Extracellular acidification attenuates bronchial contraction via an autocrine activation of EP2 receptor: Its diminishment in murine experimental asthma.

PURPOSE: Extracellular acidification is a major component of tissue inflammation, including airway inflammation in asthmatics. However, its physiological/pathophysiological significance in bronchial function is not fully understood. Currently, the functional role of extracellular acidification on bronchial contraction was explored. METHODS: Left main bronchi were isolated from male BALB/c mice. Epithelium-removed tissues were exposed to acidic pH under submaximal contraction induced by 10-5 M acetylcholine in the presence or absence of a COX inhibitor indomethacin (10-6 M). Effects of AH6809 (10-6 M, an EP2 receptor antagonist), BW A868C (10-7 M, a DP receptor antagonist) and CAY10441 (3×10-6 M, an IP receptor antagonist) on the acidification-induced change in tension were determined. The release of prostaglandin E2 (PGE2) from epithelium-denuded tissues in response to acidic pH was assessed using an ELISA. RESULTS: In the bronchi stimulated with acetylcholine, change in the extracellular pH from 7.4 to 6.8 caused a transient augmentation of contraction followed by a sustained relaxing response. The latter inhibitory response was abolished by indomethacin and AH6809 but not by BW A868C or CAY10441. Both indomethacin and AH6809 significantly increased potency and efficacy of acetylcholine at pH 6.8. Stimulation with low pH caused an increase in PGE2 release from epithelium-denuded bronchi. Interestingly, the acidic pH-induced bronchial relaxation was significantly reduced in a murine asthma model that had a bronchial hyperresponsiveness to acetylcholine. CONCLUSION: Taken together, extracellular acidification could inhibit the bronchial contraction via autocrine activation of EP2 receptors. The diminished acidic pH-mediated inhibition of bronchial tone may contribute to excessive bronchoconstriction in inflamed airways such as asthma.

Transcriptomic (DNA Microarray) and Metabolome (LC-TOF-MS) Analyses of the Liver in High-Fat Diet Mice after Intranasal Administration of GALP (Galanin-like Peptide).

The aim of this research was to test the efficacy and potential clinical application of intranasal administration of galanin-like peptide (GALP) as an anti-obesity treatment under the hypothesis that GALP prevents obesity in mice fed a high-fat diet (HFD). Focusing on the mechanism of regulation of lipid metabolism in peripheral tissues via the autonomic nervous system, we confirmed that, compared with a control (saline), intranasally administered GALP prevented further body weight gain in diet-induced obesity (DIO) mice with continued access to an HFD. Using an omics-based approach, we identified several genes and metabolites in the liver tissue of DIO mice that were altered by the administration of intranasal GALP. We used whole-genome DNA microarray and metabolomics analyses to determine the anti-obesity effects of intranasal GALP in DIO mice fed an HFD. Transcriptomic profiling revealed the upregulation of flavin-containing dimethylaniline monooxygenase 3 (Fmo3), metallothionein 1 and 2 (Mt1 and Mt2, respectively), and the Aldh1a3, Defa3, and Defa20 genes. Analysis using the DAVID tool showed that intranasal GALP enhanced gene expression related to fatty acid elongation and unsaturated fatty acid synthesis and downregulated gene expression related to lipid and cholesterol synthesis, fat absorption, bile uptake, and excretion. Metabolite analysis revealed increased levels of coenzyme Q10 and oleoylethanolamide in the liver tissue, increased levels of deoxycholic acid (DCA) and taurocholic acid (TCA) in the bile acids, increased levels of taurochenodeoxycholic acid (TCDCA), and decreased levels of ursodeoxycholic acid (UDCA). In conclusion, intranasal GALP administration alleviated weight gain in obese mice fed an HFD via mechanisms involving antioxidant, anti-inflammatory, and fatty acid metabolism effects and genetic alterations. The gene expression data are publicly available at NCBI GSE243376.

Altered renin-angiotensin system gene expression in airways of antigen-challenged mice: ACE2 downregulation and unexpected increase in angiotensin 1-7.

