[Annexin A1 activates the G protein-coupled formyl peptide receptor type 2-dependent endothelial nitric oxide synthase pathway to alleviate sepsis associated acute lung injury].

OBJECTIVE: To investigate whether annexin A1 (ANXA1) improves sepsis-induced lung injury by activating G protein-coupled formyl peptide receptor type 2 (FPR2)-dependent endothelial nitric oxide synthase (eNOS) pathway. METHODS: Twenty-four male SD rats were randomly divided into normal group (Control group), lipopolysaccharide (LPS) induced lung injury model group (LPS group), LPS+ANXA1 mimetic peptide group (LPS+Ac2-26 group) and LPS+ANXA1 mimetic peptide+FPR2 inhibitor group (LPS+Ac2-26+WRW4 group), with 6 rats in each group. On the third day before modeling, rats of the LPS+Ac2-26 group were injected with 1 mg/kg Ac2-26 by the tail vein and rats of LPS+Ac2-26+WRW4 group were injected with 1 mg/kg Ac2-26 and 2.2 mg/kg WRW4 by the tail vein. The rats of control group and LPS group were injected same volume of physiological saline. After 48 hours of modeling, the rats were anesthetized and the carotid blood was taken to detect the oxygenation index (OI). Lung tissue was taken from the euthanized rats. The wet/dry (W/D) ratio was determined. The pathological changes of lung tissue were observed under light microscope and pathological score was performed. The levels of tumor necrosis factor-α (TNF-α), interleukins (IL-1ß, IL-6, IL-10), malondialdehyde (MDA) and myeloperoxidase (MPO) were detected by enzyme-linked immunosorbent assay (ELISA). The protein expressions of eNOS, inducible nitric oxide synthase (iNOS) and nuclear factor-κB (NF-κB) were detected by Western blotting. RESULTS: Under light microscope, compared with LPS group, the infiltration degree of inflammatory cells in the lung tissue of LPS+Ac2-26 group was reduced, and the thickness of the alveolar septum was improved. The degree of inflammatory cell infiltration in the lung tissue of LPS+Ac2-26+WRW4 group was more severe than that of LPS+Ac2-26 group, and the thickness of the alveolar septum increased. These findings suggested that ANXA1 significantly inhibited inflammatory cell infiltration and improved alveolar septal thickness, WRW4 reversed the lung improvement effects of ANXA1. Compared with control group, OI in LPS group was significantly decreased, and W/D ratio, pathological score and TNF-α, IL-1ß, IL-6, MDA and MPO levels in lung tissue were significantly increased. Compared with LPS group, OI and IL-10 levels in lung tissue were significantly increased in LPS+Ac2-26 group, while W/D ratio, pathological score, TNF-α, IL-1ß, IL-6, MDA and MPO levels in lung tissue were significantly decreased. These results indicated that ANXA1 can improve the oxygenation capacity, improve lung tissue leakage, reduce edema, and inhibit lung tissue inflammation in rats with lung injury. Compared with LPS+Ac2-26 group, the LPS+Ac2-26+WRW4 group showed significant decreases in OI and lung tissue IL-10 level [OI (mmHg, 1 mmHg ≈ 0.133 kPa): 132.16±24.00 vs. 248.67±18.70, IL-10 (ng/L): 27.30±3.04 vs. 36.10±3.92, both P < 0.05], the lung tissue W/D ratio, pathological score and levels of TNF-α, IL-1ß, IL-6, MDA and MPO were significantly increased [W/D ratio: 5.29±0.02 vs. 4.83±0.02, pathological score: 5.00±0.28 vs. 2.67±0.52, TNF-α (ng/L): 39.80±4.36 vs. 32.10±2.15, IL-1ß (ng/L): 200.00±15.68 vs. 152.60±9.74, IL-6 (ng/L): 181.50±18.02 vs. 148.50±7.34, MDA (mmol/mg): 82.01±8.22 vs. 70.43±5.69, MPO (pg/mg): 6.50±0.32 vs. 4.60±0.56, all P < 0.05]. These results suggested that WRW4 could block the above improvement of ANXA1. Western blotting results showed that compared with control group, the expression of eNOS, iNOS and NF-κB in LPS group was significantly up-regulated. Compared with LPS group, the protein expression of eNOS in LPS+Ac2-26 group was significantly up-regulated (eNOS/ß-actin: 0.25±0.01 vs. 0.14±0.01, P < 0.05), and the protein expression of iNOS and NF-κB was significantly down-regulated (iNOS/ß-actin: 0.09±0.02 vs. 0.12±0.02, NF-κB/ß-actin: 0.35±0.06 vs. 0.59±0.13, both P < 0.05). These findings suggested that ANXA1 might activate the eNOS pathway and down-regulate the expression of NF-κB. Compared with LPS+Ac2-26 group, the protein expression of eNOS in LPS+Ac2-26+WRW4 group was significantly down-regulated (eNOS/ß-actin: 0.17±0.02 vs. 0.25±0.01, P < 0.05), while the protein expression of iNOS and NF-κB was significantly up-regulated (iNOS/ß-actin: 0.12±0.02 vs. 0.09±0.02, NF-κB/ß-actin: 0.52±0.10 vs. 0.35±0.06, both P < 0.05). These results suggested that WRW4 blocked the activation of the eNOS pathway by ANXA1. CONCLUSIONS: ANXA1 can improve lung injury associated with sepsis by activating FPR2-dependent eNOS pathway.

