Mir-21 Mediates the Inhibitory Effect of Ang (1–7) on AngII-induced NLRP3 Inflammasome Activation by Targeting Spry1 in lung fibroblasts

MicroRNA-21 (mir-21) induced by angiotensin II (AngII) plays a vital role in the development of pulmonary fibrosis, and the NLRP3 inflammasome is known to be involved in fibrogenesis. However, whether there is a link between mir-21 and the NLRP3 inflammasome in pulmonary fibrosis is unknown. Angiotensin-converting enzyme 2/angiotensin(1–7) [ACE2/Ang(1–7)] has been shown to attenuate AngII-induced pulmonary fibrosis, but it is not clear whether ACE2/Ang(1–7) protects against pulmonary fibrosis by inhibiting AngII-induced mir-21 expression. This study’s aim was to investigate whether mir-21 activates the NLRP3 inflammasome and mediates the different effects of AngII and ACE2/Ang(1–7) on lung fibroblast apoptosis and collagen synthesis. In vivo, AngII exacerbated bleomycin (BLM)-induced lung fibrosis in rats, and elevated mir-21 and the NLRP3 inflammasome. In contrast, ACE2/Ang(1–7) attenuated BLM-induced lung fibrosis, and decreased mir-21 and the NLRP3 inflammasome. In vitro, AngII activated the NLRP3 inflammasome by up-regulating mir-21, and ACE2/Ang(1–7) inhibited NLRP3 inflammasome activation by down-regulating AngII-induced mir-21. Over-expression of mir-21 activated the NLRP3 inflammasome via the ERK/NF-κB pathway by targeting Spry1, resulting in apoptosis resistance and collagen synthesis in lung fibroblasts. These results indicate that mir-21 mediates the inhibitory effect of ACE2/Ang(1–7) on AngII-induced activation of the NLRP3 inflammasome by targeting Spry1 in lung fibroblasts.

IL-1R-deficient mice are protected from thioacetamide (TAA)-induced fibrogenesis. Therefore, the NLRP3 inflammasome/IL-1β secretion axis is a promising therapeutic target for the prevention and treatment of lung fibrosis.
The NLRP3 inflammasome can be activated by diverse stimuli and a 2-signal model has been proposed for NLRP3 inflammasome activation. Signal 1 is provided by microbial molecules or endogenous cytokines, in which activation of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) leads to up-regulation of NLRP3 and pro-IL-1β 16 . Ling et al. 17 and Ning et al. 18 have confirmed that mir-21 activates ERK/NF-κB by promoting degradation of the target gene Spry1. Hence, it can be inferred that mir-21 activates the NLRP3 inflammasome via the ERK/NF-κB pathway by targeting Spry1 in lung fibroblasts.
In this study, we investigated whether ACE2/Ang(1-7) antagonizes AngII-mediated pathophysiological activation of processes that lead to pulmonary fibrosis both in vitro and in vivo. We demonstrated that AngII-induced mir-21 activated the NLRP3 inflammasome in lung fibroblasts by targeting Spry1, resulting in apoptosis resistance and collagen synthesis. In contrast, ACE2/Ang(1-7) prevented AngII-induced collagen synthesis in lung fibroblasts and BLM-induced pulmonary fibrosis by inhibiting the expression of mir-21.
AngII-up-regulated mir-21 led to apoptosis resistance and collagen synthesis in lung fibroblasts. It is now becoming apparent that AngII is involved in lung fibrogenesis 23 . However, the precise mechanism of this effect needs to be further explored. In 2012, Adam et al. 24 reported that AngII up-regulates the expression of mir-21 in cardiac fibroblasts. To test whether AngII promotes mir-21 expression in lung fibroblasts, the cells were treated with AngII (10 −7 mmol/L) at different time points, and the expression of mir-21 was determined. As shown in Fig. 2A, the mir-21 level increased significantly in a time-dependent manner after treatment with AngII. Consistent with our previous findings, AngII treatment inhibited apoptosis and promoted a-collagen I synthesis in lung fibroblasts (Fig. 2E,F). Furthermore, protein levels of ERK/NF-κB and the NLRP3 inflammasome were increased, but Spry1 was decreased after AngII treatment (Fig. 2C,E). We therefore explored whether the effects of AngII are mediated by mir-21. Mir-21 antisense probes were used to reduce mir-21 expression level in AngII-treated cells (Fig. 2B). AngII-induced changes of protein levels of the Spry1/ERK/NF-κB/NLRP3 inflammasome pathway and a-collagen I were restored by mir-21 antisense probes (Fig. 2C,E). Similarly, knocking down mir-21 inhibited the effect on apoptosis resistance of AngII (Fig. 2F). Immunofluorescent staining of NLRP3 and caspase-1 produced the same result (Fig. 2D). Thus, mir-21 mediated AngII-induced apoptosis resistance and a-collagen I synthesis in lung fibroblasts.
