The Effects And Mechanisms Of Angâ…¡ And Ang(1-7) On Lung Fibrosis Via Regulating The NOX4/NALP3 Inflammasome Signal Pathway

Background and objectivePulmonary fibrosis, characterized by diffuse alveolar inflammation, thickened alveolar wall, and excessive accumulation of extracellular matrix is a serious and progressive chronic interstitial lung disease with currently limited therapeutic options available. The main histological characteristic of pulmonary fibrosis is persistent injury of alveolar epithelial cells, hyperplasia and accumulation of fibroblast and myofibroblast, and excessive accumulation of extracellular matrix (ECM), the exact mechanism of which is still not well understood. Persistent and chronic injury may activate fibroblast, which secretes larger amounts of cytokines, chemokines and ECM leading to progression of pulmonary fibrosis. Therefore, to study how fibroblast secreting ECM and how to block this secretion may be of great importance to delay the development of pulmonary fibrosis.Renin Angiotensin System (RAS) participate in the development of pulmonary fibrosis. Angiotensin Ⅱ (AngⅡ), the main effector molecules in RAS, accelerates the deposition of ECM, promotes hyperplasia and migration of fibroblast via binding with AngⅡ type Ⅰ receptor (AT1R). Angiotensin converting enzyme 2 (ACE2)/ Angiotensin(1-7) (Ang(1-7))/Mas receptor axis is a newly discovered components in RAS, which may counterbalance the ACE/AngⅡ/AT1R axis while binding with Mas receptor.In recent years, studies found that oxidative stress took part in the development of pulmonary fibrosis. When human body suffer various noxious stimuli, leading to oxidative imbalance, and excessive production of reactive oxygen species (ROS) beyond the body’s removal ability, will result to cell dysfunction and tissue damage, this call oxidative stress. Study has proved that a significant inverse correlation between lung function and oxidative stress level in pulmonary fibrosis patients. Besides, lipid peroxide and hydrogen peroxide (H2O2) in bronchoalveolar lavage fluid of idiopathic pulmonary fibrosis (IPF) patients were significantly higher than that of normal controls, indicating that oxidative imbalance might have a important role in the progression of pulmonary fibrosis. Recent reports have shown that Ang Ⅱ could activate NADPH oxidase (NOX), induce abundant ROS, leading to diffuse vascular inflammation and fibrogenesis. Thus, we suppose that Ang Ⅱ may cause pulmonary fibrosis by activating NOX4. Study the target spot of Ang Ⅱ in regulating lung fibrosis, and block its way to work, may provide new theoretical basis for the treatment of pulmonary fibrosis.Recently, the NALP3 inflammasome activation is identified as a novel mechanism in lung fibrosis. The NALP3 inflammasome, a multiprotein complex, consists of NALP3, apoptosis-associated speck-like protein containing a CARD (ASC), and pro-caspase-1. This multiprotein complex acts to mediate the cleavage and activation of caspase-1, resulting in the maturation and secretion of inflammatory cytokines such as IL-1β and IL-18 after stimulating by ROS and pathogen, etc. Recent studies have shown that NALP3-deficient mice significantly alleviated the inflammation and collagen deposition induced by silica, while the collagen deposition in NALP3 overexpression mice was more apparent comparing with the wild type mice. All these indicated that the NALP3 inflammasome play a role in the pathogenesis of lung fibrosis. However, losartan, AT1 receptor blocker, could inhibit the secretion of IL1β and the mRNA expression of NALP3 and caspasel induced by LPS. Therefore, we suppose that AngⅡ might activate the NALP3 inflammasome via binding with AT1R.Our group’s precedent experiment has proved that Ang Ⅱ promoted the synthesis of