Non-invasive administration of AAV to target lung parenchymal cells and develop SARS-CoV-2-susceptible mice.

Adeno-associated virus (AAV)-mediated gene delivery holds great promise for gene therapy. However, the non-invasive delivery of AAV for lung tissues has not been adequately established. Here, we revealed that the intratracheal administration of an appropriate amount of AAV2/8 predominantly targets lung tissue. AAV-mediated gene delivery that we used in this study induced the expression of the desired protein in lung parenchymal cells, including alveolar type II cells. We harnessed the technique to develop severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-susceptible mice. Three kinds of immune function-relevant gene knockout (KO) mice were transduced with AAV encoding human angiotensin-converting enzyme 2 (hACE2) and then injected with SARS-CoV-2. Among these mice, type I interferon receptor (IFNAR) KO mice showed increased viral titer in the lungs compared to that in the other KO mice. Moreover, nucleocapsid protein of SARS-CoV-2 and multiple lesions in the trachea and lung were observed in AAV-hACE2-transduced, SARS-CoV-2-infected IFNAR KO mice, indicating the involvement of type I interferon signaling in the protection of SARS-CoV-2. In this study, we demonstrate the ease and rapidness of the intratracheal administration of AAV for targeting lung tissue in mice, and this can be used to study diverse pulmonary diseases.

Evaluation of 28-day repeated oral dose toxicity of aluminum chloride in rats.

The present study investigated the potential adverse effects of aluminum chloride (AlCl3) following a 4-week repeated oral administration in Sprague-Dawley rats. The test article was administered once daily by gavage to male and female rats at dose levels of 0, 100, 300, and 900 mg/kg/day for 4 weeks. After administration of AlCl3 at 900 mg/kg/day, treatment-related systemic toxicity manifested as significant increases in salivation incidence, neutrophil percentage, reticulocytes, serum triglyceride, adrenal gland and liver weights, and single-hepatocyte necrosis, as well as significant decreases in body weight gain, food intake, hemoglobin, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration (MCHC), lymphocyte percentage, serum total protein and albumin, and thymus weight in male rats; and significant increases in salivation incidence, serum triglyceride, and liver weight, as well as a significant decrease in lymphocyte percentage in female rats. At 300 mg/kg/day, a significant decrease in MCHC was found in male rats, but not in female rats. However, this finding was not toxicologically significant because the reduction was minimal and was not accompanied by changes in any other parameters. No treatment-related effects were observed in the 100 mg/kg/day group of both genders. Under the experimental conditions of this study, the target organs of AlCl3 were determined to be the blood, liver, and thymus in rats. The no-observed-adverse-effect level was found to be 300 mg/kg/day in rats of both genders.

Silibinin Attenuates Silica Dioxide Nanoparticles-Induced Inflammation by Suppressing TXNIP/MAPKs/AP-1 Signaling.

Silica dioxide nanoparticles (SiONPs) have been applied to several fields, such as drug delivery and gene therapy. However, SiONPs are a constituent of fine dust and can induce excessive inflammatory responses in the lungs via the airways. Silibinin, a major component of silymarin, has been known for its anti-oxidant and anti-inflammatory effects. In the present study, we explored the protective effects of silibinin against SiONPs-induced airway inflammation and explored its underlying mechanism of action, focusing on thioredoxin-interacting protein (TXNIP)/mitogen-activated protein kinases (MAPKs) in vitro and in vivo. In SiONPs-stimulated NCI-H292 airway epithelial cells, silibinin treatment effectively suppressed the elevation of the mRNA expression of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-1ß, which was accompanied by the reduction in the expression of TXNIP, MAPKs, and activator protein-1 (AP-1). In SiONPs-treated mice, silibinin administration inhibited the increase in inflammatory cell counts and proinflammatory mediators, and it alleviated airway inflammation by SiONPs exposure. In addition, silibinin administration effectively suppressed the elevation of TXNIP/MAPKs/AP-1 signaling by SiONPs exposure. Taken together, silibinin effectively inhibited SiONPs-induced inflammatory responses, and this effect was closely related to the inhibition of TXNIP/MAPK/AP-1 signaling. These results suggested that silibinin might be useful for reducing pulmonary inflammation induced by SiONPs.

Silica dioxide nanoparticles aggravate airway inflammation in an asthmatic mouse model via NLRP3 inflammasome activation.

