Acetylated α-tubulin Regulated By Ac-SDKP Exerts The Anti-fibrosis Effect In Rat Exposed To Slica

Part one The expression and pathological significance of α-Ac-Tub in patients with silicosisObjective: To explore the expression and clinic pathological significance of α-Ac-Tub in patients with silicosis.Methods:1 A total of 59 cases of paraffin-embedded sections were collected from autopsy of patients with silicosis, with an average age of 52 years(30 to 77 years). These patients were come from coal mines(such as Neimenggu, Shanxi, Hunan, Hebei, etc.), cement mills, ceramics factories and refractory plants. There were 22 cases stage I silicosis, 8 cases stageⅡ silicosis, 7 cases silicosis, and 22 cases coal dust pneumonia, with an average exposure history of 22 years(7 to 46 years). The control group was selected from 13 cases surgical sections without dust exposure and with similar age.2 The pathological change was observed by HE staining and Masson staining. The expression of α-SMA, vimentin and α-Ac-Tub were measured by immunohistochemical staining. The co-expression of α-Ac-Tub/vimentin and α-Ac-Tub/SP-A were analyzed by immunofluorescence.Results:1 The dynamic variation were observed by HE and Masson staining in a gradually deepens manner, which were macrophages alveolitis, cellulous nodules, cellulous and fibrotic nodules and fibrotic nodules(including hyaline change).2 Myofibroblasts marked by α-SMA were observed in silicotic nodules and interstitial fibrosis areas.3 The positive expression of α-Ac-Tub was observed in vascular endothelial cells, alveolar epithelial cells and lung fibroblasts of normal lung tissue, and loss expression in silicotic nodules and interstitial fibrosis areas in silicotic spections.4 Immunofluorescence results showed that the expression of vimentin was increased in fibrotic lesions, accompanied with loss expression of α-Ac-Tub and SP-A in silicosis nodules and interstitial fibrosis disease areas. Part two The anti-fibrosis effect of Ac-SDKP on rat exposed to silica via regulating α-Ac-TubObjective: Silicosis model was made by exposing to silica(SiO2) and treatment with Ac-SDKP in pre- or post-profile. To explore the mechanisms of α-Ac-Tub regulated by Ac-SDKP in silicosis rats, the role and expression of α-Ac-Tub, HDAC6 and α-SMA were analyzed in the present study.Methods:1 Briefly, 60 rats were divided into 6 groups(10 per group): 1) control 4 w(induced with 0.9% saline; then treated with 0.9% saline for 4 weeks); 2) silicosis model 4 w [silicosis induced with 50 mg SiO2(s5631, Sigma-Aldrich, St. Louis, MO, USA) in rat trachea, then treated with 0.9% saline for 4 w]; 3) control 8 w(pre-induced with 0.9% saline 48 h before 0.9% saline induction, then treated with 0.9% saline for 8 w); 4) silicosis model 8 w(pre-treated with 0.9% saline 48 h before 50 mg SiO2 induction, then treated with 0.9% saline for 8 w); 5) Ac-SDKP post-treatment(induced with SiO2, treated with 0.9% saline for 4 w, and then treated with Ac-SDKP for an additional 4 w); and 6) Ac-SDKP pre-treatment(pre-treated with Ac-SDKP 48 h before induction with SiO2, then continued Ac-SDKP treatment for 8 w).2 The pathological change was observed by HE staining and Masson staining. The expression of α-SMA and α-Ac-Tub were measured by immunohistochemical staining. The co-expression of α-Ac-Tub/α-Tub, α-Ac-Tub/vimentin, α-Ac-Tub/SP-A, HDAC6/α-SMA and HDAC6/α-Ac-Tub were measured by immunofluorescence. The level of α-SMA, α-Ac-Tub, HDAC6 and collagen type I were analyzed by western blot.Results:1 HE staining showed that in the control group(including 4 weeks and 8 weeks group) the structure of lungs