Transcriptomic Analysis of Metformin's Effect on Bovine Viral Diarrhea Virus Infection.

(1) Background: Bovine viral diarrhea virus (BVDV) causes calf diarrhea, bovine respiratory syndrome, and cow abortion, resulting in substantial economic losses in the cattle industry. Owing to its persistent infection mechanism, BVDV is a major challenge in the treatment of cattle. (2) Methods: To determine how metformin (Met) inhibits the interaction between BVDV and host cells, we treated BVDV-infected cells with Met. We then performed an RNA sequencing (RNA-seq) analysis of Met-treated cells infected with BVDV to identify differentially expressed genes (DEGs). Consequently, the RNA-seq results were validated through real-time quantitative PCR (qPCR). (3) Results: Our analysis revealed 3169 DEGs in the Met-treated cells (Met group) vs. the negative controls (NC group) and 2510 DEGs in the BVDV-infected cells after pretreatment with Met (MetBVDV group) vs. the BVDV-infected cells (BVDV group). The DEGs were involved in MDBK interactions during BVDV infection, as indicated by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. The potential interactions of the DEGs were confirmed via a protein-protein interaction (PPI) network. Met treatment induced autophagy signaling activity and the expression of the autophagy-related genes ATG2A, ATG4B, ATG10, and ATG12 in BVDV-infected Met-pretreated cells. (4) Conclusions: We found that the host transcriptomic profile was affected by BVDV infection and Met pretreatment. These findings offer valuable new insights and provide support for future studies on the inhibition of BVDV replication by Met.

Berbamine, a bioactive alkaloid, suppresses equine herpesvirus type 1 in vitro and in vivo.

Equine herpesvirus type 1 (EHV-1) poses a global threat to equines. The anticancer agent berbamine (BBM), a bioactive alkaloid, has been shown to inhibit viral infection. However, whether BBM can inhibit EHV-1 infection remains unclear. This study investigated the effect of BBM treatment on EHV-1 infection. Quantitative PCR (qPCR), immunoblotting, the Reed-Muench method, and pathological examination were employed to study the ability of BBM to inhibit EHV-1 infection, viral DNA replication, viral protein production, virion secretion, and cytopathogenesis in vitro and in vivo. The in vitro studies revealed that 10 µM BBM effectively suppressed EHV-1 viral entry into cells, viral DNA replication, and virion secretion, while the in vivo studies verified the ability of BBM to suppress EHV-1-induced damage of brain and lung tissues and animal mortality. These findings strongly suggest that BBM could be a serious contender in the therapeutic control of EHV-1 infection of equines.