Silica dioxide nanoparticles (SiONPs) are mainly used in the rubber industry; however, they are a major air pollutant in Asia. Thus, extensive research on this issue is required. In this study, we investigated the effects of SiONPs on asthma aggravation and elucidated the underlying mechanism using ovalbumin (OVA)-induced asthmatic mice model and in NCI-H292 cells. Mice exposed to SiONPs showed markedly increased Penh values, inflammatory cell counts, and inflammatory cytokine levels compared to OVA-induced asthmatic mice. Exposure to SiONPs also induced additional airway inflammation and mucus secretion with increases in protein expression levels of thioredoxin-interacting protein (TXNIP), NOD-like receptor pyrin domain-containing 3 (NLRP3) inflammasome, and interleukin (IL)-1ß compared to those in OVA-induced asthmatic mice. Treatment of SiONPs in NCI-H292 cells also significantly increased mRNA expression levels of inflammatory cytokines accompanied with elevation in the levels of TXNIP, NLRP3 inflammasome, and IL-1ß proteins in a concentration-dependent manner. Taken together, exposure to SiONPs aggravated asthma development, which is closely related to inflammasome activation. Our results provide useful information about the toxicological effects of SiONPs on asthma exacerbation and suggest the need to avoid SiONP exposure especially in individuals with respiratory diseases.

Scrophularia buergeriana attenuates allergic inflammation by reducing NF-κB activation.

BACKGROUND: Scrophularia buergeriana Miq. (Scrophulariaceae) (SB) has been used as an oriental medicine for the treatment of inflammatory diseases, such as neuritis and pharyngolaryngitis. PURPOSE: We explored the therapeutic effects of S. buergeriana ethanol extract (SBE) on airway inflammation in ovalbumin (OVA)-induced asthmatic mice and lipopolysaccharide (LPS)-stimulated RAW264.7 cells. METHODS: Mice were intraperitoneally injected with OVA on days 0 and 14 to elevate the immune response. On days 21 to 23, the mice were challenged with OVA solution and SBE (20 and 40 mg/kg) was administered daily by oral gavage from days 18 to 23. RAW264.7 cells were pretreated with SBE 1 h before LPS stimulation. RESULTS: SBE administration effectively suppressed inflammatory cell infiltration, the expression of interleukin (IL)-5, IL-13, and IL-17, immunoglobulin E, and airway hyperresponsiveness in an OVA-induced allergic asthma model. A reduction in histological alterations, including airway inflammation and mucus hypersecretion, was observed. These effects of SBE were accompanied by a decrease in matrix metalloproteinase-9 (MMP-9) expression and nuclear factor kappa B (NF-κB) phosphorylation. These responses were observed in LPS-stimulated RAW264.7 cells. SBE treatment reduced the mRNA expression of tumor necrosis factor (TNF)-α, IL-6, and MMP-9, and NF-κB phosphorylation, in LPS-stimulated RAW264.7 cells. CONCLUSION: Our results indicated that SBE effectively attenuated airway inflammation in an OVA-induced allergic asthma model. These properties of SBE were thought to be involved in the suppression of NF-κB phosphorylation, suggesting that the material has the potential to regulate the development of allergic asthma.

Cisplatin-induced ototoxicity involves interaction of PRMT3 and cannabinoid system.

This study investigated whether protein arginine methyltransferase (PRMT) and the cannabinoid system are involved in cisplatin-induced ototoxicity. Cisplatin increased cytosine-cytosine-adenosine-adenosine-thymidine-enhancer-binding protein homologous protein expression. This effect is indicative of an increase in endoplasmic reticulum (ER) stress, and apoptosis signaling including cleavage of caspase-3, caspase-9, poly-adenosine diphosphate-ribose polymerase, and phospho-p53, as well as expression of PRMT3, PRMT4 and fatty acid amide hydrolase (FAAH)1 in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells. In addition, overexpression of PRMT3 or PRMT4 increased the expression of FAAH1 expression, apoptosis, and ER stress signaling in HEI-OC1 cells, whereas PRMT3 or PRMT4 knockdown had the opposite effect. Furthermore, overexpression of FAAH1 increased apoptosis and ER stress, but expression of the PRMTs was unchanged. In addition, a cannabinoid 1 receptor agonist and FAAH inhibitor attenuated apoptosis and ER stress, while cisplatin increased the binding of PRMT3 with FAAH1. In the in vivo experiments, cisplatin was injected intraperitoneally at 6 mg/kg/day into C57BL/6 mice, and 7 days later, this study confirmed that PRMT3 and PRMT4 were upregulated in the organ of Corti of the mice. These results indicate that cisplatin-induced ototoxicity was correlated with PRMT3, PRMT4 and the cannabinoid system, and PRMT3 binding with FAAH1 was increased by cisplatin in HEI-OC1 cells. Therefore, this study suggests that PRMT3 mediates cisplatin-induced ototoxicity via interaction with FAAH1 in vitro and in vivo.

Melatonin attenuates cisplatin-induced acute kidney injury in rats via induction of anti-aging protein, Klotho.

