| Objective To reserch N-acetyl-seryl-aspartyl-lysyl-proline(Ac-SDKP) whether by actvation of c AMP dependent protein kinase(protein kinase A, PKA) signal suppression of angiotensin(angiotensin, Ang) II/ROCK(Rho-associated coiled-coil-forming protien kinase) mediated pulmonary myofiroblast differentiation,and then play a role in anti fibrosis.Methods Estabishment of an animal model of silicosis of rats,and Ac-SDKP anti fibrosis or prevention treatment,to observe the expression and distribution of PKA protein and smooth muscle actin(α-SMA) by immunofluorescence staining of lung tissue, To detect PKA,ROC K,p-MBS, α-SMA,collagen type I by Western blot. Cell culture experiments using Ang II stimulated human embryonic lung fibroblasts(MRC-5),and Ac-SDKP,Valsartan,Y-27632,6-Bnz-c AMP pretreatment,to detect the expression and localization of PKA and α-SMA by immunofluorescence staining;to detect the expression of PKA,ROCK, p-MBS, α-SMA,collagen type I protein by Western blot.Statistical analysis was performed using SPSS17.0 software,P<0. 05.The difference was statistically significant.Results In the animal model of silicosis group, immunofluorescence staining showes a high expression of α-SMA protein in the control group, while PKA is low expression.Detection of Western blot in silicosis 4 weeks and control 8 weeks, PKA protein expression is down-regulation respectively control 4 weeks and 8 weeks of 12.50% and 10.25%.AcSDKP anti fibrosis treatment or prevention treatment can up regulate the expression of PKA protein, which is 3.50 times and 4.62 times compared by silicosis 8 week group.ROCK,P-MBS, α-SMA, type I collagen protein expression is just the opposite. The expression of ROCK protein in silicosis 4 weeks and 8 weeks is up-regulated respectively control 4 weeks and 8 weeks group of 3.82 times and 3.62 times, ROCK expression is significantly down regulated respectively silicosis 8 weeks of 21.83% and 22.98% in anti fibrosis treatment or prevention treatment of Ac-SDKP; The expression of P-MBS protein in silicosis 4 weeks and 8 weeks is up-regulated respectively control 4 weeks and 8 weeks group of 3.0 7 times and 2.78 times, p-MBS expression is significantly down regulated respectively silicosis 8 weeks of 48.31% and 50.56% in anti fibrosis treatment or prevention treatment of Ac-SDKP; type I collagen is significantly increased in silicosis 4 weeks and 8 weeks respectively control 4 weeks and 8 weeks of 6.88 times and 5.53 times, the expression of type I collagen is significantly down regulated respectively silicosis 8 weeks of 52.77% and 65.2 7% in Ac-SDKP anti fibrosis treatment or prevention treatment; α-SMA protein in silicosis 4 week and 8 is up-regulated respectively control 4 weeks and 8 weeks of 6. 26 times and 4.05 times, given Ac-SDKP anti fibrosis treatment or prevention treatment, the expression of a-SMA protein is 43.20% and 28.39% compared silicosis 8 week group. The differences were statistically significant(P<0.05). In vitro experiment, stimulated MRC-5 cells by Ang- II, the expression of α-SMA and type I collagen protein showed a time dependent effect. Immunofluorescence staining showed that Ang II induced expression of PKA was weaker than control group, while the expression of α-SMA protein was significantly increased. At the same time,Western blot showed that PKA protein expression was down regulated with Ang II induced,and was 21.13% compared by control group, when given SDKP, Valsartan or Y-27632, expression of PKA protein significantly increased by 5.23 times,5.96 times,3. 30 times compared by Ang II group, to be an activator of PKA named 6-Bnz-c AMP, the results was 3.88 times compared by Ang II stimulation group. Given Ang II induction,ROCK,p- MBS, α- SMA, type I collagen protein expression were raised, among them, the ROCK protein in Ang II stimulation group was 2.46 times compared by control group when given Ac-SDKP,Valsartan, Y-27632 or PKA activation 6-Bnz-c AMP, ROCK protein expression significantly decreased, respectively compared by Ang II stimulation group of 46.87-%, 52.50%, 56.87%, 51.87%; the p-MBS protein in Ang II stimulation group was 4.57 times compared by the control group when given Ac-SDKP, Valsartan, Y-27632 or PKA activation 6-Bnz-c AMP, p–MBS protein expression significantly decreased, respectively compared by Ang II stimulation group of 29.14%, 14.97%, 12.95%,; The α-SMA protein expression was significantly higher in Ang II group, 2.67 times that of the control group, when given Ac-SDK P,Valsartan, Y-27632 or PKA activation 6-Bnz- c AMP after pretreatment, α-SMA protein expression significantly decreased,respectively was 39.66%,27.37%,44.1-, 3%compared by Ang II sitmulation group; In Ang II stimulus group,expressionof collagen I was 4.02 times that in the control group when given Ac-SD KP, Valsartan, Y-27632 or PA activation 6-Bnz-c AMP after pretreatment, type I collagen expression significantly decreased, respectively was 45.22%, 36.94%, 43.94%, compared by Ang II stimulation group; the difference had statistical significance(P < 0.05);The above results show that the Ac-SDKP can increase the PKA protein expression, inhibition of ROCK signal pathway by Ang II mediated, which obstruct muscle differentiation of fibroblasts.Conclusion Ac-SDKP can through the regulation of PKA and ROCK signaling interaction, inhibition of Ang II induced myofibroblast differentiation so as to exert anti silicosis fibrosis. |
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