Time-dependent effect of inhaled cigarette smoke exposure in the bleomycin-induced lung injury rat model.

Cigarette smoke (CS) substances are known to induce diverse ailments such as cancer, decreased immunity, and lung diseases. Although some studies have been actively conducted to evaluate cigarette toxicity, the current animal exposure methods, that is, exposure of 28- or 90-days, require considerable research cost and lead to obscure results of the CS effects. In a previous study, we compared the effects of CS in a rat model of bleomycin (BLM) and lipopolysaccharide (LPS) induced lung disease. We determined that compared to the LPS-induced rat model, the BLM-induced rat model was more sensitive to alterations in secreting cytokines and total cell number. In the current study, we further confirmed the time-point of effective inhalation exposure by CS in the BLM-induced lung injury rat model. Using an automatic video instillator, rats were administered a single dose of 2.5 mg/kg BLM (day 1), and subsequently exposed to CS via inhalation (nose-only) 4 h/day, for 1, 2, 3, and 4 weeks. The bronchoalveolar lavage fluid (BALF) was obtained from the right lung lobes, total cell numbers were counted, and chemokine and cytokine expressions were evaluated using Enzyme-Linked Immunosorbent Assay. For the 1-week exposure, we observed a greater increase of neutrophils in the BLM + CS 300 µg/L group than in the BLM or CS 300 µg/L groups. Exposure of CS in the BLM-induced lung injury rat model enhanced the secretions of chemokines and cytokines, such as CCL2/MCP-1, CXCL2/MIP-2 and TNF-α, at 1 week. Immunohistochemistry and Hematoxylin and Eosin staining of lungs at 1-2 weeks after exposure clearly confirmed this tendency in the increased levels of CCL2/MCP-1 and TNF-α. Taken together, these results indicate that the rat model of BLM-induced lung injury is more sensitive to CS exposure than other rat models, and may be an appropriate model to evaluate the effect of CS exposure at 1-2 weeks.

Anti-fibrotic effect of pycnogenol® in a polyhexamethylene guanidine-treated mouse model.

Pulmonary fibrosis (PF) is a respiratory disease that causes serious respiratory problems. The effects of French marine pine bark extract (Pycnogenol®), with antioxidant and anti-inflammatory properties, were investigated on lung fibrosis in polyhexamethylene guanidine (PHMG)-treated mice. Mice were separated into four groups (n = 6): vehicle control (VC, saline 50 µl); PHMG (1.1 mg/kg); PHMG + Pycnogenol® (0.3 mg/kg/day); and PHMG + Pycnogenol® (1 mg/kg/day). PF was induced via intratracheal instillation of PHMG. Treatment with PHMG decreased body weight and increased lung weight, both of which were improved by treatment with PHMG + Pycnogenol® (1 mg/kg). Enzyme-linked immunosorbent assay, western blotting, and PCR revealed that Pycnogenol® attenuated PHMG-induced increase in inflammatory cytokines and fibrosis-related factors in a dose-dependent manner. Finally, histopathological analysis revealed reduced inflammation/fibrosis in the PHMG + Pycnogenol® (1 mg/kg) group. Collectively, the results indicate that Pycnogenol® can be used to treat PF as it hinders fibrosis progression by inhibiting inflammatory responses in the lungs of PHMG-treated mice.

Exposure to cigarette smoke exacerbates polyhexamethylene guanidine-induced lung fibrosis in mice.

Cigarette smoke (CS) is the leading cause of chronic pulmonary diseases, including lung cancer, chronic obstructive pulmonary disease, and pulmonary fibrosis. In this study, we aimed to investigate the effects of repeated CS exposure on polyhexamethylene guanidine (PHMG)-induced pulmonary fibrosis in mice. A single intratracheal instillation of 0.6 mg/kg PHMG enhanced the immune response of mice by increasing the number of total and specific inflammatory cell types in the bronchoalveolar lavage fluid. It induced histopathological changes such as granulomatous inflammation/fibrosis and macrophage infiltration in the lungs. These responses were upregulated upon exposure to a combination of PHMG and CS. In contrast, a 4-hr/day exposure to 300 mg/m3 CS alone for 2 weeks by nose-only inhalation resulted in minimal inflammation in the mouse lung. Furthermore, PHMG administration increased the expression of fibrogenic mediators, especially in the pulmonary tissues of the PHMG + CS group compared with that in the PHMG alone group. However, there was no upregulation in the expression of inflammatory cytokines following exposure to a combination of PHMG and CS. Our results demonstrate that repeated exposure to CS may promote the development of PHMG-induced pulmonary fibrosis.

