| Silicosis is caused by inhalation of silicon dioxide dust in the professional activities for a long term, and its basic pathological features include the formation of silicotic nodule and lung fibrosis. The current study shows that local renin angiotensin system(RAS) in lung tissue involved in the occurrence and development of pulmonary fibrosis. As the key enzyme of RAS, angiotensin converting enzyme(ACE) consists of two catalytic domains–the C and N domains. The C domain can catalyze angiotensin I(Ang I) into angiotensin II(Ang II). Ang II is the major active protein of RAS, its fibrotic effect is mainly mediated by angiotensin II type 1 receptor(AT1R), which include inducing inflammatory response, promoting the proliferation of fibroblasts and extracellular matrix deposition. The N domain is responsible for hydrolyzing the anti-fibrosis short peptide N-acetyl-seryl-aspartyl-lysylproline(Ac-SDKP) specifically. Ac-SDKP level increased obviously in ACE N domain defects mice, which could be manifested by the anti-organ fibrosis effect. Similarly, treatment with exogenous Ac-SDKP could reduce the degrees of ACE wild-type mice lung injuries and pulmonary fibrosis. Obviously, the interaction between Ac-SDKP and RAS may be one of the important molecular mechanisms of its anti-fibrosis effect. In the present study, we selected the Ang II signal as a breakthrough point, to observe whether Ac-SDKP could regulate the pathogenesis and development of silicotic fibrosis by inhibiting the action of Ang II. Our previous study found, the expression of β2 adrenergic receptor-stimulatory G protein(ADRB2-Gs) complexes were decreased in silicotic model, and its expression in Ac-SDKP anti-fibrotic group significantly increased, which suggested that it was one of the regulatory proteins of Ac-SDKP in silicotic fibrosis. The anti-organ fibrosis effect of ADRB2 was related to the synthesis of adenosine 9monophosphate cyclic(c AMP). Related research reported, the interaction between c AMP signal and Ang II/transforming gorwth factor-β1(TGF-β1) signal played an important role in the pathogenesis of fibrosis. c AMP signaling could inhibit the transformation of fibroblasts into myofibroblasts and collagen synthesis induced by Ang II/TGF-β1. Ang II/TGF-β1 were able to accelerate the hydrolysis of c AMP by upregulating the content of phosphodiesterase(PDE). At present there are few report about the regulatory effect of Ac-SDKP on c AMP signal and Ang II signal and wether Ac-SDKP can regulate the Ang II signal through the c AMP signal. Based on the above research, by designing the experiment of cultured rat model of silicosis bound with myofibroblasts differentiation induced by Ang II, this study was designed to explore the interaction between Ac-SDKP and c AMP signal and Ang II signal on the pathogenesis and development of silicotic fibrosis, and further analysis wether the protective effect of Ac-SDKP was related to the activation of c AMP/c AMP dependent protein kinase A/c AMP-response element binding protein pathway. To further reveal the possible mechanism of the occurrence and development of silicosis and the protective effect of Ac-SDKP. Part one The dynamic changes of Ac-SDKP, Ang II signal and c AMPsignal on the pathogenesis and development of silicotic fibrosisObjective: Through the establishment of rat model of silicosis and observing myofibroblasts differentiation induced by Ang II in vitro, to analyse the changes of Ac-SDKP, ACE/Ang II/AT1 R axis and c AMP signal and their impact on the pathogenesis and development of silicotic fibrosis.Methods:1 HOPE MED 8050 exposure control apparatus was used to create the silicosis model. Setting were as follows: exposure chamber volume 0.3m3, cabinet temperature 20~25℃, humidity 70~75%, pressure-50~+50Pa, oxygen concentration 20%, flow rate of Si O2 3.0~3.5ml/min, dust mass concentration in the cabinet 2000mg/m3, and each animal inhaled for 3h per day. 3-week-old male Wistar rats were randomly divided into 6 groups, 10 rats in each group. The rats inhaled dust respectively for 0w, 2w, 4w, 8w, 12 w, 16 w, for observing the dynamic change of