collagen, aggravated pulmonary fibrosis induced by bleomycin (BLM) via activating the NOX4/ROS/Rock2 signal pathway. Ang(1-7) attenuated pulmonary fibrosis induced by BLM via inhibiting the NOX4/ROS/Rock2 signal pathway. However, the effect of Ang Ⅱ and Ang(1-7) on the NALP3 inflammasome is still unclear, whether Ang Ⅱ could activate the NALP3 inflammasome via inducing NOX4/ROS, and so thus to promote he synthesis of collagen, whether Ang(1-7) could attenuated pulmonary fibrosis induced by BLM via inhibiting the NOX4/ROS/NALP3 inflammasome signal pathway, all these questions remain further elucidate in this study.Method1、In vitro experiment(1) In vitro, the primary lung fibroblast was separated by attachment culture and authenticated by vimentin immunofluorescent staining. (2) Primary lung fibroblasts were treated with 10"7mol/L Ang Ⅱ for 0h、12h、 24h、36h、48h, and intracellular ROS production was measured. (3) Primary lung fibroblasts were treated with 10-7mol/L Ang Ⅱ for 0h、12h、 24h、36h、48h, and intracellular H2O2 production was measured. (4) Primary lung fibroblasts were treated with different concentrations (10-9、 10-7、10-5mol/1) of Ang Ⅱ for 24h, and intracellular caspasel activity was detected。(5) Primary lung fibroblasts were treated with different concentrations (10-9、 10-7、10-5 mol/l) of Ang Ⅱ for 24h, and supernatants were harvested to measure IL1β by enzyme-linked immunosorbent assay (Elisa)(6) Immunofluorescent staining for NALP3+ caspase1、 ASC+ NALP3 was observed in primary lung fibroblasts.(7) Western blot analysis of NOX4、α-collagen Ⅰ、NALP3、pro-caspase1、 caspase1 p10、IL1β、cleave IL1β in lysate of primary lung fibroblasts was detected.2、In vivo experimentIn vivo, to determine the different effect of Ang(1-7) and Ang Ⅱ on pulmonary fibrosis induced by BLM, the animal model was established as following:(1) 48 male wistar rats were randomly divided into four groups of twelve rats each, including a control group, a BLM treatment group, a BLM+ Ang Ⅱ treatment group and a BLM +Ang (1-7) treatment group. All rats were hocus by intraperitoneal injection of sodium pentobarbital (4mg/100g), and then the BLM treatment group, BLM+Ang Ⅱ treatment group and BLM+Ang (1-7) treatment group received 200μl of sterile saline that contained 5 mg/kg of BLM sulfate via intratracheal instillation, respectively. The control group received 200μl of sterile saline that without BLM sulfate via intratracheal instillation. At the same time, the BLM+ Ang Ⅱ treatment group and BLM+ Ang (1-7) treatment group implanted micro-osmotic pumps subcutaneously which allowing 28 days of continuous infusion with Ang Ⅱ (25ug/kg-1·h-1) or Ang(1-7) (25ug/kg-1·h-1).28 days later, right lung tissues were collected, and Immunofluorescent staining for NOX4+ α-SMA, NALP3+α-SMA was observed, western blot analysis of NOX4、cc-collagen Ⅰ、NALP3、pro-caspase1、 caspase1 p10、IL1β、cleave IL1β in pulmonary tissue homogenate was detected.3、Statistical analysisAll of the data are represented as the means±standard deviation. Significant differences were evaluated by ANOVA followed by testing the homogeneity of variances using Levene method. When the variance result is homogeneous, multiple-comparison testing (with Least-significant difference tests) was performed. When the variance result is not homogeneous, Dunnett’s T3 was performed. P values less than 0.05 were considered statistically significant. All of the data were analyzed using SPSS 13.0(?).Result1、Morphological and fluorescent identification of rat primary lung fibroblasts.Lung tissue adherent 2-4 days, long spindle cells gradually climbing out from the organization could be observed by light microscope. The primary lung fibroblasts’ form a star or fusiform, cytoplasmic shining brilliantly, nuclear round or oval, with obvious nucleolus, compact