Carnosine Protects against Cerebral Ischemic Injury by Inhibiting Matrix-Metalloproteinases.

Stroke is one of the leading causes of death and disability worldwide. However, treatment options for ischemic stroke remain limited. Matrix-metalloproteinases (MMPs) contribute to brain damage during ischemic strokes by disrupting the blood-brain barrier (BBB) and causing brain edemas. Carnosine, an endogenous dipeptide, was found by us and others to be protective against ischemic brain injury. In this study, we investigated whether carnosine influences MMP activity. Brain MMP levels and activity were measured by gelatin zymography after permanent occlusion of the middle cerebral artery (pMCAO) in rats and in vitro enzyme assays. Carnosine significantly reduced infarct volume and edema. Gelatin zymography and in vitro enzyme assays showed that carnosine inhibited brain MMPs. We showed that carnosine inhibited both MMP-2 and MMP-9 activity by chelating zinc. Carnosine also reduced the ischemia-mediated degradation of the tight junction proteins that comprise the BBB. In summary, our findings show that carnosine inhibits MMP activity by chelating zinc, an essential MMP co-factor, resulting in the reduction of edema and brain injury. We believe that our findings shed new light on the neuroprotective mechanism of carnosine against ischemic brain damage.

Discovery of a novel highly potent broad-spectrum heterocyclic chemical series of arenavirus cell entry inhibitors.

We identified and explored the structure-activity relationship (SAR) of a novel heterocyclic chemical series of arenavirus cell entry inhibitors. Optimized lead compounds, including diphenyl-substituted imidazo[1,2-a]pyridines, benzimidazoles, and benzotriazoles exhibited low to sub-nanomolar potency against both pseudotyped and infectious Old and New World arenaviruses, attractive metabolic stability in human and most nonhuman liver microsomes as well as a lack of hERG K + channel or CYP enzyme inhibition. Moreover, the straightforward synthesis of several lead compounds (e.g., the simple high yield 3-step synthesis of imidazo[1,2-a]pyridine 37) could provide a cost-effective broad-spectrum arenavirus therapeutic that may help to minimize the cost-prohibitive burdens associated with treatments for emerging viruses in economically challenged geographical settings.

Inhalation exposure by cigarette smoke: Effects on the progression of bleomycin- and lipopolysaccharide-induced lung injuries in rat models.

The toxic substances of cigarette smoke (CS) induce inflammatory responses in the lung by recruiting inflammatory cells. In this study, we investigated the effects of CS on the progression of lung disease in bleomycin (BLM) and lipopolysaccharide (LPS)-induced lung injury rat models. Briefly, rats were exposed to CS via inhalation (nose-only) for 28 consecutive days, for 4 h per day. Using an automatic video instillator, rats were administered a single dose of 2.5 mg/kg BLM (day 1) or 0.5 mg/kg LPS (day 26), prepared in 50 µL phosphate-buffered saline (PBS) solution. Examination of the bronchoalveolar lavage fluid (BALF) revealed that the number of neutrophils increased in a concentration-dependent manner of CS. Exposure to CS also enhanced the expression of cytokines, i.e., CCL2 (MCP-1), CCL3 (MIP-1α), CXCL2 (CINC3), CXCL10 (IP-10), TNF-α, IFN-γ, IL-2, IL-4 in the BALF of the vehicle (VC) and BLM groups in a concentration-dependent manner. In particular, the expressions of CCL2, CXCL10 and TNF-α were remarkably upregulated in the BLM + CS 300 treatment as compared to VC, while there were no differences in these cytokine levels in the serum following CS exposure. Exposure to CS resulted in compacted alveolar spaces and macrophage aggregation in the lung tissues following BLM and LPS treatments. Compared to VC, pulmonary fibrosis and chronic inflammation of bronchioloalveoli were observed in the BLM + CS treatment and inflammatory cell infiltration of bronchioloalveoli was observed in the LPS + CS treatment in a concentration-dependent manner by CS. The expression levels of CCL2 and IFN-γ in the lung tissues were increased similar to the levels obtained in BALF, in a concentration-dependent manner by CS. Taken together, these results indicate that repeated exposure to CS may exacerbate the lung injury initially caused by BLM and LPS.