silicosis.2 To observe the induction effect of Ang II(10-7mol/L) on the transformation of lung fibroblast into myofibroblast at different time points(0, 5min, 15 min, 30 min, 1h, 3h, 6h, 12 h, 24 h, 48h).3 HE staining and Masson trichromatic dyeing method were used to observe the pathology change of lung tissue.The expression of α smooth muscle actin(α-SMA), Collagen I, fibronectin(Fn), Vimentin, Gαs, inhibitory G protein(Gαi2/3), ACE and AT1 R were detected by immunohistochemistry and Western blot respectively. The content of Ac-SDKP, Ang II and c AMP were detected by enzyme immunoassay(EIA) and enzyme linked immunosorbent assay(ELISA).Results:1 The results of HE staining showed that normal lung tissue structure was clear and the alveolar wall was thin, and there was no obvious inflammatory cell infiltration. In the silicosis 2w group, macrophages were found in alveolar and alveolar wall became widening. In silicosis 4w group showed isolated cell nodules which composed of macrophages. The alveolar wall widened obviously and the number of silicotic nodules increased in the lung tissue of silicosis 8w and 12 w group. In silicosis 16 w group the silicotic nodule volume increased, nodule fusion and the formation of interstitial fibrosis could be seen, and cell fibrous nodules were visible. Masson trichrome staining showed, in the nodules and interstitial fibrosis area appeared blue purple collagen deposition and gradually increased with the extension of the model preparation time, which were most significant in the silicosis 16 weeks group.2 Immunohistochemical staining indicated α-SMA marked myofibroblasts were located among the nodules and distributed in the interstitial fibrotic area. Vimentin expressed in the cell nodule region. Western blot showed that, compared with control group the expression of α-SMA, Collagen I, Fn and Vimentin protein in silicosis 4w, 8w, 12 w, 16 w group increased gradually.3 Western blot and ELISA results showed that in silicosis 2w, 4w, 8w, 12 w, 16 w group, the expression of ACE, Ang II, AT1 R gradually increased compared to control. The level of Ac-SDKP in lung tissue of silicosis 2w group was significantly higher than that of control group, afterwards in the silicosis 4w, 8w, 12 w and 16 w group Ac-SDKP level decreased.4 Western blot assay displayed that the expression of Gαi2/3 in silicosis 4w, 8w, 12 w, 16 w group increased gradually compared to control group. Compared with control group, the expression of Gαs and c AMP in silicosis 4w began to decrease significantly, the content were the lowest in the silicosis 16 w group.5 Ang II could induce myofibroblast differentiation. After Ang II induction, the expression of α-SMA, Collagen I, Fn significantly increased in cultured lung fibroblasts compared to control. The expression of Gαs, c AMP decreased gradually with the extension of Ang II stimulation time, while the expression of Gαi2/3 increased gradually. Part two Function and mechanism of Ac-SDKP on activating the c AMP/PKA signal and restraining silicotic fibrosisObjective: Given the rats with silicosis Ac-SDKP and db-c AMP(c AMP analogue) respectively as anti-fibrosis therapy and prevention therapy. Using Ang II to induce the transition of pulmonary fibroblasts into myofibroblast in vitro, by giving Ac-SDKP, db-c AMP and the selective PKA blocking agent H89 treatment, we observed the effect the c AMP/PKA signal activation has on the lung fibrosis inhibiting function of the Ac-SDKP.Methods:1 HOPE MED 8050 exposure control apparatus was used to create the silicosis model. Rats were randomly divided into 6 groups: 1) controls for 16w; 2) silicosis for 16w; 3) Ac-SDKP post-treatment; 4) Ac-SDKP pre-treatment; 5) db-c AMP post-treatment; 6) db-c AMP pre-treatment.2 The primary lung fibroblasts were cultured and the cells were given the following treatment respectively :1) control; 2) Ang II(10-7mol/L); 3) Ang II +Ac-SDKP(10-8mol/L); 4) Ang II +Ac-SDKP + H89(10-6mol/L); 5) Ang II + db-c AMP(10-4mol/L); 6) Ang II+ db-c AMP+ H89.3 The expression of α-SMA, Collagen I, Vimentin, Fn, ACE, Ang II, AT1 R, Gαs、Gαi2/3, c AMP and PKA protein were