Angiotensin-converting enzyme 2 prevents lipopolysaccharide-induced rat acute lung injury via suppressing the ERK1/2 and NF-κB signaling pathways

Acute respiratory distress syndrome (ARDS) caused by severe sepsis remains a major challenge in intensive care medicine. ACE2 has been shown to protect against lung injury. However, the mechanisms of its protective effects on ARDS are largely unknown. Here, we report that ACE2 prevents LPS-induced ARDS by inhibiting MAPKs and NF-κB signaling pathway. Lentiviral packaged Ace2 cDNA or Ace2 shRNA was intratracheally administrated into the lungs of male SD rats. Two weeks after gene transfer, animals received LPS (7.5 mg/Kg) injection alone or in combination with Mas receptor antagonist A779 (10 μg/Kg) or ACE2 inhibitor MLN-4760 (1 mg/Kg) pretreatment. LPS-induced lung injury and inflammatory response were significantly prevented by ACE2 overexpression and deteriorated by Ace2 shRNA. A779 or MLN-4760 pretreatment abolished the protective effects of ACE2. Moreover, overexpression of ACE2 significantly reduced the Ang II/Ang-(1-7) ratio in BALF and up-regulated Mas mRNA expression in lung, which was reversed by A779. Importantly, the blockade of ACE2 on LPS-induced phosphorylation of ERK1/2, p38 and p50/p65 was also abolished by A779. Whereas, only the ERK1/2 inhibitor significantly attenuated lung injury in ACE2 overexpressing rats pretreated with A779. Our observation suggests that AEC2 attenuates LPS-induced ARDS via the Ang-(1-7)/Mas pathway by inhibiting ERK/NF-κB activation.

PVDF membranes. Viral titer was evaluated by gradient dilution. The packaged lentiviruses were designated as Lv-ACE2 and Lv-ACE2-RNAi.

Lentiviral transduction. For delivery of the Lenti-Ace2 (recombinant lentivirus carrying Ace2 cDNA) and
Lenti-Ace2-RNAi (recombinant lentivirus carrying Ace2 shRNA) into the lungs, rats underwent a midline incision to expose the trachea under isoflurane anesthesia. Briefly, anesthetized rats were placed in a supine position on an inclined platform (approximately 45°) and then the trachea was exposed surgically on the ventral side of neck. A 26# needle was inserted through the tracheal wall into the lumen just blow the larynx. Empty virus (control), the Lenti-Ace2, or Lenti-Ace2-RNAi viral particles (1 × 10 8 if u/μ l in 100 μ l of phosphate-buffered saline) were directly injected into the trachea followed by 300 μ l of air to enhance the spread of virus in the rat lungs. The same injection was performed 7 days later. One week after the last lentiviral treatment, animals were subjected to LPS administration.
LPS-induced acute lung injury. Acute lung injury (ALI) was induced by single intravenous injection of LPS (7.5 mg/Kg) as previously described 28 . Control rats received 0.9% NaCl solution (500 μ l) through the tail vein. In treatment groups, A779 (10 μ g/Kg) or MLN-4760 (1 mg/Kg) was injected via rat tail vein 30 min before the induction of ALI. All animals were breathing spontaneously during the experimental protocol.
Eight hours after LPS administration, animals were anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/Kg) and euthanized by exsanguination. Serum sample were collected and stored at − 80 °C. Broncho-alveolar lavage fluid (BALF) was collected from three lavage samples from the left lung with aliquots of 3 ml normal saline. Greater than 90% recovery of the saline was achieved. The retrieved BALF was pooled and centrifuged (300 g at 4 °C for 10 min). The supernatant was stored in aliquots at − 80 °C. The right lung was fixed immediately in 4% paraformaldehyde.
To further investigate the role of MAPKs pathways in development of ARDS and the effects of ACE2 overexpression, MAPKs specific inhibitors (SB203580, PD98059 and SP600125, 10 mg/Kg) were pretreated intraperitoneally at the time 10 min before LPS administration.
Histopathology. The embedded lung tissues were cut into 4 μ m-thick sections and stained with hematoxylin-eosin for microscopic observation. The degree of lung injury was semi-quantitatively scored as described by Murakami and colleagues 29 . Briefly, the lung injury score, including edema, inflammation, and hemorrhage, which was graded on the following scale: normal (0), light (1), moderate (2), strong (3), and severe (4). Analysis was conducted by a pathologist blinded to the experimental group. The values of each of the three parameters analyzed were added. The final lung injury score was the average score calculated within each experimental group.