A humidifier disinfectant biocide, polyhexamethylene guanidine phosphate, inhalation exposure during pregnancy induced toxicities in rats.

Biocides are widely used for their effective antiseptic and disinfectant properties, including polyhexamethylene guanidine phosphate (PHMG-P), which is also used as a biocide as it selectively disrupts bacterial cell membrane. It is used to clean humidifiers commonly used in the dry winter season in South Korea, which exposes people to PHMG-P inhalation. However, comprehensive toxicological data on PHMG-P inhalation exposure, including in pregnant women, and the potential occurrence of lung disease is lacking. Therefore, in this study, we investigated PHMG-P inhalation exposure-induced toxicities in pregnant rats and prenatal development of their conceptus. Pregnant rats were exposed to PHMG-P via inhalation at target concentrations of 0, 0.14, 1.60, and 3.20 mg/m3 from implantation to nearly parturition (from gestation day 6-20) and then analyzed for relevant abnormalities. Results showed systemic toxicities in the pregnant rats including respiratory function abnormalities, decreased body weight gain, and decreased food consumption at ≥1.60 mg/m3. Prenatal development toxicities, including decreased fetal weight with ossification retardations of fetal bones, were observed at 3.20 mg/m3. These results will contribute to clarifying the PHMG-P inhalation exposure-induced toxicities during pregnancy and support its risk assessment in humans.

CORM-2-entrapped ultradeformable liposomes ameliorate acute skin inflammation in an ear edema model via effective CO delivery.

The short release half-life of carbon monoxide (CO) is a major obstacle to the effective therapeutic use of carbon monoxide-releasing molecule-2 (CORM-2). The potential of CORM-2-entrapped ultradeformable liposomes (CORM-2-UDLs) to enhance the release half-life of CO and alleviate skin inflammation was investigated in the present study. CORM-2-UDLs were prepared by using soy phosphatidylcholine to form lipid bilayers and Tween 80 as an edge activator. The deformability of CORM-2-UDLs was measured and compared with that of conventional liposomes by passing formulations through a filter device at a constant pressure. The release profile of CO from CORM-2-UDLs was evaluated by myoglobin assay. In vitro and in vivo anti-inflammatory effects of CORM-2-UDLs were assessed in lipopolysaccharide-stimulated macrophages and TPA-induced ear edema model, respectively. The deformability of the optimized CORM-2-UDLs was 2.3 times higher than conventional liposomes. CORM-2-UDLs significantly prolonged the release half-life of CO from 30 s in a CORM-2 solution to 21.6 min. CORM-2-UDLs demonstrated in vitro anti-inflammatory activity by decreasing nitrite production and pro-inflammatory cytokine levels. Furthermore, CORM-2-UDLs successfully ameliorated skin inflammation by reducing ear edema, pathological scores, neutrophil accumulation, and inflammatory cytokines expression. The results demonstrate that CORM-2-UDLs could be used as promising therapeutics against acute skin inflammation.

Safety assessment of cerium oxide nanoparticles: combined repeated-dose toxicity with reproductive/developmental toxicity screening and biodistribution in rats.

Cerium oxide nanoparticles (CeO2 NPs) are widely used in various commercial applications because of their characteristic properties. People can be easily exposed to CeO2 NPs in real life, but the safety assessment of CeO2 NPs has not been fully investigated. Therefore, in this study, we conducted a combined repeated-dose and reproductive/developmental toxicity screening study (OECD testing guideline 422) to investigate the potential hazards on human health, including reproductive/developmental functions, after repeated daily CeO2 NPs oral gavage administration to both males and females. In addition, tissues from parental animals and their pups were collected to analyze the internal accumulation of cerium. CeO2 NPs were orally administered to Sprague-Dawley rats at doses of 0, 100, 300 and 1000 mg/kg during their pre-mating, mating, gestation and early lactation periods. In the general systemic and reproductive/developmental examinations, no marked toxicities were observed in any in-life and terminal observation parameters in this study. In the biodistribution analysis, cerium was not detected in either parental or pup tissues (blood, liver, lungs and kidneys). Repeated oral exposure of CeO2 NPs did not induce marked toxicities affecting general systemic and reproductive/developmental functions up to the dose level of 1000 mg/kg and the CeO2 NPs were not systemically absorbed in parental animals or their pups. This result could be used in risk assessment for humans, and additional toxicity studies with CeO2 NPs will be necessary considering various physicochemical properties and exposure probabilities of these nanoparticles.