OBJECTIVE: Evidence suggest that the renin-angiotensin system (RAS) is activated in people with asthma, although its pathophysiological role is unclear. Angiotensin-converting enzyme 2 (ACE2) is the major enzyme that converts angiotensin II to angiotensin 1-7 (Ang-1-7), and is also known as a receptor of SARS-CoV-2. The current study was conducted to identify the change in RAS-related gene expression in airways of a murine asthma model. METHODS: The ovalbumin (OA)-sensitized mice were repeatedly challenged with aerosolized OA to induce asthmatic reaction. Twenty-four hours after the last antigen challenge, the main bronchial smooth muscle (BSM) tissues were isolated. RESULTS: The KEGG pathway analysis of differentially expressed genes in our published microarray data revealed a significant change in the RAS pathway in the antigen-challenged mice. Quantitative RT-PCR analyses showed significant increases in the angiotensin II-generating enzymes (Klk1, Klk1b3 and Klk1b8) and a significant decrease in Ace2. Surprisingly, ELISA analyses revealed a significant increase in Ang-1-7 levels in bronchoalveolar lavage (BAL) fluids of the antigen-challenged animals, while no significant change in angiotensin II was observed. Application of Ang-1-7 to the isolated BSMs had no effect on their isometrical tension. CONCLUSION: The expression of Ace2 was downregulated in the BSMs of OA-challenged mice, while Klk1, Klk1b3 and Klk1b8 were upregulated. Despite the downregulation of ACE2, the level of its enzymatic product, Ang-1-7, was increased in the inflamed airways, suggesting the existence of an unknown ACE2-independent pathway for Ang-1-7 production. The functional role of Ang-1-7 in the airways remains unclear.

Association between Plasma Ascorbic Acid Levels and Postoperative Delirium in Older Patients Undergoing Cardiovascular Surgery: A Prospective Observational Study.

BACKGROUND: The incidence of delirium is high in older patients undergoing cardiovascular surgery with cardiopulmonary bypass (CPB). Intraoperative tissue hypoperfusion and re-reperfusion injury, which generate reactive oxygen species (ROS), are suggested to induce delirium. Ascorbic acid is an excellent antioxidant and may reduce organ damage by inhibiting the production of ROS. This prospective observational study aimed to measure pre- and postoperative plasma ascorbic acid levels and examine their association with delirium. METHODS: Patients older than 70 years of age scheduled for elective cardiovascular surgery using CPB were enrolled. From September 2020 to December 2021, we enrolled 100 patients, and the data of 98 patients were analyzed. RESULTS: In total, 31 patients developed delirium, while 67 did not. Preoperative plasma ascorbic acid levels did not differ between the non-delirium and delirium groups (6.0 ± 2.2 vs. 5.5 ± 2.4 µg/mL, p = 0.3). Postoperative plasma ascorbic acid levels were significantly different between the groups (2.8 [2.3-3.5] vs. 2.3 [1.6-3.3] µg/mL, p = 0.037). CONCLUSIONS: In patients who undergo cardiovascular surgery with CPB, lower postoperative plasma ascorbic acid levels may be associated with the development of delirium.

Platinum-based anticancer drugs-induced downregulation of myosin heavy chain isoforms in skeletal muscle of mouse.

Cisplatin, a platinum-based anticancer drug used frequently in cancer treatment, causes skeletal muscle atrophy. It was predicted that the proteolytic pathway is enhanced as the mechanism of this atrophy. Therefore, we investigated whether a platinum-based anticancer drug affects the expression of the major proteins of skeletal muscle, myosin heavy chain (MyHC). Mice were injected with cisplatin or oxaliplatin for four consecutive days. C2C12 myotubes were treated using cisplatin and oxaliplatin. Administration of platinum-based anticancer drug reduced quadriceps mass and muscle strength compared to the control group. Protein levels of all MyHC isoforms were reduced in the platinum-based anticancer drug groups. However, only Myh2 (MyHC-IIa) gene expression in skeletal muscle of mice treated with platinum-based anticancer drugs was found to be reduced. Treatment of C2C12 myotubes with platinum-based anticancer drugs reduced the protein levels of all MyHCs, and treatment with the proteasome inhibitor MG-132 restored this reduction. The expression of Mef2c, which was predicted to act upstream of Myh2, was reduced in the skeletal muscle of mice treated systemically with platinum-based anticancer drug. Degradation of skeletal muscle MyHCs by proteasomes may be a factor that plays an important role in muscle mass loss in platinum-based anticancer drug-induced muscle atrophy.

Effects and Mode of Action of Oleic Acid and Tween 80 on Skin Permeation of Disulfiram.

Oral disulfiram (DSF) has been used clinically for alcohol dependence and recently has been found to have antitumor activity. A transdermal delivery system would be useful for maintaining drug concentration and reducing the frequency of administration of DSF for cancer treatment. Penetrating the stratum corneum (SC) barrier is a challenge to the transdermal delivery of DSF. Therefore, we investigated the promoting effects and mechanism of action of the combination of oleic acid (OA) and Tween 80 on the skin permeation of DSF. Hairless mouse skin was exposed to OA and Tween 80, combined in various ratios (1 : 0, 2 : 1, 1 : 1, 1 : 2, and 0 : 1). A permeation experiment was performed, and total internal reflection IR spectroscopic measurements, differential scanning calorimetry, and synchrotron radiation X-ray diffraction measurements were taken of the SC with each applied formulation. The combination of OA and Tween 80 further enhanced the absorption-promoting effect of DSF, compared with individual application. The peak of the CH2 inverse symmetric stretching vibration near the skin surface temperature was shifted by a high frequency due to the application of OA, and DSF solubility increased in response to Tween 80. We believe that the increased fluidity of the intercellular lipids due to OA and the increased solubility of DSF due to Tween 80 promoted the absorption of DSF. Our study clarifies the detailed mechanism of action of the skin permeation and promoting effect of DSF through the combined use of OA and Tween 80, contributing to the development of a transdermal preparation of DSF.