Measurement of protein concentration and inflammatory mediators in BALF.
For assessment of lung permeability in rats, the protein concentration in the BAL fluid was measured. Briefly, BALF samples (100 μ l) from the left lung were centrifuged at 4 °C, 1500 g for 5 min, and protein concentration in the supernatant was quantified by BCA protein assay (Pierce, IL, USA).
Statistical analyses. Statistical analyses were performed with the Prism software package (GraphPad v5, San Diego, CA, USA). All values are presented as mean ± standard deviation (SD). Data were analyzed using Scientific RepoRts | 6:27911 | DOI: 10.1038/srep27911 one-way ANOVA and the Newman-Keuls test for multiple comparisons. A P-value less than 0.05 was considered statistically significant.

Results
Lentiviral-mediated gene transfer efficiently increases or down-regulates Ace2 mRNA and ACE2 protein expression in rat lung tissue. Compared with control animals, ACE2 mRNA and protein levels in the lung tissue of the Lv-ACE2 group (rat lung transfected with Lenti-Ace2) were increased by 9.47 and 4.21 fold, respectively; in contrast, the ACE2 mRNA and protein levels in Lenti-Ace2-RNAi infected rat lungs (Lv-ACE2-RNAi group) were decreased by 52% and 80%. There were no significant differences in ACE2 mRNA and protein expression between the control group and Lv-NC group (rat lung transfected with recombinant lentivirus carrying negative control shRNA). These results showed high efficiency of Ace2 or Ace2-shRNA gene transfer into rat lung via intratracheal injection. (Fig. 1A-C) ACE2 prevents rat from LPS-induced acute lung injury. After 8 hours, LPS treatment resulted in acute lung injury as demonstrated by edema, inflammation, and hemorrhage ( Fig. 2A). The lung injury score (Fig. 2B) and BALF protein level (Fig. 2C) of the LPS group were significantly increased as compared with the control group. ACE2 overexpression in rat lung markedly attenuated LPS-induced lung injury and decreased the lung injury score and BALF protein level. In sharp contrast, silencing ACE2 noticeably deteriorated LPS-induced lung injury as evidenced by increased lung injury score and BALF protein level. Furthermore, pretreatment with either A779, a potent and selective antagonist of Ang-(1-7) via competitively binding with its specific Mas receptor, or MLN-4760, the ACE2 inhibitor, significantly inhibited the protective effects of ACE2 on LPS-induced lung injury. The lung injury score and BALF protein level of the LPS+ ACE2+ A779 group were significantly higher than that of LPS+ ACE2 group, suggesting that ACE2 protects rat from LPS-induced acute lung injury, partially via Mas-mediated signaling.

ACE2 attenuates LPS-induced inflammatory response and reverses Ang II/Ang-(1-7) ratio in BALF.
After LPS administration, the levels of both Ang II (Fig. 3A) and Ang-(1-7) (Fig. 3B) in BALF significantly increased as compared with the control group. ACE2 overexpression significantly reduced the BALF Ang II concentration and enhanced Ang-(1-7) level. The ratio of Ang II to Ang-(1-7) markedly dropped from 2.12 in the LPS group to 0.68 in the LPS+ ACE2 group. A779 or MLN-4760 pretreatment in Lv-Ace2 transduced rat both reversed the decrease of Ang II level, and increasing Ang-(1-7) level in BALF. The ratio of Ang II to Ang-(1-7) notably increased to 5.63 and 6.83 in LPS+ ACE2+ A779 group and LPS+ ACE2+ MLN4760 group, respectively. In addition, ACE2 RNAi also significantly increased Ang II level and decreased Ang-(1-7) level, with the ratio of Ang II to Ang-(1-7) increased to 9.48, which was the highest level in all groups.
The TNF-α (Fig. 3C), IL-1β ( Fig. 3D) and IL-6 ( Fig. 3E) concentrations in the BALF of the rats exposed to LPS were significantly higher than that in control group. LPS-induced secretion of TNF-α , IL-1β and IL-6 in BALF were dramatically inhibited by ACE2 overexpression, but further promoted by ACE2 knockdown in rat BALF. Treatment with A779 or MLN-4760 prior to LPS injection resulted in an obvious increase of TNF-α , IL-1β and IL-6 in BALF of rat transduced with Lenti-Ace2. The concentration of TNF-α and IL-1β in BALF of the LPS+ ACE2+ A779 group or the LPS+ ACE2+ MLN-4760 group were similar to that of the LPS group. These results demonstrated that LPS mainly promoted the generation of Ang II and enhanced the cytokines secretion, all of which were abolished by ACE2 overexpression.