Autophagy-mediated occludin degradation contributes to blood-brain barrier disruption during ischemia in bEnd.3 brain endothelial cells and rat ischemic stroke models.

BACKGROUND: The blood-brain barrier (BBB) maintains homeostasis of the brain environment by tightly regulating the entry of substances from systemic circulation. A breach in the BBB results in increased permeability to potentially toxic substances and is an important contributor to amplification of ischemic brain damage. The precise molecular pathways that result in impairment of BBB integrity remain to be elucidated. Autophagy is a degradation pathway that clears damaged or unnecessary proteins from cells. However, excessive autophagy can lead to cellular dysfunction and death under pathological conditions. METHODS: In this study, we investigated whether autophagy is involved in BBB disruption in ischemia, using in vitro cells and in vivo rat models. We used brain endothelial bEnd.3 cells and oxygen glucose deprivation (OGD) to simulate ischemia in culture, along with a rat ischemic stroke model to evaluate the role of autophagy in BBB disruption during cerebral ischemia. RESULTS: OGD 18 h induced cellular dysfunction, and increased permeability with degradation of occludin and activation of autophagy pathways in brain endothelial cells. Immunostaining revealed that occludin degradation is co-localized with ischemic autophagosomes. OGD-induced occludin degradation and permeability changes were significantly decreased by inhibition of autophagy using 3-methyladenine (3-MA). Enhanced autophagic activity and loss of occludin were also observed in brain capillaries isolated from rats with middle cerebral artery occlusion (MCAO). Intravenous administration of 3-MA inhibited these molecular changes in brain capillaries, and recovered the increased permeability as determined using Evans blue. CONCLUSIONS: Our findings provide evidence that autophagy plays an important role in ischemia-induced occludin degradation and loss of BBB integrity.

LRP-1 functionalized polymersomes enhance the efficacy of carnosine in experimental stroke.

Stroke is one of the commonest causes of death with limited treatment options. L-Carnosine has shown great promise as a neuroprotective agent in experimental stroke, but translation to the clinic is impeded by the large doses needed. We developed and evaluated the therapeutic potential of a novel delivery vehicle which encapsulated carnosine in lipoprotein receptor related protein-1 (LRP-1)-targeted functionalized polymersomes in experimental ischemic stroke. We found that following ischemic stroke, polymersomes encapsulating carnosine exhibited remarkable neuroprotective effects with a dose of carnosine 3 orders of magnitude lower than free carnosine. The LRP-1-targeted functionalization was essential for delivery of carnosine to the brain, as non-targeted carnosine polymersomes did not exhibit neuroprotection. Using Cy3 fluorescence in vivo imaging, we showed that unlike non-targeted carnosine polymersomes, LRP-1-targeted carriers accumulated in brain in a time dependent manner. Our findings suggest that these novel carriers have the ability to deliver neuroprotective cargo effectively to the brain.

Immediate effects of ankle eversion taping on gait ability of chronic stroke patients.

OBJECTIVES: The purpose of this study is to assess the immediate effects of applying ankle eversion taping using kinesiology tape in patients with foot drop after stroke. DESIGN: Randomized cross-over trial. METHOD: In this study, fifteen subjects with stroke underwent three interventions in a random order. Subjects were randomly initially assigned to an ankle balance taping, placebo taping, and no taping each group. The ankle eversion taping was used for mechanical correction. Ankle eversion taping is involved in ankle dorsiflexion and eversion. The placebo taping began from both malleolus and was applied up to the middle point of the lower limb. Gait ability was assessed by the GAITRite System. The measured gait variables are gait velocity, step length, stride length, H-H base support, and cadence. All of the measurements were performed immediately after intervention. RESULTS: Our results showed gait function in chronic stroke patients was improved after ankle eversion taping. Velocity, step length, stride length and cadence under the ankle eversion taping conditions significantly increased (p < 0.05) compared to the placebo and no taping conditions. Ankle eversion taping significantly reduced (p < 0.05) H-H base support compared to the no taping condition. CONCLUSIONS: We conclude that the application of ankle eversion taping that uses kinesiology tape instantly increased the gait ability of chronic stroke patients with foot drop. However, more research is necessary to identify the long-term effects of the ankle eversion taping.