A lncRNA MALAT1 is a positive regulator of RhoA protein expression in bronchial smooth muscle cells.

AIMS: Augmented smooth muscle contractility of the airways associated with an increased expression of RhoA, a monomeric GTPase responsible for Ca2+ sensitization of contraction, is one of the causes of airway hyperresponsiveness. However, the mechanism of the altered properties of airway smooth muscle cells, including the RhoA upregulation, is not fully understood. This study aims to define functional role of a long non-coding RNA MALAT1 in the RhoA expression and development of bronchial smooth muscle (BSM) hyper-contractility. MAIN METHODS: Cultured human BSM cells were transfected with MALAT1 antisense oligonucleotide (AS), miR-133a-3p mimic, and/or inhibitor, and then stimulated with interleukin-13 (IL-13). In animal experiments, the ovalbumin (OA)-sensitized mice were repeatedly challenged with aerosolized OA to induce asthmatic reaction. KEY FINDINGS: Treatment of the cells with IL-13 induced an increase in RhoA protein. Either MALAT1 AS or miR-133a-3p mimic transfection inhibited the IL-13-induced upregulation of RhoA. The inhibitory effect of MALAT1 AS was abolished by co-transfection with miR-133a-3p inhibitor. In BSMs of the murine asthma model, upregulations of Malat1 and RhoA protein were observed concomitantly with downregulation of miR-133a-3p. SIGNIFICANCE: These findings suggest that MALAT1 positively regulates RhoA protein expression by inhibiting miR-133a-3p in BSM cells, and that its upregulation causes the RhoA upregulation, resulting in an augmented BSM contractility.

Downregulation of Sparc-like protein 1 during cisplatin-induced inhibition of myogenic differentiation of C2C12 myoblasts.

Patients with cancer often experience muscle atrophy, which worsens their prognosis. Decreased muscle regenerative capacity plays an important role in the complex processes involved in muscle atrophy. Administration of cisplatin, a cancer chemotherapeutic agent, has been implicated as a cause of muscle atrophy. In this study, we examined whether cisplatin affects the differentiation of myoblasts into myotubes. We treated C2C12 myoblasts with a differentiation medium containing cisplatin and its vehicle during for 8 days and observed the changes in the expression of myosin heavy chain (MyHC) and myogenin in the myoblasts. Cisplatin was injected in mice for 4 consecutive days; on Day 5, the mice quadriceps muscles were sampled and examined. The expression of MyHCs increased and that of myogenin decreased after cisplatin treatment. The secretion of acidic cysteine-rich proteins (e.g., Sparc proteins) reportedly promotes C2C12 myoblast differentiation. Therefore, we investigated the Sparc family gene expression during myogenesis in C2C12 myoblasts after cisplatin treatment. Of all the genes investigated, Sparc-like protein 1 (Sparcl1) expression was significantly suppressed by cisplatin on Days 4-8. Simultaneous treatment with recombinant mouse Sparcl1 almost inhibited the cisplatin-induced suppression of total MyHC and myogenin protein levels. Moreover, Sparcl1 expression decreased in the skeletal muscles of mice, leading to cisplatin-induced muscle atrophy. Our results suggest that cisplatin-induced myogenesis suppression causes muscle atrophy and inhibits the expression of Sparcl1, which promotes C2C12 cell differentiation during myogenesis.

Hyperresponsiveness to Extracellular Acidification-Mediated Contraction in Isolated Bronchial Smooth Muscles of Murine Experimental Asthma.

PURPOSE: Extracellular acidification is a major component of tissue inflammation, including airway inflammation. The extracellular proton-sensing mechanisms are inherent in various cells including airway structural cells, although their physiological and pathophysiological roles in bronchial smooth muscles (BSMs) are not fully understood. In the present study, to explore the functional role of extracellular acidification on the BSM contraction, the isolated mouse BSMs were exposed to acidic pH under contractile stimulation. METHODS AND RESULTS: The RT-PCR analyses revealed that the proton-sensing G protein-coupled receptors were expressed both in mouse BSMs and cultured human BSM cells. In the mouse BSMs, change in the extracellular pH from 8.0 to 6.8 caused an augmentation of contraction induced by acetylcholine. Interestingly, the acidic pH-induced BSM hyper-contraction was further augmented in the mice that were sensitized and repeatedly challenged with ovalbumin antigen. In this animal model of asthma, upregulations of G protein-coupled receptor 68 (GPR68) and GPR65, that were believed to be coupled with Gq and Gs proteins respectively, were observed, indicating that the acidic pH could cause hyper-contraction probably via an activation of GPR68. However, psychosine, a putative antagonist for GPR68, failed to block the acidic pH-induced responses. CONCLUSION: These findings suggest that extracellular acidification contributes to the airway hyperresponsiveness, a characteristic feature of bronchial asthma. Further studies are required to identify the receptor(s) responsible for sensing extracellular protons in BSM cells.