ACE2 overexpression in rat lung significantly enhanced LPS-induced IL-10 secretion in BALF, which was abolished by A779 or MLN4760 pretreatment. Interestingly, ACE2 knockdown in rat lung also promoted IL-10 level in BALF after LPS administration. These results suggest that the anti-inflammatory response may be also triggered in lung by LPS exposure to counter the effects of pro-inflammatory cytokines, and ACE2 promotes anti-inflammatory response via Ang-(1-7)/Mas pathway.
ACE2 overexpression reversed LPS-induced ACE2 decrease and enhanced Mas mRNA increase in lung. LPS administration resulted in a significant decrease of ACE2 protein in rat lungs, which was up-regulated by Lenti-Ace2 transduction and attenuated by ACE2-RNAi (Fig. 4). The up-regulated ACE2 protein expression in rat lung by Lenti-Ace2 was suppressed by A779 or MLN-4760 pretreatment before LPS injection. The ACE2 expression in LPS+ ACE2+ MLN-4760 group was similar to that in the LPS group.   -7), which was further up-regulated by ACE2 overexpression and reduced by ACE2 knockdown. Pretreatment with A779 or MLN-4760 both reversed the changes of Ang II and Ang-(1-7) that resulted from ACE2 overexpression. (C-F) The levels of TNF-α , IL-1β , IL-6 and IL-10 were significantly increased after 8 hours of LPS injection. ACE2 overexpression markedly suppressed the TNF-α , IL-1β and IL-6 secretion and increased IL-10 level, which were noticeably abolished by pretreatment with A779 or MLN-4760. The increased levels of TNF-α , IL-1β IL-6 and IL-10 caused by LPS administration were all further promoted by ACE2 knockdown. Data are represented as mean ± SD. * p < 0.05, versus control group; # p < 0.05, versus LPS group; $ p < 0.05, versus ACE2 group (n = 6, per group).
Mas mRNA was up-regulated in lung tissue of rats that were exposed to LPS. ACE2 overexpression caused a significant further increase of Mas mRNA in LPS stimulated rat lung, while ACE2 knockdown markedly suppressed the Mas mRNA expression. Treatment with A779 or MLN-4760 before LPS administration significantly reduced the expression of Mas mRNA in Lenti-Ace2 transduced rat lung. There was no significant difference in Mas mRNA expression between LPS+ ACE2+ MLN-4760 group and control group (Fig. 5).  blot results showed that LPS administration induced the phosphorylation of p38 MAPK (Fig. 6A), ERK1/2 (Fig. 6B) and JNK (Fig. 6C). ACE2 overexpression significantly suppressed the phosphorylation levels of p38 MAPK, ERK1/2 and JNK in lung tissue of rats receiving LPS. The inhibitory effects of ACE2 overexpression on LPS-induced p38 MAPK and ERK1/2 phosphorylation were completely abolished by pretreatment with A779 or MLN-4760. Meanwhile, there was no significant difference in the JNK phosphorylation in LPS+ ACE2+ A779 , which was significantly suppressed by ACE2 overexpression in rat lung. Pretreatment with A779 or MLN-4760 completely abolished the inhibitory effects of ACE2 overexpression on LPS-induced p38 MAPK and ERK1/2 phosphorylation, but did not affect the level of JNK phosphorylation. ACE2 RNAi in rat lung significantly enhanced the LPS-induced ERK1/2 and JNK phosphorylation but did not change p38 MAPK phosphorylation. Data are represented as mean ± SD. * p < 0.05, versus control group; # p < 0.05, versus LPS group; $ p < 0.05, versus ACE2 group (n = 6, per group).
Scientific RepoRts | 6:27911 | DOI: 10.1038/srep27911 or LPS+ ACE2+ MLN-4760 as compared to the LPS+ ACE2 group. Additionally, ACE2 knock down in rat lung significantly enhanced the LPS-induced increase of ERK1/2 and JNK phosphorylation in rat lung and there was no significant difference in p38 MAPK phosphorylation level between the LPS+ ACE2-RNAi group and the LPS group (Fig. 6).