Mir-21 mediated the contravariant response of ACE2/Ang(1-7) and AngII in BLM-induced lung fibrosis. Compared with control rats, BLM treatment alone was associated with higher Ashcroft scores, higher levels of hydroxyproline, and severe fibrosis with marked mononuclear infiltration and thickened alveolar septa throughout the lung parenchyma. Infusion of Ang(1-7) or over-expression of ACE2 was associated with markedly lower scores and a reduced degree of fibrotic lesions. However, the pro-fibrotic effect was more evident in AngII-treated rats (Fig. 5A,B,C). These data suggest that AngII exacerbated and ACE2/Ang(1-7) attenuated BLM-induced pulmonary fibrosis in rats.
We then investigated the role of mir-21 in this rat model. The mir-21 level was markedly enhanced in the BLM group compared with the control group. Although the mir-21 level was markedly reduced by infusion of Ang(1-7) or over-expression of ACE2, it was augmented by Ang II treatment (Fig. 5D,E). Moreover, protein levels of ERK/NF-κB/NLRP3 and collagen were increased, but the protein level of Spry1 was decreased in BLM-induced pulmonary fibrosis. This response was abolished by ACE2 or Ang(1-7) treatment and exacerbated by Ang II (Fig. 5F,G,H). Similarly, ACE2 or Ang(1-7) reduced the a-collagen I protein levels induced by BLM, which were aggravated by AngII treatment (Fig. 5F). Together with the in vitro results, we concluded that ACE2/ Ang(1-7) attenuated and AngII exacerbated BLM-induced lung fibrosis by modulation of mir-21.
Mir-21 is recognized as an oncomiR due to its activity on cellar proliferation, differentiation, and apoptosis. It has been extensively researched and serves as a potential biomarker for diagnosing cancer and determining its prognosis 27 . Increasing additional roles [28][29][30] in other diseases suggest that elevated mir-21 expression may represent a common feature of pathological progression, including fibrotic diseases. It has been reported that mir-21 levels are up-regulated in fibrosis of the kidneys 31 , heart 32 , liver 9 , and lungs 7 , and that targeting mir-21 therapeutically may have the ability to prevent development of organ fibrosis 4 . Furthermore, recent studies have shown that mir-21 is involved in the promotion of fibrogenic activation of fibroblasts in different organs 2 . Consistent with these results, we found that mir-21 l was markedly up-regulated in the lung of BLM-treated rats in vivo, and that over-expression of mir-21 inhibited lung fibroblast apoptosis and promoted collagen synthesis in vitro. As it has also been shown that the pro-fibrotic factors transforming growth factor (TGF)-β 33 and platelet-derived growth factor (PDGF) 4 promote fibrogenesis by up-regulating mir-21, we investigated whether other pro-fibrotic factors can activate mir-21. AngII, the principal effector peptide of the vasoconstrictor arm of the renin-angiotensin system (RAS), has a key role in the initiation and perpetuation of inflammation and fibrosis in experimental lung fibrosis 34 , and it is now becoming apparent that mir-21 is transcriptionally activated in cardiac fibrosis related to AngII 32 . Adam et al. 24 have also reported that AngII up-regulates the expression of mir-21 in cardiac fibroblasts, resulting in their activation and atrial fibrosis. Thus, we propose that the pro-fibrotic effect of AngII may be associated with its augmentation of mir-21. Consistent with previous findings, we found that mir-21 levels were significantly increased in lung fibroblasts treated with AngII. Moreover, the effects of AngII in inhibiting apoptosis and promoting collagen synthesis can be reversed by silencing mir-21 in lung fibroblasts. In BLM-treated rats, constant infusion of exogenous AngII significantly up-regulated the expression of mir-21 and aggravated lung fibrosis. Hence, AngII-induced mir-21 promoted collagen synthesis in lung fibroblasts and exacerbated BLM-induced lung fibrosis.
Although the pro-fibrotic effect of mir-21 is clear, the precise molecular mechanism by which it exerts this effect needs to be elucidated. A number of signaling pathways have been identified as being involved in the fibrogenensis mediated by mir-21 in different organs, including PTEN/Akt, NF-κB, ERK, TGF-β1/Smad, and IL-13/Smad signaling pathways 35 . Ning et al. 18 discovered that Spry1, an important target gene of mir-21, can inhibit the ERK/NF-κB pathway, which is involved in liver fibrosis 18 . In this study, we have shown that mir-21  over-expression or AngII-induced mir-21 significantly increased p-ERK and NF-κB nucleoprotein levels, but decreased the Spry1 protein level in lung fibroblasts. Moreover, mir-21 silencing inhibited the Spry1/ERK/ NF-κB pathway activation. Collectively, we demonstrated that AngII-induced mir-21 mediated ERK/NF-κB pathway activation by targeting Spry1 in lung fibroblasts. NF-κB is a nuclear transcription factor that regulates the expression of a large number of genes, including the NLRP3 inflammasome.