connection between cells, and smooth edges. Immunofluorescent staining for primary lung fibroblasts was observed. All primary lung fibroblasts expressed vimentin, unique landmark protein for fibroblast.2、Ang Ⅱ induced the synthesis of collagen in primary lung fibroblasts.Primary lung fibroblasts were treated with 10-7mol/L Ang Ⅱ for 0h、12h、24h、 36h、48h, and the expression of CTGF and α-collagen Ⅰ were detected. Ang Ⅱ could obviously promote the production of CTGF and α-collagen Ⅰ (both P<0.05), the time to reach protein content peak was 36 h.3、The oxidation reaction was induced by Ang Ⅱ.Primary lung fibroblasts were treated with 10-7mol/L Ang Ⅱ for Oh、12h、24h、 36h、48h, and intracellular ROS production, H2O2 level and NOX4 expression were measured. Measurement of intracellular ROS shown that in response to Ang Ⅱ, ROS was markedly raise at 12 h, and to the peak of ROS was at 36 h (both P<0.05). Measurement of intracellular ROS shown that in response to Ang Ⅱ, H2O2 was markedly raise at 12 h, and to the peak of ROS was at 36 h (both P<0.05). In Western blotting, the protein expression of NOX4 was remarkable increased comparing with the controls at the time of 24 and 36 h (both P<0.05).4、AngⅡ-activated NOX4 induced the synthesis of collagen in primary lung fibroblasts.In Western blotting, the protein expression of NOX4 was significantly enhanced after treatment with Ang Ⅱ (P<0.05), while pretreated with NAC (10-3mol/L) or DPI (10-5mol/L) for 1 hour before exposure to Ang Ⅱ (10"7 mol/L) could significantly inhibit the expression of NOX4, as well as a-collagen Ⅰ (both P<0.05). In addition, transfected with siRNA targeting NOX4 (NOX4 siRNA) for 48 hour to inhibit the translation of NOX4, the protein level of a-collagen Ⅰ was significantly reduced in comparison with the NT siRNA+Ang Ⅱ group (P<0.05).5、NALP3 inflammasome was activated by Ang Ⅱ and was co-localized with mitochondria.Immunofluorescent staining shown that after treatment with Ang Ⅱ for 24 h, the fluorescence intensity of NALP3, Caspasel, and ASC increased obviously, meanwhile, the co-localization of NALP3+ caspasel, ASC+ NALP3 was more obviously. Moreover, the co-localization of NALP3+ caspasel, ASC+ NALP3 with mitochondria (blue fluorescence) was also more obviously, indicating that mitochondria might take part in the activation of the NALP3 inflammasome. In Western blotting, Ang Ⅱ could significantly increase the protein level of NALP3, pro-caspasel, caspase1 p10, IL1β and cleave IL1β (both P<0.05).. In addition, the caspase1 activity in intracellular and the level of IL1β in supernatants apparently increased compared to the control group (both P<0.05).6、Inhibited NALP3 inflammasome activation attenuated Ang Ⅱ-induced α-collagen Ⅰ synthesis in lung fibroblasts.Comparing with the control group, the protein level of α-collagen-Ⅰ, NALP3, pro-caspasel, caspasel p10, IL1β and cleave IL1β were significantly enhanced in the Ang Ⅱ group (both P< 0.05). However, pre-treatment of caspasel inhibitor AC-YVAD-CMK could significantly inhibited the protein level of a-collagen-Ⅰ induced by Ang Ⅱ (both P<0.05). In addition, transfected with siRNA targeting NALP3 (NALP3 siRNA) for 48 hour to inhibit the translation of NALP3, the protein level of α-collagen Ⅰ was significantly reduced in comparison with the NT siRNA +AngⅡ group (P<0.05).7、Ang Ⅱ activated NALP3 inflammation via inducing NOX4. In Western blotting, the protein expression of NALP3, IL1β, and cleave IL1β was significantly enhanced after treatment with Ang Ⅱ (P<0.05), while pretreated with NAC (10-3mol/L) or DPI (10-5mol/L) for 1 hour before exposure to Ang Ⅱ (10-7 mol/L) could significantly inhibit the expression of NALP3, IL1β, and cleave IL1β (both P<0.05). In addition, transfected with siRNA targeting NOX4 (NOX4 siRNA) for 48 hour