Inhibition of the MAPKs pathways reverses the blockade of A779 or MLN4760 on the protective effects of ACE2 overexpression. Pretreatment with specific MAPKs inhibitors in Lenti-Ace2-RNAi
infected rats attenuated LPS-induced lung injury as evidenced by reduced lung injury score and BALF protein level (Fig. 7A,B and E). The BALF protein levels in rats of the LPS+ ACE2+ A779 group and LPS+ ACE2+ MLN4760 group were significantly reduced by SB203580, PD98059 or SP600125 pretreatment. The LPS-induced lung injury was also attenuated by MPAKs inhibitors prior to LPS administration in rats of these two groups (Fig. 7A,F and G). However, the evaluation of lung injury score showed that there was no statistical difference between the rats with or without MPAKs inhibitor pretreatment in the LPS+ ACE2+ A779 group and LPS+ ACE2+ MLN4760 group, except that the rats of LPS+ ACE2+ A779 group pretreated with PD98059 showed a significant decrease in lung injury score (Fig. 7C,D). In addition, the BALF cytokine TNF-α and IL-1β were dramatically reduced by specific MAPKs inhibitors pretreatment in the rats from the LPS+ ACE2-RNAi, LPS+ ACE2+ A779 and LPS+ ACE2+ MLN4760 groups (Fig. 8). These results indicate that blocking the MAPKs pathway can remarkably attenuate LPS-induced inflammatory response in Lenti-Ace2-RNAi infected rats and A779 or MLN-4760 pretreated rats with ACE2 overexpression. Furthermore, the histological evaluation of whole lung injury demonstrated that pretreatment with the specific MAPKs inhibitors significantly attenuated LPS-induced lung injury in Lenti-Ace2-RNAi infected rats, but did not significantly reverse the blockade of MLN4760 on the effects of ACE2 overexpression in preventing LPS-induced lung injury. Specially, inhibiting the ERK1/2 pathway significantly attenuated both LPS-induced cytokine secretion and lung injury in ACE2 overexpression rats pretreated with Mas receptor antagonist.

ACE2 inhibits LPS-induced activation of NF-κB pathway in rat lung. Eight hours after LPS
administration, the NF-κ B p50 and p65 phosphorylation level significantly increased and the Iκ Bα expression clearly decreased in lung as compared to control rats. ACE2 overexpression in the rat lung markedly suppressed LPS-induced NF-κ B p50 and p65 phosphorylation and enhanced the expression of Iκ Bα , which were completely abolished by A779 or MLN4760 pretreatment. Furthermore, the NF-κ B p65 phosphorylation level was higher in the LPS+ ACE2+ A779 group and LPS+ ACE2+ MLN4760 group than the LPS group. As compared with the LPS group, ACE2 down-regulation further enhanced LPS-induced increase of NF-κ B p65 phosphorylation level in lung tissue. There was no difference in NF-κ B p50 phosphorylation level between the LPS group and LPS+ ACE2-RNAi group. Meanwhile, the Iκ Bα expression level in the LPS+ ACE2-RNAi group was significantly lower than that in the control group, but still higher as compared with the LPS group (Fig. 9).

Discussion
In this study, we demonstrated that LPS administration evoked severe lung injury, characterized by increased inflammatory cell infiltration, edema, and hemorrhage in the interstitium and alveolus, as well as increased TNF-α and IL-β levels in BALF. Our results also showed that LPS administration significantly reduced ACE2 expression in the rat lung, whereas overexpression ACE2 significantly attenuated LPS-induced lung injury and suppressed inflammatory response, which was abolished by ACE2 inhibitor. Further, ACE2 knockdown caused a marked deterioration of lung injury and increase of cytokine secretion in rats receiving LPS injection. Together, these findings suggest that ACE2 in local lung tissue prevents LPS-induced lung injury and inflammation, and may be useful as a therapeutic agent targeting ARDS.