Recent studies have elucidated the important role of the NLRP3 inflammasome in the development of fibrosis in various organs, including the heart 36 , kidneys 37 , liver 10 , and lungs 38 . Artlett & Thacker 39 consider that activation of the NLRP3 inflammasome is a common thread linking divergent fibrogenic diseases. Furthemore, our previous studies have demonstrated NLRP3 inflammasome mediated AngII-induced pulmonary fibrosis in animal models. In accordance with these results, we found the NLRP3 inflammasome/IL-1β secretion axis was activated by AngII or mir-21 over-expression in lung fibroblasts, and that this can be reversed by anti-mir-21 probes. Thus, mir-21 has a potential role in activation of the NLRP3 inflammasome/IL-1β secretion axis. However, how mir-21 exerts this effect remains to be elucidated.
The ACE2/Ang(1-7)/Mas pathway is involved in many physiological and pathophysiological processes in several systems and organs, notably by opposing the detrimental effects of inappropriate over-activation of the ACE/ AngII/AT1R axis 40 . Li et al. 41 consider that Ang(1-7) protects against experimental lung fibrosis by limiting the local tissue accumulation of AngII that occurs in response to BLM-induced lung injury, which is consistent with our previous studies. However, the precise molecular mechanism of the anti-fibrotic effect of ACE2/Ang(1-7) remains to be elucidated. We found that the effect of AngII-induced mir-21 was abolished by ACE2/Ang(1-7) in lung fibroblasts in vitro, and that exogenous Ang(1-7) infusion or lentiACE2 intratracheal instillation significantly decreased mir-21 expression and attenuated lung fibrosis induced by BLM in vivo. Furthermore, in primary lung fibroblasts, ACE2/Ang(1-7) suppressed AngII-induced ERK phosphorylation, NF-κB nuclei translocation, and degradation of the targeting gene Spry1, and blocked AngII-induced activation of the NLRP3 inflammasome. In exploring whether these responses of ACE2/Ang1-7 were mediated by mir-21, we found that activation of the Spry1/ERK/ NF-κB pathway and the NLRP3 inflammasome/IL-1β secretion axis by over-expression of mir-21 could not be reversed by ACE2/ Ang(1-7). Overall, these data suggest that mir-21 mediates the inhibitory effect of ACE2/ Ang (1-7) on AngII-induced activation of the NLRP3 inflammasome by targeting Spry1 in lung fibroblasts. Figure 6. Schematic working hypothesis. It has been demonstrated local RAAS is activated in lung tissue of fibrosis. AngII binds with AT1R in lung fibroblasts to enhance the expression level of mir-21, the recognized pro-fibrotic factor. Mir-21 promotes ERK phosphorylation and NF-κB nuclei translocation by targeting Spry1 degradation, following NLRP3 inflammasome activation. The released proinflammatory factors (Caspase-1/ IL-1) by the inflammasome pathway lead to a-colllagen I synthesis and apoptosis resisitance. ACE2 degrades AngII to produce Ang(1-7), which reverses the AngII's profibrotic effect via downregulating mir-21. As a result, mir-21 and NLRP3 inflammasome play a key in local RAAS imbalance induced lung fibrosis.
SCieNtifiC RepoRts | 7: 14369 | DOI:10.1038/s41598-017-13305-3 Excessive accumulation of mesenchymal cells, especially fibroblasts, is a feature of pulmonary fibrosis, and fibroblasts from fibrotic lungs are resistant to a variety of apoptotic stimuli 42 . Meng et al. 43 found that AngII activates MAPK/NF-κB and promotes apoptosis resistance, resulting in accumulation of fibroblasts and augmented pulmonary fibrosis. Similarly, mir-21 has been shown to be involved in apoptosis resistance in a variety of disease states 29,30,44,45 . In accordance with these results, we found that AngII-induced mir-21 inhibited fibroblast apoptosis by the Spry1/ERK/ NF-κB pathway by targeting Spry1 degradation. Furthermore, ACE2/Ang(1-7) reversed the anti-apoptosis effect of AngII by down-regulating mir-21. However, the mechanism of this anti-apoptotic effect needs to be further elucidated.
In conclusion, our study has demonstrated that AngII induction of mir-21 is a crucial factor that mediates pulmonary fibrosis by activating the NLRP3 inflammasome/IL-1β secretion axis via the Spry1/ERK/NF-κB pathway. Exogenous ACE2/Ang(1-7) over-expression protected against BLM-induced pulmonary fibrosis by down-regulating mir-21. These findings suggest a critical role of mir-21 in both the pro-fibrotic effect of AngII and the anti-fibrotic effect of ACE2/ Ang(1-7) in BLM-induced pulmonary fibrosis. Consequently, down-regulation of mir-21 may be a promising strategy for the prevention and treatment of pulmonary fibrosis.