Reproductive and developmental toxicity screening of polyhexamethylene guanidine phosphate by oral gavage in rats.

Polyhexamethylene guanidine phosphate (PHMG-P) has effective antimicrobial activity against various microorganisms and has been widely used as a biocide in commercial products. However, its use as a humidifier disinfectant has provoked fatal idiopathic lung disease in South Korea, especially in pregnant or postpartum women and their young children. PHMG-P-related toxicological studies of reproduction and development in experimental animals have not been identified, and thus, we investigated the potential effects of early-stage oral exposure to PHMG-P by assessing its toxicological properties. PHMG-P was repeatedly administered by oral gavage at dose levels of 0, 13, 40 and 120 mg/kg to Sprague-Dawley rats during the pre-mating, mating, gestation and early lactation periods, and then general systemic and reproductive/developmental toxicities were investigated. At 120 mg/kg, PHMG-P-related toxicities including subdued behavior, thin appearance, decreased body weight, decreased food consumption and decreased F1 pup body weight were observed. Based on the results of this study, the no-observed-adverse-effect levels (NOAELs) of PHMG-P for both general systemic effects and development are considered to be 40 mg/kg/day.

Inhibitory Effects of a Novel Chrysin-Derivative, CPD 6, on Acute and Chronic Skin Inflammation.

The skin is an important physiological barrier against external stimuli, such as ultraviolet radiation (UV), xenobiotics, and bacteria. Dermal inflammatory reactions are associated with various skin disorders, including chemical-induced irritation and atopic dermatitis. Modulation of skin inflammatory response is a therapeutic strategy for skin diseases. Here, we synthesized chrysin-derivatives and identified the most potent derivative of Compound 6 (CPD 6). We evaluated its anti-inflammatory effects in vitro cells of macrophages and keratinocytes, and in vivo dermatitis mouse models. In murine macrophages stimulated by lipopolysaccharide (LPS), CPD 6 significantly attenuated the release of inflammatory mediators such as nitric oxide (NO) (IC50 for NO inhibition: 3.613 µM) and other cytokines. In cultured human keratinocytes, CPD 6 significantly attenuated the release of inflammatory cytokines induced by the combination of IFN-γ and TNF-α, UV irradiation, or chemical irritant stimulation. CPD 6 inhibited NFκB and JAK2/STAT1 signaling pathways, and activated Nrf2/HO-1 signaling. In vivo relevancy of anti-inflammatory effects of CPD 6 was observed in acute and chronic skin inflammation models in mice. CPD 6 showed significant anti-inflammatory properties both in vitro cells and in vivo dermatitis animal models, mediated by the inhibition of the NFκB and JAK2-STAT1 pathways and activation of Nrf2/HO-1 signaling. We propose that the novel chrysin-derivative CPD 6 may be a potential therapeutic agent for skin inflammation.

Study on the design development of a multi joint ankle foot orthosis.

[Purpose] The purpose of this study was to investigate the effects of a newly designed multi joint ankle-foot orthosis on the gait and dynamic balance of stroke patients having foot drop. [Participants and Methods] This study was investigated 10 participants who were diagnosed with stroke. Patients were evaluated based on a 10-meter walk test, timed up and go test and Berg balance scale after each participant wore a plastic ankle-foot orthosis and a multi joint ankle-foot orthosis (AFO) that consisted of orthosis joints (having poster-stop joint and Klenzak joint functions). [Results] The 10-meter walk test, timed up and go test and Berg balance scale showed significant differences in the orthosis with the Klenzak joint function. [Conclusion] The appropriate use of Klenzak AFO of the newly designed multi joint AFO is expected to have a positive effect on improving the gait and balancing ability of stroke patients having foot drop.

Effect of mirror use on lower extremity muscle strength of patients with chronic stroke.

