Ac-SDKP group,(4)TGF-β1 and Ac-SDKP group.Immunohistochemical detection of α-SMA,PTC,SHH,SMO,GLI1,GLI2,GLI3 protein expression;Western blot method was used to detect the expression of α-SMA,ColI,PTC,SHH,SMO,GLI1,GLI2 and GLI3;Cell Scratch test was used to detect the migration ability of skin fibroblasts.Results 1 Western Blot stimulation of fibroblasts by TGF-β1showed that compared with the control group,the expression of ColI,α-SMA,SHH,SMO,GLI1 and GLI2 in the TGF-β1 group was up-regulated by 4.78 times,4.08 times,1.43 times,2.19 times and 1.91 times,respectively.1.47 times.GLI3 and PTC expression were down-regulated to 41.46% and 42.23%(P<0.05).2 Immunohistochemical using TGF-β1 and GDC-0449 on skin fibroblasts showed that((1)Compared with the control group,α-SMA was strongly expressed in the cytoplasm of the TGF-β1 group.After TGF-β1 and GDC-0449 intervention,α-SMA expression was higher than that of TGF-β1 group weakened.(2)Western Blot results showed that compared with the control group,the expressions of ColI,α-SMA,SHH,SMO,GLI1,and GLI2 in the TGF-β1 group were up-regulated by 1.53 times,1.74 times,1.42 times,2.08 times,1.92 times,1.60 times.The expressions of PTC and GLI3 were down-regulated to 67.76% and 58.63%,respectively.Compared with the TGF-β1 group,the expressions of ColI,α-SMA,SHH,SMO,GLI1,and GLI2 were down-regulated in the TGF-β1 and GDC-0449 group,and decreased to 77.68%,73.32%,and 80.68% of the TGF-β1 group,respectively.71.93%,67.13%,77.18%.The expressions of PTC and GLI3 were up-regulated by 1.45 times and 1.33 times(P<0.05).(3)The results of cell scratches showed that compared with the control group,the area of the cells in the TGF-β1 group migrating to the central scratch area is significantly increased;after the intervention of TGF-β1 and GDC-0449,the cells migrate to the area of the central scratch area Compared with TGF-β1 group.3 The results of immunohistochemistry using TGF-β1 and GANT61 on skin fibroblasts showed that(1)Compared with the control group,α-SMA was strongly expressed in the cytoplasm of TGF-β1 group.After TGF-β1 and GANT61 intervention,the expression of α-SMA was higher than TGF-β1 Group weakened.(2)Western Blot results showed that compared with the control group,the expression of ColI,α-SMA,GLI1,and GLI2 in the TGF-β1 group were up-regulated by 5.09 times,4.53 times,2.60 times,and 2.89 times,respectively.GLI3 expression was down-regulated to 58.63%.Compared with TGF-β1,the expression of ColI,α-SMA,GLI1 and GLI2 in TGF-β1 and GANT61 group decreased to 49.81%,37.83%,65.99% and 46.763% in TGF-β1 group,respectively.GLI3 expression was up-regulated by 1.33 times(P <0.05).(3)The results of cell scratches showed that compared with the control group,the area of cells in the TGF-β1 group migrating to the central scratch area is significantly increased;the area of cells migrating to the central scratch area after stimulation with TGF-β1 and GANT61 is greater than that of the TGF-β1 group decreases.4 The results of immunohistochemistry using TGF-β1 and Ac-SDKP on skin fibroblasts showed that(1)α-SMA,SHH,SMO,GLI1,and GLI2 positive expressions were significantly increased in TGF-β1 group,while PTC and GLI3 positive expressions were missing.After TGF-β1 and Ac-SDKP stimulation,the positive expressions of α-SMA,SHH,SMO,GLI1,and GLI2 were significantly reduced compared with TGF-β1 group,and the positive expressions of PTC and GLI3 were significantly in increased.(2)Western Blot results showed that compared with the control group,the expression of ColI,α-SMA,SHH,SMO,GLI1,and GLI2 in the TGF-β1 group were up-regulated by 8.28 times,4.75 times,3.66 times,3.50 times,2.71 times,respectively.2.78 times.The expressions of PTC and GLI3 were down-regulated to 14.73% and 58.63%,respectively.Compared with the TGF-β1 group,the expression of ColI,α-SMA,SHH,SMO,GLI1,and GLI2 in the TGF-β1 and Ac-SDKP group decreased to 79.07%,41.67%,52.25%,68.38%,62.10%,68.44%.The expressions of PTC and GLI3 were up-regulated by 2.69 times and 1.33 times(P<0.05).(3)Cell scratch experiments showed that the area of cells in the TGF-β1 group migrating to the central scratch area was significantly larger than that of the control group;the area of cells migrating to the center scratch area after TGF-β1 and Ac-SDKP stimulation was greater than that of TGF β1 group decreases.Conclusions 1 TGF-β1 induces the transformation of skin fibroblasts to myofibroblasts and the activation of HH signaling pathway.2 Ac-SDKP can inhibit the transformation and migration of skin fibroblasts to myofibroblasts mediated by TGF-β1 by regulating HH signaling pathway,thereby exerting its anti-fibrotic effect.Figure12;Table3;Reference 111... |
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