| Objectives To study the role of Ac-SDKP in regulating the transdifferentiation and apoptosis of fibroblasts through ?-TAT1.Methods The silicosis rat model was established by HPOE-ED8050 dynamic dust control system in the animal central barrier laboratory of North China University of Science and Technology.Experimental groups: the control group of silicosis model(rats intraperitoneal embedded trace sustained-release capsules containing 2ml saline and freely feeded for 24 weeks),the silicosis model group(rats intraperitoneal embedded trace sustained-release capsules containing 2ml saline with dust exposure 3h/d for 24weeks),the Ac-SDKP treatment group(rats intraperitoneal embedded trace sustainedrelease capsules containing 2ml saline with dust exposure 3h/d for 24 weeks,and saline was replacement by Ac-SDKP 12 weeks after dust exposure).Lung fibroblasts of Wistar rats were cultured by adherent method and treated with TGF-? and Ac-SDKP at different time points(0 ? 6h ? 12 h ? 24 h ? 48h).Fibroblasts of ?-TAT1 overexpression and Silent expression were devided into 6 groups:(1)Control group(Sham),(2)TGF-beta induced group(TGF-?),and(3)Ac-SDKP treatment group(TGF-?+ Ac-SDKP),(4)?-TAT1 overexpression group(?-TAT1-pc DNA),(5)overexpression of ?-TAT1 induced by TGF-?group(?-TAT1 pc DNA+ TGF-?),(6)?-TAT1 silencing group(?-TAT1 si RNA+ TGF-?+Ac-SDKP).HE staining and Sirius red staining were used to detect collagen deposition.Immunohistochemical staining was used to detect the expression of ?-TAT1.Expression of ?-smooth muscle actin(?-SMA),Type I collagen(Col I),acetylated tubulin alpha(AcTub ?),Caspase-3,?-TAT1,Bcl-2 and Bax in lung tissue were analyzed by Western blot assay.Results 1 Animal model experiments:(1)Typical fibrous nodules were observed in model group by HE staining.The number and area of silica nodules in Ac-SDKP treatment group were significantly lower than those in model group.Result of Sirius red staining revealed that collagen deposition abundantly in silicosis model group,and the deposition of collagen was decreased obviously in Ac-SDKP treated group.(2)Western blot results showed that the expressions of type I collagen and ?-SMA in silicosis group were 3.97 times and 2.72 times higher than that in control group,and the expression of type I collagen and ?-SMA were decrease after Ac-SDKP treatment,which were 41.9%and 47.1% of the Silicosis group.(3)Immunohistochemical staining results showed that?-TAT1 was strongly expressed in bronchial cilia,alveolar type II epithelium and fibroblasts,however,?-TAT1 was absent in the silicotic nodules,composed of interstitial cells and myofibroblasts,in silicosis model group.The positive expression of ?-TAT1 in Ac-SDKP group was higher than that in silicosis model group.(4)The results of Western blot showed that the protein expression of Ac-Tub? and ?-TAT1 were consistent with that of immunohistochemical staining.2 Cell culture experiment:(1)The results of Western blot showed that the expression of ?-TAT1 was down-regulated and the expression of ?-SMA was up-regulated by TGF-? at different time points.However,the expression of Caspase-3 apoptosis protein reached the highest level at 12 h and then decreased gradually,and it was found that the expression of ?-TAT1 could be upregulated by Ac-SDKP at different time points,thus increasing the acetylation level of tubulin and maintaining the stability of tubulin skeleton.(2)The results of Western blot showed that the target plasmids were transfected into lung fibroblasts,and then the ?-TAT1 overexpression and silencing expression plasmids were constructed and screened.(3)The results of Western blot showed that when ?-TAT1 overexpression plasmid was transfected into pulmonary fibroblasts(?-TAT1 pc DNAs),there was no significant difference in expression of type I collagen,?-SMA and Caspase-3 between the two groups.However,compared with TGF-? group,?-TAT1 overexpressed lung fibroblasts received TGF-? stimuli could down-regulated the expression of type I collagen and ?-SMA,accompanied by the upregulation of Caspase-3 protein.(4)The results of Western blot showed that the expression of type I collagen,?-SMA and Bcl-2 were increased by4.56 times,1.99 times and 2.11 times respectively,and the expression of Caspase-3 and Bax protein were down-regulated to 35.53% and 44.14% of the control group,respectively.After treatment with Ac-SDKP,it was found that the expression of type I collagen,?-SMA and Bcl-2 protein decreased to 45.22%,34.50% and 30.47% of TGF-?group respectively,but the level of Caspase-3 and Bax protein was higher than that of TGF-? group.After ?-TAT1 silencing,it was found that Ac-SDKP could not decrease the expression of type I collagen,?-SMA and Bcl-2 protein induced by TGF-?,meanwhile,?-TAT1 silencing could increase the expression of Caspase-3 and Bax.Conclusions Ac-SDKP exerts its anti-silicosis effect via regulating the expression of ?-TAT1 to influence the acetylation level of tubulin and differentiation and apoptosis resistance of myofibroblast mediated by TGF-?. |
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