[Purpose] This study examines the effect on muscle strength of lower extremity muscle strength exercise while using a mirror on the non-paretic side in patients with chronic stroke. [Subjects and Methods] Subjects were randomly assigned to a non-mirror lower extremity exercise group (n=10), a mirror lower extremity exercise group (n=10), or a mirror lower extremity muscle strength exercise group (n=10). Subjects were asked to do the exercise assigned to their group (5 sets 30 times a day, 5 times weekly for 4 weeks) with general physical therapy in the hospital. Muscle strength in the knee extensor and flexor of paretic and non-paretic side were measured using electrical muscle testing device before and after the intervention. [Results] Muscle strength significantly increased within each group after intervention. No significant differences were found among the three groups. [Conclusion] This study showed that the lower extremity muscle strength exercise of the non-paretic side using a mirror has a positive effect on muscle strength in patient with chronic stroke.

Methylglyoxal induced advanced glycation end products (AGE)/receptor for AGE (RAGE)-mediated angiogenic impairment in bone marrow-derived endothelial progenitor cells.

Endothelial cells (ECs) maintain the structure and function of blood vessels and are readily exposed to exogenous and endogenous toxic substances in the circulatory system. Bone marrow-derived endothelial progenitor cells (EPCs) circulate in the blood and differentiate to EC, which are known to participate in angiogenesis and regeneration of injured vessels. Dysfunction in EPC contributes to cardiovascular complications in patients with diabetes, but the precise molecular mechanisms underlying diabetic EPC abnormalities are not completely understood. The aim of this study was to investigate the mechanisms underlying diabetic EPC dysfunction using methylglyoxal (MG), an endogenous toxic diabetic metabolite. Data demonstrated that MG decreased cell viability and protein expression of vascular endothelial growth factor receptor (VEGFR)-2 associated with functional impairment of tube formation in EPC. The generation of advanced glycation end (AGE) products was increased in EPC following exposure to MG. Blockage of receptor for AGE (RAGE) by FPS-ZM1, a specific antagonist for RAGE, significantly reversed the decrease of VEGFR-2 protein expression and angiogenic dysfunction in MG-incubated EPC. Taken together, data demonstrated that MG induced angiogenic impairment in EPC via alterations in the AGE/RAGE-VEGFR-2 pathway which may be utilized in the development of potential therapeutic and preventive targets for diabetic vascular complications.

The relationship between anterior pelvic tilt and gait, balance in patient with chronic stroke.

[Purpose] The aim of this study is to find out the association between anterior pelvic tilt and gait and balance in chronic stroke. [Subjects and Methods] Fourteen chronic stroke patients were included in this study. A palpation meter was employed to measure the anterior inclination of the pelvis. A GAITRite system automates measuring temporal and spatial gait parameters. A 10-Meter Walk test was used to measure gait speed. The Timed Up and Go test was used to measure the dynamic balance ability and gait ability of the participants. A BioRescue was used to assess balance by measuring the moving distance and area of the center of pressure. [Results] There were significant negative correlations between pelvic anterior tilt and velocity, step length, and stride. There were significant positive correlations between velocity and cadence, step length, and stride length. There were significant negative correlations between velocity and cycle time, H-H base, TUG, and 10MWT. There was significant negative correlation between cadence and cycle time and H-H base. [Conclusion] This study showed a negative correlation between pelvic anterior tilt and gait function including gait speed and step length.

The effect of newly designed multi joint ankle foot orthosis on the gait and dynamic balance of stroke patients with foot drop.

[Purpose] The purpose of this study is to investigate the effect of the newly designed multi joint ankle-foot orthosis on the gait and dynamic balance of stroke patients having foot drop. [Subjects and Methods] This study was conducted with 15 subjects who were diagnosed with stroke. 10-meter walk test, functional reaching test and timed up and go test were measured after each subjects wore a plastic ankle-foot orthosis and a multi joint ankle-foot orthosis that consists of orthosis joints (having free joint, anterior-stop joint, poster-stop joint, and Klenzak joint functions). In the case of the newly developed multi joint ankle-foot orthosis, the experiments were performed using posterior-stop joint and Klenzak joint. [Results] 10-meter walk test, functional reaching test and timed up and go test showed significant differences in the orthosis using posterior joint-stop function and Klenzak joint function. [Conclusion] The appropriate use of the four functions of the newly designed multi joint ankle-foot orthosis is expected to have a positive effect on improving the gait and balancing ability of stroke patients having foot drop.