This study investigated the protective effects of melatonin (MT) against cisplatin (CP)-induced acute kidney injury in rats as well as its possible mechanism of action associated with anti-aging protein Klotho. The following four experimental groups were evaluated: vehicle control, CP (7 mg/kg), CP&MT20 (20 mg/kg/day), and CP&MT40 (40 mg/kg/day). The concomitant administration of MT significantly ameliorated CP-induced acute kidney injury in rats, as evidenced by increased kidney weight, increased serum levels of blood urea nitrogen and creatinine, and increased incidence of histopathological alterations with renal tubular cell apoptosis. In addition, MT treatment protected kidney tissue against oxidative damages and significantly upregulated the expression level of Klotho decreased by CP treatment, resulting in reduced phosphorylation of protein kinase B (AKT) and forkhead box O (FOXO) as well as reduced expression levels of B-cell lymphoma 2-associated X protein (Bax) and caspase-3. MT not only partially regulated oxidative stress via AKT/FOXO signaling, but also reduced apoptosis caused by CP by inhibiting the Bax/caspase-3 pathway. Our results indicated that MT could prevent acute kidney injury induced by CP in rats, presumably through upregulating the expression of Klotho, resulting in elevated anti-oxidant and anti-apoptotic properties.

Protective effect of water extract of guibi-tang against pulmonary inflammation induced by cigarette smoke and lipopolysaccharide.

Water extract of guibi-tang (GB), a traditional Chinese, Japanese, and Korean herbal medicine, is used to treat memory impairment, insomnia, and peptic ulcers. The aim of this study was to investigate the protective effects of GB on pulmonary inflammation induced by cigarette smoke (CS) and lipopolysaccharide (LPS). C57BL/6 mice were used to develop a pulmonary inflammation model by exposing them to CS for 1 h per day for 7 days. LPS was intranasally administered to mice under mild anesthesia on day 5. GB was administered 1 h before CS exposure at doses of 50 or 100 mg/kg for 7 days. Our results showed that GB suppressed the CS and LPS induced elevation in inflammatory cell counts in the bronchoalveolar lavage fluid (BALF), with significant reductions in protein, tumor necrosis factor (TNF)-α, and interleukin (IL)-6 levels. Histological studies revealed that GB decreased the inflammatory cell infiltration into lung tissue caused by CS- and LPS-exposure. GB also significantly decreased the CS and LPS-induced expression of inducible nitric oxide synthase (iNOS) in the lung tissue. Taken together, GB effectively attenuated airway inflammation caused by CS and LPS. These results indicate that GB is a potential therapeutic herbal formula for pulmonary inflammatory disease.

Huntingtin's spherical solenoid structure enables polyglutamine tract-dependent modulation of its structure and function.

The polyglutamine expansion in huntingtin protein causes Huntington's disease. Here, we investigated structural and biochemical properties of huntingtin and the effect of the polyglutamine expansion using various biophysical experiments including circular dichroism, single-particle electron microscopy and cross-linking mass spectrometry. Huntingtin is likely composed of five distinct domains and adopts a spherical α-helical solenoid where the amino-terminal and carboxyl-terminal regions fold to contain a circumscribed central cavity. Interestingly, we showed that the polyglutamine expansion increases α-helical properties of huntingtin and affects the intramolecular interactions among the domains. Our work delineates the structural characteristics of full-length huntingtin, which are affected by the polyglutamine expansion, and provides an elegant solution to the apparent conundrum of how the extreme amino-terminal polyglutamine tract confers a novel property on huntingtin, causing the disease.

Identification of a novel ligand binding site in phosphoserine phosphatase from the hyperthermophilic archaeon Thermococcus onnurineus.

Phosphoserine phosphatase (PSP) catalyzes the final and irreversible step of L-serine synthesis by hydrolyzing phosphoserine to produce L-serine and inorganic phosphate. Developing a therapeutic drug that interferes with serine production is of great interest to regulate the pathogenicity of some bacteria and control D-serine levels in neurological diseases. We determined the crystal structure of PSP from the hyperthermophilic archaeon Thermococcus onnurineus at 1.8 Å resolution, revealing an NDSB ligand bound to a novel site that is located in a fissure between the catalytic domain and the CAP module. The structure shows a half-open conformation of the CAP 1 module with a unique protruding loop of residues 150-155 that possesses a helical conformation in other structures of homologous PSPs. Activity assays indicate that the enzyme exhibits marginal PSP activity at low temperature but a sharp increase in the k(cat)/K(M) value, approximately 22 fold, when the temperature is increased. Structural and biochemical analyses suggest that the protruding loop in the active site might be an essential component for the regulation of the activity of PSP from hyperthermophilic T. onnurineus. Identification of this novel binding site distantly located from the catalytic site may be exploited for the development of effective therapeutic allosteric inhibitors against PSP activity.