Proteolytic cleavage of influenza A virus (IAV) hemagglutinin by host proteases is crucial for virusinfectivity and spread. The transmembrane serine protease TMPRSS2 was previously identified as the essential protease that can cleave hemagglutinin of many subtypes of influenza virus and spike protein of coronavirus. Herein, we found that a guanine rich tract, capable of forming intramolecular G-quadruplex in the presence of potassium ions, in the promoter region of human TMPRSS2 gene was quite important for gene transcriptional activity, hence affecting its function. Furthermore, 7 new synthesized benzoselenoxanthene analogues were found to enable stabilizing such G-quadruplex. More importantly, compounds can down-regulate TMPRSS2 gene expression, especially endogenous TMPRSS2 protein levels, and consequently suppress influenza A virus propagation in vitro. Our results provide a new strategy for anti-influenza A virus infection by small molecules targeting the TMPRSS2 gene G-quadruplex and thus inhibiting TMPRSS2 expression, which is valuable for developing small molecule drugs against influenza A virus and also may be a potential candidate as anti-SARS-CoV-2 (Severe Acute Respiratory Syndrome CoV 2) lead molecules.Figure 5. Benzoselenoxanthene analogues inhibit influenza virus propagation. (A) Compounds inhibit IAV (A/ PR8/34) in vitro.Firstly, Calu-3 cells were treated with compounds or Oseltamivir and Camostat as references for 24 h, then, infected with IAV and followed by culture in compounds contained medium, finally, supernatants were collected at 48 h after infection and viral titers were determined by plaque assay. (B) Compounds suppressed cytopathic effect. After Calu-3 cells were treated with compounds (8 μM) and infected with IAV (A/ PR8/34, herein, abbr.: PR8) as described above, photos were collected at 48 h after infection. (C) The inhibitory effects of Se1, Se3 and Se5 on IAV spread can be rescued by trypsin. Firstly, Calu-3 cells were treated with compounds or Oseltamivir as references for 24 h, then, infected with IAV and followed by culture in compounds and trypsin (4 μg/mL) contained medium, finally, supernatants were collected at 48 h after infection and viral titers were determined by plaque assay. All of experiments were repeated three times. Statistical significance was determined using Student's two-tailed t-test (A,C). *P < 0.05, **P < 0.01, ***P < 0.001. Error bars represent the SEM of three independent experiments. Scientific RepoRtS | (2020) 10:7635 | https://doi.PCR stop assay. PCR reaction system contained 1 μM of TMPRSS2-L and TMPRSS2-Rev, 0.2 mM dNTP, 0.1 U/μL taq polymerase and different concentrations of compounds. PCR setting was as follow: 94 °C for 5 min, followed by 10 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s. Amplified products were run on 12% native PAGE and stained by SYBR Gold.
Toll-like receptors (TLRs) are involved in the progression of ischemic brain injury and hence vascular dementia; however, the underlying mechanisms are largely unknown. Here, we have investigated the interrelationship between stress-responsive heme oxygenase (HO)-1 isoenzyme and TLR4 during chronic brain hypoperfusion. The right unilateral common carotid artery occlusion was performed by ligation of the right common carotid artery in C57BL/6J mice. The brain cortex or hippocampus was removed for western blotting and confocal immunofluorescence analysis. The link between HO-1 and TLR4 was further examined by silencing TLR4 and pharmacological inhibition of HO-1 in primary cultured cortical neurons. Cognitive dysfunction and decrease in cerebral blood flow in mice were observed 4 weeks after the occlusion. Our data further show that common carotid artery occlusion induced an increase in TLR4 and HO-1 protein levels. Although the administration of CoPP (10 mg/kg), HO-1 agonist, improved the cognitive dysfunction in a mice model of occlusion, western blot analysis in primary cultured cortical neurons showed that HO-1 was upregulated after lipopolysaccharide treatment; this was partially abolished by the TLR4 siRNA interference. The flow cytometry analysis showed that pharmacological inhibition of HO-1 by ZnPP (100 μM) further exaggerated lipopolysaccharide-induced neuronal cell death. Hence, stress-responsive HO-1 isoenzyme participates in TLR4-induced inflammation during chronic brain ischemia. The pharmacological manipulation of TLR4 or the HO-1 antioxidant defense pathway may represent a novel treatment strategy for neuronal protection in vascular dementia.
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