Figure 4: Recombinant hACE2 reduces the severity of H5N1-induced acute lung injury.

BALB/c mice were injected with recombinant hACE2 (i.p., 100 μg kg⁻¹) or allantoic fluid (AF) as a vehicle control (+BSA i.p., 100 μg kg⁻¹) 3 h prior to, as well as 8 h and 3 days after intratracheal instillation of AF or live H5N1 virus (10⁶ TCID₅₀). (a) Kaplan-Meier survival curves. n=21 for the Control+H5N1 and ACE2+H5N1 groups, n=5 for the Control+AF and ACE2+AF groups. *P<0.05 when comparing the Control+H5N1 group with the ACE2+H5N1 group (Log-rank test). (b) Relative mRNA expression levels of influenza A M2 in the lungs of mice at the indicated time points using real-time PCR (mean±s.e.m.). n=3–5 mice per time point. **P<0.01 (two-tailed t-test). (c) Angiotensin II levels in the serum of mice were determined using radioimmunoassays at the indicated time points (mean±s.e.m.). n=3-4 mice per time point. *P<0.05 (two-tailed t-test). (d) Wet to dry lung weight ratios of the mice were assessed 4 days after AF or H5N1 instillation in the presence or absence of recombinant hACE2 (mean±s.e.m.). n=4-5 mice per group. *P<0.05, **P<0.01 (two-tailed t-test). (e) Representative lung histopathology of AF and H5N1-challenged BALB/c mice left untreated or treated with recombinant ACE2 (i.p., 100 μg kg⁻¹). Scale bar=100 μm. The numbers of infiltrating cells per microscopic field (mean±s.e.m.; top panel) and lung injury scores (mean±s.e.m.; bottom panel) are shown for day 4 after infection in the bar graphs. n=100 fields analyzed for three mice for each treatment. *P<0.05, **P<0.01 (two-tailed t-test). Each experiment was repeated at least three times.