Activation of renin-angiotensin system plays a central role in the pathophysiology of ARDS, and suppression of the ACE/Ang II/AT1R axis has been shown to improve the symptoms of ARDS 28,[30][31][32] . In different ARDS models, loss of ACE2 in mice resulted in more aggravated lung injury, whereas recombinant ACE2 attenuated the symptoms of ARDS in Ace2-knockout mice, especially in wild type mice 15 . The lung injury in ARDS mouse model caused by limb ischemia-reperfusion (LIR) was deteriorated by Ace2 depletion while protected by Ace2 transgene, and the changes of ACE2 expression in lung tissue were accompanied by alteration of Ang II/Ang-(1-7) ratio 33 . Prior studies have shown that down-regulation of ACE2 expression in lung and increased serum Ang II level are associated with severity of respiratory syncytial virus H7N9 or H5N1-induced ARDS, which is also ameliorated by administration of recombinant human ACE2 [34][35][36] . Moreover, the decrease of ACE2 activity in BALF of ventilated animals exposed to LPS was correlated with enhanced BALF Ang II but reduced Ang-(1-7) levels 11 . Similar to these previous studies, our results showed that ACE2 overexpression in rat lung reduced the BALF Ang II level and further increased Ang-(1-7) level in animals treated with LPS. The ratio of Ang II to Ang-(1-7) in BALF was significantly decreased by ACE2 overexpression while markedly increased by ACE2 knockdown. Despite the most of evidences suggest an decrease of Ang-(1-7) level in lung tissue during the development of ARDS, there are some conflicting results from published literatures. The Ang-(1-7) level in BALF increases during mechanical ventilation-induced rat lung injury but decreases after 24 hours of LPS administration 11 . In a mouse ARDS model, Ang-(1-7) in lung tissue was shown a decrease after 24 hours of LPS challenge 37 . In the early stage of LIR-induced ARDS, the lung tissue Ang-(1-7) level significantly increased at 1 hour and persisted up to 12 hours after reperfusion 33 . In the present study, we found that LPS administration caused a significant increase of Ang II and also an unexpected increase of Ang-(1-7) in BALF. In renin-angiotensin system, Ang-(1-7) is mainly generated by ACE2 hydrolyzing Ang II, but it also can be generated by ACE catalyzing Ang-(1-9). It is possible that the increased levels of Ang-(1-7) in lung tissue may play a compensatory role in the initial development of ARDS.
The discovery that Ang- (1-7), the major product of ACE2, stimulates downstream Mas receptors to oppose the effects of Ang II/AT1R 38,39 , suggesting that Ang-(1-7) may contribute to the protective effects of ACE2. In  (1)(2)(3)(4)(5)(6)(7) or Mas agonist, AVE0991, prevent ventilator-or acid aspiration-induced lung injury, which was reversed by a Mas receptor antagonist 41 . In the present study, we demonstrated that Mas receptor antagonist effectively abolished the protective effects of ACE2 overexpression in the lung of rats exposed to LPS. In addition, Mas receptor antagonist also reversed the decrease of Ang II and increase of Ang-(1-7) in BALF of Lenti-Ace2 transduced rats, elevating the Ang II/Ang-(1-7) ratio. These results indicate that the protective effects of ACE2 on LPS-induced lung injury primarily depend on the Ang-(1-7)/Mas pathway.
Recent study has found that infusion of Ang-(1-7) enhances Mas mRNA expression in a bleomycin-induced lung injury model 42 . Furthermore, overexpression of ACE2 in rostral ventrolateral medulla of spontaneously hypertensive rats increased the expression of Mas 43 . In this study, we found that ACE2 overexpression resulted in a significant further increase of Mas mRNA expression in LPS-stimulated rat lungs, while ACE2 knockdown remarkably suppressed Mas mRNA expression. Moreover, blockade of Mas receptor not only reduced the expression of ACE2 in lung tissue but also significantly suppressed Mas mRNA expression. These data suggest that the protective effects of ACE2/Ang-(1-7) against lung injury not only depend on Mas signaling cascade but also up-regulating Mas receptor expression.
The underlying molecular mechanism of ACE2 protects against ARDS remains elusive. Previous studies indicated that activation of MAPKs signaling promoted the progression of ARDS 17 , but the functional linkage of the MAPKs pathways with ACE2/Ang-(1-7)/Mas signaling axis in LPS-induced lung injury has not been elucidated. Our current study showed that LPS administration stimulated the phosphorylation of P38 MAPK, ERK1/2 and JNK, which was significantly reduced by ACE2 overexpression in lung tissue. Moreover, Ace2 shRNA enhanced the phosphorylation of ERK1/2 and JNK in rat lung. Either Mas receptor antagonist or ACE2 inhibitor completely abolished the inhibitory effects of AEC2 on LPS-induced p38 MAPK and ERK1/2 phosphorylation. Combined prevented Ang II-mediated myocardial injury through blocking the activation of ERK1/2 45 . In addition, it has been shown that ACE2 regulates the balance of AngII/Ang(1-7) to prevent alveolar epithelial cells apoptosis and JNK phosphorylation, while pretreatment with A779 abolishes the anti-apoptotic effect of Ang-(1-7) and its inhibition of JNK phosphorylation 46 . ACE2 over-expression in PMVECs protects LPS-induced apoptosis and suppresses p38 MAPK/JNK phosphorylation, which was attenuated by pretreatment with A779 25 . These data indicate that the suppression of MAPKs activation by the ACE2/Ang-(1-7)/Mas signaling pathway is cell type dependent.