detected by Western blot and ELSIA in vivo and vitro. Immunofluorescence was performed on lung tissue sections and fibroblasts to check the co-staining of α-SMA/Gαi3, α-SMA/PKA.Results:1 Western blot results showed that, compared to silicosis 16 w group, α-SMA, Collagen I, Fn and vimentin expression were significantly down-regulated in Ac-SDKP and db-c AMP treatment group(post-treatment and pre-treatment group).2 Western blot data indicated that in Ac-SDKP and db-c AMP post-treatment group and pre-treatment group, AT1 R expression was significantly lower than that of silicosis 16 w group, the expression of ACE and Ang II in the Ac-SDKP and db-c AMP treatment group decreased, but the difference was not statistically significant.3 The results of immunofluorescence staining indicated that a large number of green fluorescence labeled α-SMA positively expressed in the area of fibrosis. The expression of Gαi3 with red fluorescence was also significantly increased. Combined two fluorescence marker, the coexpression of Gαi3 and α-SMA were visible in the nodules and alveolar wall broadening area. In Ac-SDKP, db-c AMP post-treatment group and pre-treatment group, the positive expression of Gαi3 and α-SMA decreased. Western blot results showed that compared with the silicosis 16 w group, Gαi2/3 expression decreased, Gαs, c AMP and PKA expression were significantly up-regulated in Ac-SDKP and db c AMP treatment group.4 Immunohistochemical staining showed that following the induction of Ang II for 24 h, the positive staining of α-SMA in cytoplasm increased. After pre-treatment with Ac-SDKP and db-c AMP, the positive staining of α-SMA showed attenuated. H89 could significantly inhibit the effects of Ac-SDKP and db-c AMP. Western blot analysis showed that Ac-SDKP, db-c AMP intervention could significantly reduce the expression of α-SMA, collagen I and Fn induced by Ang II. While in Ac-SDKP + H89 and db-c AMP + H89 intervention group, the expression of α-SMA, Collagen I, Fn protein were significantly increased.5 Western blot results indicated that pretreatment with Ac-SDKP and db-c AMP, the expression of Gαi2/3 protein induced by Ang II were significantly down-regulated, the expression of Gαs protein was up-regulated than that of Ang II group. In Ac-SDKP+H89, db-c AMP+H89 group Gαi2/3 protein expression were significantly increased, the expression of Gαs protein was significantly reduced.6 Immunofluorescent data from PKA/α-SMA coexpression showed that PKA were observed in the cytoplasm and nuclei in the control group, in cytoplasm α-SMA showed a weak expression. After induced by Ang II for 24 h, the expression of PKA protein was weaken and the positive expression of α-SMA was significantly enhanced compared to control group. After pretreatment with Ac-SDKP and db-c AMP, the positive staining of α-SMA showed attenuated, and the positive expression of PKA increased than that of Ang II group. These were reversed with H89. Western blot data indicated that c AMP and PKA protein expression significantly increased in both Ac-SDKP and db-c AMP pretreatment group. In Ac-SDKP + H89 group and db-c AMP + H89 group, the expression of c AMP and PKA protein decreased. Part three Function and mechanism of Ac-SDKP on regulating p CREBsignal and p Smad2/3 signal in order to restrain silicotic fibrosisObjective: Using the animal model of silicosis bounded with cell culture in vitro, to explore whether the inhibiting effect of Ac-SDKP on silicotic fibrosis was related to the regulation of p CREB, p Smad2/3 signal.Methods:1 HOPE MED 8050 exposure control apparatus was used to create the silicosis model. Rats were randomly divided into 6 groups: 1) controls for 16w; 2) silicosis for 16w; 3) Ac-SDKP post-treatment; 4) Ac-SDKP pre-treatment; 5) db-c AMP post-treatment; 6) db-c AMP pre-treatment.2 The fibroblasts were given the following treatment respectively: 1) control; 2) Ang II(10-7mol/L); 3) Ang II + Ac-SDKP(10-8mol/L); 4) Ang II +Ac-SDKP + H89(10-6mol/L); 5) db-c AMP(10-4mol/L)+Ang II; 6) Ang II+ db-c AMP + H89.3 The expression of p CREB, CREB, p Smad2/3 and Smad2/3 in the lung tissue and fibroblast