ACE2 inhibitors(University of Chinese Academy of Sciences)

Acute lung injury (ALI) and its most severe manifestation of acute respiratory distress syndrome (ARDS) is a disease with a clinical mortality rate of up to 40% and is one of the most dangerous and common complications of severe coronavirus disease 2019 (COVID-19). Sivelestat (SIV) is the only licensed therapeutic medicine in the world for ALI/ARDS treatment. The angiotensin-converting enzyme 2 (ACE2)/angiotensin (Ang)-(1-7)/Mas receptor axis is critical in the prevention of ALI/ARDS. This study aims to investigate whether SIV alleviates lipopolysaccharides (LPS)-induced ALI by inhibiting the down-regulation of ACE2/Ang-(1-7)/Mas receptor axis expression.

In vivo, 90 male Sprague-Dawley rats were randomized into 5 groups. Then, we pretreated different groups of rats with dexamethasone (DEX) or SIV. Rats were sacrificed at three different time points (3, 6, and 12 hours) following LPS instillation. In vitro, RAW264.7 cells were divided into 11 groups. Different groups of cells were pretreated with DEX or SIV. And then added with LPS for 3, 6, and 12 hours. Next, we introduced A779, a potent Ang-(1-7) receptor antagonist, and DX600 as the ACE2 antagonist in different groups. Then the protein and messenger RNA (mRNA) expression levels of ACE2 in rat lung tissue and the expression levels of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and Ang-(1-7) in the rat serum and the cell culture supernatant were measured. And the data were statistically analyzed.

In vivo, the rats pretreated with SIV or DEX had significantly lower lung wet/dry (W/D) ratios and lung pathological alterations than those exposed to LPS only. Both in vivo and in vitro, we observed that SIV or DEX significantly attenuated the LPS-induced up-regulation of IL-6 and TNF-α levels, and the down-regulation of ACE2 and Ang-(1-7) levels. In vitro, the pretreatment of the RAW264.7 cells with DX600 and A779 significantly reduced and even abolished the protective effects of SIV.

Therefore, it was concluded that SIV protected against LPS-induced ALI and decreased inflammatory cytokine release by up-regulating the ACE2/Ang-(1-7)/Mas receptor axis. Our results enrich the theoretical foundation for the clinical application of SIV and provide fresh ideas for the treatment of ALI/ARDS.

P32 protein serves as a crucial structural component of Goat pox virus (GTPV), which causes a highly virulent infectious disease in sheep and goats. Despite the fact that P32 has been widely expressed in the previous studies, it is difficult to obtain recombinant P32 efficiently. This study aimed to achieve soluble expression of P32 recombinant protein and to develop its specific monoclonal antibody. The gene fragment of P32Δ (GP32Δ) was synthesized by optimizing the coding sequence of amino acids 1-246 of the known goatpox P32 protein. Subsequently, GP32Δ was cloned into a prokaryotic expression vector for expression and purification, resulting in the successful production of soluble recombinant protein rP32Δ. Utilizing rP32Δ, an indirect ELISA method was established by immunizing 6-week-old BALB/c mice with inactivated GTPV as the antigen. Through hybridoma technology, three monoclonal antibody hybridoma cell lines secreting anti-goat pox virus rP32Δ were screened, designated as 2F3, 3E8, and 4H5, respectively. These monoclonal antibodies, classified as IgG1, IgG2a, and IgG2b, respectively, with κappa light chains, were characterized following ascites preparation and purification. Indirect ELISA results demonstrated that the ELISA potency of the three monoclonal antibodies exceeded 1:12800. Furthermore, Western blot analysis revealed specific reactivity of both 3E8 and 4H5 with rP32Δ, while immunofluorescence assays confirmed 3E8's ability to specifically recognize GTPV in cells. The preceding findings demonstrate the successful acquisition of the soluble expressed recombinant P32 protein and its specific monoclonal antibody 3E8 in this study, thereby laying a foundational material basis for the establishment of a GTPV detection method.