| Objectives The purpose of the present study is to observe the role of the renin-angiotensin system(RAS)in the formation of silicosis,which is for exploring the effects of blocking RAS on silicotic fibrosis in mice,and to study the effect and mechanism of which N-acetyl-seryl-aspartyl-lysyl-proline(Ac-SDKP)regulates RAS to antagonize silicosis fibrosis in mice.Methods 1 Silicotic mice(C57BL/6)models were established by the one-time orotracheal installation of Si O2 suspension,and cell models of epithelial-mesenchymal transition(EMT)were induced by Ang ? in MLE-12 cells.2 The changes in the lung histopathology were observed using HE,VG and Sirius red staining.The expression and co-localization of alpha-smooth muscle actin(?-SMA)and the surfactant protein-D(SP-D)in lung tissue were observed through immunohistochemistry/immunofluorescent chemical staining.Western blot was used to detect the protein expression of ACE,ACE2,Ang ?,Ang-(1-7),AT1,Mas,?-SMA,vimentin(Vim),collagen I(Col I),collagen ?I(Col ?I)and E-cadherin(E-cad)in the lung tissue and MLE-12 cells,while the expression of ACE and ACE2 m RNA was detected by real-time quantitative PCR.The proliferation of MLE-12 cells was detected using the CCK-8 method,and Fine Pointe whole-body plethysmography(WBP)was used to detect the lung function of the mice.3The experimental protocol was as follows:1)In order to observe the effect of ACE-Ang ?-AT1 axis blockage on RAS,EMT and silicotic fibrosis,the mice and MLE-12 cells were treated with captopril and losartan,respectively.2)To observe the effect of ACE2-Ang-(1-7)-Mas R axis blockage on RAS,EMT and silicotic fibrosis,the mice and MLE-12cells were treated with MLN-4760 and A779,respectively.3)The silicosis model of the ACE gene(ACE+/-)and ACE2 gene(ACE2+/-)knockdown mice were prepared,and the effect of the RAS target gene knockdown on EMT and silicosis was observed.4)The silicosis model of ACE+/-and ACE2+/-mice and the cell model of silencing ACE(si ACE)and ACE2(si ACE2)genes were separately treated with Ac-SDKP to observe the effect of Ac-SDKP on EMT and silicosis by regulating RAS.Results 1 By analyzing the relationship between the Si O2 dose(Ang ? concentration)/time effect and the degree of silicosis,we concluded that the optimal conditions for the experiment were one-time orotracheal instillation of 2.5 mg/mouse silica suspension for 4weeks to prepare the mouse silicosis models and 10-6M Ang ? treatment for 48 h to prepare the cell EMT models.2 We observed the following effects of blocking the ACE-Ang ?-AT1R axis by captopril and losartan:1)Compared with the model group,the use of captopril and losartan reduced the number and area of silicotic nodules,positive expression of?-SMA and the co-expression of?-SMA and SP-D in the lung tissue.The protein expression of ACE,Ang ?,AT1,?-SMA,Vim,Col I and Col ?I was decreased and that of ACE2,Ang-(1-7),Mas and E-cad was increased in the lung tissue.In addition,the expression of ACE m RNA was decreased,and that of ACE2 m RNA was increased.Besides,the use of these substances reduced Tvb,Mvb,Ti,PIF,PEF and EF50,which improved the lung function.2)Compared with the Ang ? group,captopril and losartan could reduce the cell proliferation and the protein expression of ACE,Ang ?,AT1,?-SMA,Vim,Col I and Col ?I,and increase the protein expression of ACE2,Ang-(1-7),Mas and E-cad in MLE-12 cells.3 Regarding the blocking of the ACE2-Ang-(1-7)-Mas R axis by MLN-4760 and A779,we found the following effects:1)Compared with the model group,MLN-4760 and A779 aggravated the number and area of nodules,positive expression of?-SMA and co-expression with SP-D in the lung tissue.Furthermore,the protein expression of ACE,Ang ?,AT1,?-SMA,Vim,Col I and Col ?I was further increased and that of ACE2,Ang-(1-7)and Mas was further decreased in the lung tissue.In addition,the medicines also aggravated the changes of the elevated ACE m RNA and declined ACE2 m RNA expression,and they could further reduce Tvb,Mvb,Ti,PIF,PEF and EF50 and elevate F,Te,Penh and PAU.2)Ang-(1-7)could alleviate the cell proliferation,decrease the protein expression of ACE,Ang ?,AT1,?-SMA,Vim,Col I and Col ?I and increase that of ACE2,Ang-(1-7),Mas and E-cad in MLE-12 cells.However,compared with the Ang-(1-7)treatment group,the use of MLN-4760 and A779 resulted in increasing the cell proliferation and protein expression of ACE,Ang ?,AT1,?-SMA,Vim,Col I and Col ?I,and decreasing the protein expression of ACE2,Ang-(1-7),Mas and E-cad in MLE-12 cells.4 silicosis in mice can be alleviated through knockdown of the ACE gene and aggravated by knockdown of the ACE2 gene.5 We observed the following effects of Ac-SDKP:1)Compared with the model group,Ac-SDKP could reduce the number and area of nodules and mitigate the positive expression of?-SMA and its co-expression with SP-D in the lung tissue.Besides,Ac-SDKP also reduced the protein expression of ACE,Ang ?,AT1,?-SMA,Vim,Col I and Col ?I and increased that of ACE2,Ang-(1-7),Mas and E-cad.It also decreased the expression of ACE m RNA and increased that of ACE2 m RNA in the lung tissue of silicotic mice.Additionally,Ac-SDKP reduced the elevated Tvb,Mvb,Ti,PIF,PEF and EF50 indices induced by silica.2)Compared with the Ang ? group,Ac-SDKP could reduce the cell proliferation,decrease the protein expression of ACE,Ang ?,AT1,?-SMA,Vim,Col I and Col ?I and increase that of ACE2,Ang-(1-7),Mas and E-cad in MLE-12 cells.6 Ac-SDKP could further reduce the degree of EMT and silicotic fibrosis in ACE+/-mice,showing an incremental regulatory effect,while low expression or silencing of the ACE2gene can exacerbate Si O2-induced silicotic fibrosis in mice and the synthesis of collagen and EMT in MLE-12 cells induced by Ang ?,which partially offsets the inhibitory effect of Ac-SDKP on silicosis.Conclusions 1 RAS plays an important role in the occurrence and development of silicosis in mice.Blocking the ACE-Ang ?-AT1R axis inhibits EMT and reduces silicotic fibrosis,while blocking the ACE2-Ang-(1-7)-Mas R axis promotes EMT and aggravates silicotic fibrosis in mice.2 Silicotic fibrosis in mice can be alleviated by the low expression of the ACE gene and aggravated by the low expression of the ACE2 gene.3 Ac-SDKP inhibits EMT and the synthesis of collagen by regulating RAS in the lung tissue of silicotic mice,thereby exerting its antagonistic effect on silicotic fibrosis.Figure 84;Table 4;Reference 290... |
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