to inhibit the translation of NOX4, the protein level of caspasel, caspase1 p10, IL1β, and cleave IL1β was significantly reduced in comparison with the NT siRNA+ Ang Ⅱ group (P<0.05).8、Ang(1-7) suppressed Ang Ⅱ-induced α-collagen Ⅰ synthesis by regulating the NOX4/NALP3 inflammasome pathway.Western blotting shown that the expression of α-collagen Ⅰ protein in the Ang Ⅱ +Ang(1-7) group and the Ang Ⅱ+Irbesartan group was remarkable less than the Ang Ⅱ group, however, the Ang Ⅱ+Ang(l-7)+ A779 group was markedly enhanced than the Ang Ⅱ+Ang(1-7) group (both P<0.05). Similarly, both the Ang Ⅱ+Ang(1-7) group and the Ang Ⅱ+Irbesartan group could significantly decrease the protein expression of NOX4, NALP3, and cleave IL1β compared to the AngⅡ group (both P<0.05). And pre-treatment with the Mas receptor blocker A779 could abolish these effect, showing an increase of NOX4, NALP3, and cleave IL1β expression, in comparison with the AngⅡ+Ang(1-7) group (both P<0.05) However, unlike the Ang Ⅱ+Ang(1-7) group, Ang(1-7) alone may also promote the expression of α-collagen I, NOX4, NALP3, and cleave IL1β protein.9、The different effect of Ang(1-7) and Ang Ⅱ on BLM induced lung fibrosis.9.1 The NOX4 expression in BLM-induced lung fibrosis in rats was differently affected by Ang(1-7) and Ang Ⅱ.Immunofluorescent staining shown that after treatment with BLM, the fluorescence intensity of NOX4 increased obviously compared with the control group, meanwhile, the co-localization of NOX4 and αSMA was more obviously. Moreover, in the BLM+ Ang Ⅱ group, the co-localization of NOX4 and aSMA was more obviously than the BLM group. However, in the BLM+Ang(1-7) group, the degree of fibrosis had a great improvement, and the fluorescence intensity of NOX4 was more lower than the BLM group, with less co-localization with aSMA.In Western blotting, the BLM group could significantly increase the protein level of NOX4 and α-collagen Ⅰ (both P<0.05). In addition, the protein level of NOX4 and α-collagen Ⅰ in the BLM+ Ang Ⅱ group had a more obviously increase, while that in the BLM+ Ang(1-7) group had a significantly decrease in comparison with the BLM group (both P<0.05).9.2 The NALP3 inflammasome in BLM-induced lung fibrosis in rats was differently affected by Ang(1-7) and Ang Ⅱ.Immunofluorescent staining shown that after treatment with BLM, the fluorescence intensity of NALP3 increased obviously compared with the control group, meanwhile, the co-localization of NALP3 and aSMA was more obviously. Moreover, in the BLM+ Ang II group, the co-localization of NALP3 and aSMA was more obviously than the BLM group. However, in the BLM+ Ang(1-7) group, the degree of fibrosis had a great improvement, and the fluorescence intensity of NALP3 was more lower than the BLM group, with less co-localization with aSMA.In Western blotting, the BLM group could significantly increase the protein level of NALP3, caspase1 p10 and cleave IL1β (both P<0.05). In addition, the protein level of NALP3, caspase1 p10 and cleave IL1β in the BLM+AngⅡ group had a more obviously increase, while that in the BLM+ Ang(1-7) group had a significantly decrease in comparison with the BLM group (both P<0.05).Conclusion:1. AngⅡ promoted the α-collagen Ⅰ synthesis in primary lung fibroblasts via activating the NOX4/NALP3 inflammasome signal pathway.2. Irbesartan and Ang(1-7) decreased the synthesis of α-collagen Ⅰ in primary lung fibroblast via inhibiting the activation of NOX4/NALP3 inflammasome signal pathway induced by Ang Ⅱ.3. Ang(1-7) alone may also promote the NOX4/NALP3 inflammasome signal pathway.4. Ang Ⅱ aggravated BLM-induced pulmonary fibrosis by activating the NOX4/NALP3 inflammasome signal pathway, while Ang(1-7) protected against BLM-induced pulmonary fibrosis by inhibiting the NOX4/NALP3 inflammasome signal pathway.