was clear and the alveolar wall was thin, there were no significant interstitial infiltration of inflammatory cells. Widen alveolar septum and silicosis nodules composed by macrophages were observed in silicosis 4w group, which were serious in silicosis 8w group. The volume and number of silicosis nodules were reduced in Ac-SDKP post- or pre-treatment group.2 The majority of detected α-Ac-Tub was expressed in the airway epithelium, also appeared in multiple cell types throughout the alveolar cells of the lung. To eliminate the potential confounding effects of these antibodies, two different commercial antibodies were used, and similar expression patterns of α-Ac-Tub in rat lung tissue were obtained.3 Immunohistochemical staining results showed that specific expression of α-SMA was observed in silicosis nodules and interstitial fibrotic regions in the silicosis model, with loss expression of α-Ac-Tub. The immunostaining results were further confirmed by using a Western blot analysis, which showed that Ac-SDKP post- and pre-treatment should reversed highexpression of α-SMA, collagen type I and down-regulation of α-Ac-Tub induced by silica.4 Positive expression of HDAC6 was observed in bronchial epithelial cells(cytoplasm) and part of alveolar type Ⅱ epithelial cells(nuclear) in control group. But in silicosis groups, HDAC6 positive expression was seen in macrophages and interstitial fibrosis area(cytoplasm). Co-expression of HDAC6 and α-SMA were mainly located in silicosis lesions without positive expression of α-Ac-Tub. Ac-SDKP post- and pre-treatment should reverse high-expression of HDAC induced by silica. Part three The effect and mechanism of α-Ac-Tub regulated by Ac-SDKP in inhibiting myofibroblast differentiation induced by AngⅡObjective: myofibroblast differentiation from fibroblasts was induced by AngⅡ. To explored the mechanism of anti-fibrotic effect of Ac-SDKP via regulating α-Ac-Tub, myofibroblasts were pre-treated with Ac-SDKP, valsartan, TCS HDAC6 20 b and Y-27632.Methods: Primary rat lung fibroblasts cultured in vitro and divided into 6 groups: 1) control group; 2) AngⅡinduced group; 3) AngⅡ+ Ac-SDKP group; 4) Ang Ⅱ+ valsartan group; 5) AngⅡ+ TCS HDAC6 20 b group; 6) AngⅡ+ Y-27632 groupResults:1 Immunofluorescence results showed that after induction of AngⅡ, the expression of α-Ac-Tub was down-regulated in fibroblasts induced by AngⅡ.2 Upon treatment with AngⅡfor 24 h, rat lung fibroblasts showed morphological changes, including cell widening and strong positive expression of α-SMA accompanied by decreased expression of α-Ac-Tub. Significantly up-regulated levels of α-SMA and col I protein, as well as down-regulated levels of α-Ac-Tub were observed in response to AngⅡ treatment measured by western blot. Treatment with Ac-SDKP, valsartan(AT1 inhibitor), TCS HDAC6 20b(a specific HDAC6 inhibitor) and Y-27632( a ROCK inhibitor) caused α-Ac-Tub to be redistributed, and it attenuated the up-regulation of α-SMA and col I induced by AngⅡ.3 The expression of HDAC6 was up-regulated in fibroblasts induced by Ang II. Moreover, treatment with Ac-SDKP, valsartan, TCS HDAC6 20 b, and Y-27632 alleviated the elevated expression of HDAC6 induced by AngⅡin fibroblasts.Conclusions:1 The lost expression of α-Ac-Tub may be a new mechanism in rat silicosis.2 Ac-SDKP inhibits myofibroblast differentiation and collagen deposition accompanied by stabilizing the expression of α-Ac-Tub in vivo and in vitro, which is at least in part related with blocking angiotensinⅡ(AngⅡ) type 1 receptor(AT1), Rho-associated coiled coil-forming protein kinase(ROCK) and histone deacetylase family member 6(HDAC6) signals.