Animals. Male
Production of lentiACE2 viral particles. Lentiviral particles containing enhanced green fluorescent protein (pGC-FU-GFP, lenti-GFP/lentiNC) or human ACE2 (pGC-FU-ACE2-GFP, lentiACE2) were prepared. Viral medium containing lenti-GFP or lentiACE2 was then collected, concentrated and titered. The concentration of viral particles was determined using quantitative real-time polymerase chain reaction (qRT-PCR) technology. The efficacy of lentiACE2 in producing active ACE2 enzymes has been previously established.
Histological analysis and immunochemical assessment. The right lung was fixed using an intratracheal instillation of 4% paraformaldehyde, embedded in paraffin, and cut into 5 μm thick sections. Hematoxylin and eosin (H&E) staining was used to identify alveolitis and fibrosis. The severity of pathological changes was scored according to the Ashcroft scale, and the presence of collagen was assessed by analyzing the stained area as a percentage of the total area.
For immunohistochemical (IHC) analysis, sections were stained with anti-NF-κB and anti-NLRP3 antibodies. After incubation with streptavidin peroxidase-conjugated secondary antibody, peroxidase conjugates were visualized with diaminobenzidine and observed under a light microscope.
Hydroxyproline assay. The lung tissue was hydrolyzed, followed by derivation using the HYP analysis kit (Sigma). The collagen content was measured and analyzed by ultraviolet spectrophotometry, using the procedure recommended by the manufacturer.
In situ hybridization (ISH). Lung sections were treated with an acetylation solution and proteinase K.
The sections were then blocked with hybridization solution and incubated with digoxigenin-conjugated mir-21 probes (Exiqon, Denmark). Subsequently, the sections were incubated with the HRP-conjugated anti-digoxigenin antibody (Roche, Shanghai, China). Finally, the sections were treated with NBT/BCIP (Roche, Shanghai, China). Light blue cytoplasmic staining indicates a positive test.
Western blot analysis. Lung tissue was lysed with radioimmunoprecipitation assay (RIPA) buffer (Beyotime, China) to extract the total protein. A bicinchoninic acid (BCA) protein assay was used to determine the protein concentration. The lysates were mixed with sodium dodecyl sulfate (SDS) buffer and denatured at 100 °C for 5 min. Equal concentrations of proteins were then separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were probed with primary antibodies at 4 °C overnight, including anti-collagen I, anti-Spry1, anti-p-ERK1/2, anti-ERK1/2, anti-NF-κB, anti-β-actin, and anti-histone (1:1000; Cell Signaling Technology, Massachusetts, USA), and then incubated with anti-rabbit near-infrared secondary antibody (1:15000, Li-Cor, USA) for 1 h. The membrane was exposed to Odyssey ® CLx Imager, and Odyssey Software was used for capturing images and the data analysis. The experiment was repeated 3 times for consistency.
Quantitative real-time PCR analysis (qRT-PCR). The total RNA of cultured cells and lung tissue was isolated by using Trizol reagent (Invitrogen, Life Technologies, NY, USA). The All-in-One mir-21q-RT-PCR kit (GeneCopoeia, Guangzhou, China) was used according to the manufacturer's instructions. qRT-PCR was performed using the ABI 7500 Real-Time PCR System (Applied Biosystems). The universal primer of mir-21, UAGCUUAUCAGACUGAUGUUGA, was used during reverse transcription, and the results were normalized to U6. Flow cytometry. Cell apoptosis was examined with flow cytometry analysis. Briefly, the cells were collected and washed twice with phosphate-buffered saline (PBS), fixed in 2% paraformaldehyde for 30 minutes, and permeabilized using 0.1% Triton-X for 30 minutes. They were then stained with FITC-conjugated annexin V and propidium iodide (PI) using the Apoptosis Detection Kit (Invitrogen, USA) according to the manufacturer's instructions.
The apoptotic rate was measured with flow cytometry (FACS Calibur ™ , BD Biosciences, Franklin Lanes, NJ, USA), and the data were analyzed using CellQuest software. Each experiment was performed in triplicate.
Statistical analysis. All data were presented as means ± standard deviation (SD). Significant differences were evaluated by analysis of variance (ANOVA) with the least significant difference (LSD) for multiple comparisons. Differences were considered statistically significant at a P-value of <0.05. All data were analyzed using SPSS ® software, version 13.0 (SPSS Inc, Chicago, IL, USA).
Date availability. All data generated or analysed during this study are included in this published article.