To investigate whether the MAPKs pathway plays a role in the protective effect of ACE2 against LPS-induced lung injury, the specific MAPKs inhibitors were pretreated intraperitoneally in rats receiving LPS injection. Pretreatment with p38, ERK1/2 or JNK inhibitors significantly attenuated LPS-induced inflammatory response and injury in rat lung with ACE2 knockdown. Moreover, the Mas receptor antagonist or ACE2 inhibitor abolished the protective effects of ACE2 overexpression in LPS-induced protein and cytokine secretion in BALF, which was obviously reversed by specific MAPKs inhibitor. Whereas, only the ERK1/2 inhibitor significantly attenuated lung injury in ACE2 overexpressing rats pretreated with A779. These results suggest that there may be other signaling pathways that are involved in the mechanism of ACE2 protective effects from LPS-induced lung injury.
The NF-κ B family, including p65 (RelA), p50/p105 (NF-κ B1), p52/p100 (NF-κ B2), RelB and c-Rel, exists as homo-or heterodimers in cytoplasm and maintains inactive state by binding to Iκ B protein. Upon stimulation by LPS or pro-inflammatory cytokines, Iκ B protein is phosphorylated by activating Iκ B kinase (IKK) and degraded, leading to activation and then translocation of NF-κ B into the nucleus to upregulate transcription of inflammatory genes 47,48 . The activation of NF-κ B is pivotal to the development of endotoxin-induced ARDS in animals 49,50 . Imai et al. identified the TLR4-NF-κ B signaling pathway as a key role in controlling severity of acute lung injury 24 . In the present in vivo study, overexpressing ACE2 in the rat lung significantly suppressed LPS-induced NF-κ B p50/p65 phosphorylation and enhanced Iκ Bα expression, which was completely reversed by Mas receptor antagonist or ACE2 inhibitor pretreatment. In contrast with ACE2 overexpression, silencing ACE2 markedly enhanced LPS-induced activation of NF-κ B p50/p65 and degradation of Iκ Bα . These results indicate that NF-κ B signaling pathway is also important and may cooperate with MAPKs pathway to facilitate the protective effect of ACE2 against LPS-induced ARDS. The result of our in vitro study also suggests that ACE2 prevents PMVECs from LPS-induced apoptosis and inflammation by inhibiting JNK and NF-κ B activity 25 . Our findings are consistent Figure 9. Effects of ACE2 and different treatments on LPS-induced activation of NF-κB pathway. LPS exposure caused a marked increase in the phosphorylation of NF-κ B p50 and p65 and decrease of Iκ Bα expression, which was significantly reversed by ACE2 overexpression in the rat lung. Pretreatment with A779 or MLN-4760 completely abolished the inhibitory effects of ACE2 overexpression on LPS-induced NF-κ B p50 and p65 phosphorylation and reduced Iκ Bα expression. ACE2 RNAi in the rat lung significantly enhanced the LPS-induced NF-κ B p65 phosphorylation but did not change NF-κ B p50 phosphorylation. Data are represented as mean ± SD. * p < 0.05, versus control group; # p < 0.05, versus LPS group; $ p < 0.05, versus ACE2 group (n = 6, per group).
with previous observations that pharmacological inhibition of ERK or p38 attenuates LPS-induced lung injury via suppressing the activation of the NF-kB pathway 18,19 .
In summary, the previous studies and the data presented here suggest that AEC2 protects lung injury likely via generating Ang-(1-7), which in turn stimulates Mas-mediated signaling to inhibit ERK1/2 and NF-κ B activation during the process of ARDS (Fig. 10). Clinically, intravenous administration of recombinant human ACE2 in healthy humans results in increased, decreased or unchanged Ang-(1-7) serum concentration without cardiovascular effects 51 . In a clinical trial performed by Petty WJ et al., Ang-(1-7) administration led to a decrease of plasma placental growth factor level in cancer patient with clinical benefit 52 , suggesting that the Ang-(1-7)/Mas pathway is a promising therapeutic target of ARDS.