were assessed by Western blot. The colocalization of p CREB and α-SMA, p Smad2/3 and α-SMA were detected by immunofluorescence in vivo and vitro. Co-immunoprecipitation were performed to analyse the interaction between CREB binding protein(CBP) and p CREB and p Smad2/3 in the nuclei of fibroblasts.Results:1 Immunohistochemical detection of p CREB and p Smad2/3 showed that the positive expression of p CREB were seen in alveolar epithelial cells, its expression decreased in the silicotic nodules. While in the silicotic nodules positive expressions of p Smad2/3 increased. The results of immunofluorescence staining indicated that p Smad2/3 with red fluorescent markers were weakly expressed in the nuclei, α-SMA with green fluorescents existed positive expression only in trachea smooth muscle cells. In silicosis 16 w group, α-SMA positive expressed in silicotic nodule and in alveolar wall widen region, p Smad2/3 protein were also expressed in the nuclei of the above site. Conbined the two fluorescent marked proteins indicating much more cells in the area of fibrosis had both positive expression of α-SMA protein in cytoplasm and positive expression of p Smad2/3 protein in the nucleus. In Ac-SDKP and db-c AMP treatment groups, the positive expression of p Smad2/3 and α-SMA decreased. Western blot results showed that the expression of p CREB protein decreased in silicosis 16 w group, p Smad2/3 protein expression was significantly up-regulated. Ac-SDKP and db-c AMP treatment promoted the expression of p CREB protein, and inhibited the expression of p Smad2/3 protein. There were no significant difference in the expression of CREB and Smad2/3 protein.2 The coexpression of α-SMA and p CREB was observed by immunofluorescence staining, the results showed that positive expression of p CREB were located in the nuclei and cytoplasm, and the expression of α-SMA was weak. Following the induction of Ang II for 24 h, the positive expression of p CREB was significantly decreased, and the expression of α-SMA was significantly increased. After given Ac-SDKP and db-c AMP intervention, α-SMA expression significantly decreased, p CREB expression significantly enhanced. H89 could significantly inhibit the effect of Ac-SDKP and db-c AMP. Immunofluorescence staining results also showed that Ang II could significantly enhance the expression of p Smad2/3 in the nuclei and α-SMA expression in the cytoplasm. Pretreatment with Ac-SDKP and db-c AMP reduced the expression of p Smad2/3 and α-SMA in fibroblast. H89 could significantly block the role of Ac-SDKP and db-c AMP. The results of western blot showed that the expression of p CREB were decreased and the expression of p Smad2/3 protein were significantly increased follwing the induction of Ang II for 24 h. Ac-SDKP, db-c AMP pretreatment significantly promoted the expression of p CREB, reduced the expression of p Smad2/3. And these were reversed with H89.3 Co-immunoprecipitation analysis indicated the CBP was capable of binding p Smad2/3 and p CREB respectively. In Ang II group, the combination of p Smad2/3 and CBP increased, while the combination of p CREB and CBP decreased. After Ac-SDKP and db-c AMP pretreatment, the interaction of CBP with p CREB up-regulated, while interaction of CBP with p Smad2/3 down-regulated.Conclusions:1 Along with the pathogenesis and development of silicosis, ACE/Ang II/AT1 R in local lung tissues increased, and Ang II was capable of stimulating the transformation of lung fibroblast into myofibroblast in vitro; and during the pathogenesis and development process, the expression of Ac-SDKP and c AMP decreased. Ac-SDKP and c AMP signal and Ang II signal had participated the silicotic fibrosis process of rats.2 In the silicotic tissues and in fibroblasts induced with Ang II,by activating the signal of c AMP/PKA/p CREB, Ac-SDKP was capable of restraining the expression of p Smad2/3, and accelerating the formation of p CREB/CBP compound, reducing the integration of p Smad2/3 and CBP, so as to restrain the Smad signal and play an effective role against silicotic fibrosis. |
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