Increased 20S Proteasome Expression and the Effect of Bortezomib during Cisplatin-Induced Muscle Atrophy.

Cisplatin is a chemotherapy drug used to treat a variety of cancers. Muscle loss in cancer patients is associated with increased cancer-related mortality. Previously, we suggested that cisplatin administration increases the atrophic gene expressions of ubiquitin E3 ligases, such as atrogin-1 and muscle RING finger-1 (MuRF1), which may lead to muscle atrophy. In this study, C57BL/6J mice were treated with cisplatin (3 mg/kg, intraperitoneally) or saline for 4 consecutive days. Twenty-four hours after the final injection of cisplatin, quadriceps muscles were removed from the mice. The gene expression of Psma and Psmb, which comprise the 20S proteasome, was upregulated by cisplatin administration in the quadriceps muscle of mouse. Systemic administration of cisplatin significantly reduced not only the quadriceps muscle mass but also the diameter of the myofibers. In addition, bortezomib (0.125 mg/kg, intraperitoneally) was administered 30 min before each cisplatin treatment. The co-administration of bortezomib, a proteasome inhibitor, significantly recovered the reductions in the mass of quadriceps and myofiber diameter, although it did not recover the decline in the forelimb and forepaw strength induced by cisplatin. Increased 20S proteasome abundance may play a significant role in the development of cisplatin-induced muscle atrophy. During cisplatin-induced skeletal muscle atrophy, different mechanisms may be involved between loss of muscle mass and strength. In addition, it is suggested that bortezomib has essentially no effect on cisplatin-induced muscle atrophy.

Inhibition of Spred/Sprouty Expression in the Skin of a Contact Dermatitis-Like Model.

We have previously reported that swellings caused by haptens, such as 2,4,6-trinitrochlorobenzene (TNCB), may be associated with the extracellular signal-regulated kinase (ERK)-induced proliferation pathway. However, the involvement of the Spred/Sprouty family as critical negative regulators of the Ras/Raf/ERK signaling pathway at disease sites is not well-established. Thus, in the present study, the effects of hapten-challenge on the expression levels of genes and proteins associated with the Spred/Sprouty family in the ear of mice were investigated. The activation of ERK and epidermal growth factor receptor (EGFR) tyrosine kinase was inhibited by their selective inhibitors, namely, U0126 and PD168393, respectively. Twenty-four hours after the final challenge by the haptens TNCB, 2,4-dinitrofluorobenzene, or oxazolone, ear thickness was augmented by challenge with all haptens and the gene expression levels of Spred1, Spred2, Sprouty1, and Sprouty2 in swelling induced by all haptens were significantly decreased. Furthermore, Spred2, Sprouty1, and Sprouty2 genes were decreased in the epidermis and dermis of the TNCB-challenged ear. In conclusion, it is possible that the mechanism of hapten-challenge-induced skin thickening involves not only the enhancement of cell proliferative functions via the activation of ERK by EGFR tyrosine kinase activation but also the decreases expression of Spred/Sprouty family members.

Effect of nadir hematocrit during cardiopulmonary bypass on the early outcomes after surgical repair of acute type A aortic dissection.

OBJECTIVE: Although hemodilution during hypothermic cardiopulmonary bypass (CPB) had been thought to improve microcirculation and reduce blood viscosity, there has been no report investigating the effect of low nadir hematocrit (Hct) values caused by severe hemodilution on the surgical outcomes of patients with acute type A aortic dissection (ATAAD). METHODS: We retrospectively reviewed 112 consecutive patients who emergently underwent emergency surgical repair of ATAAD at our institution. The patients were classified into the high Hct (nadir Hct ≥ 21% during CPB; n = 51) and low Hct (nadir Hct < 21% during CPB; n = 61) groups. After propensity score matching of preoperative characteristics, surgical outcomes were compared between the groups. RESULTS: Although there was no difference in the surgical procedure, longer CPB time and more blood transfusion during surgery were needed in the low Hct group than in the high Hct group. After surgery, estimated glomerular filtration rate was significantly lower (p = .015), lactaic acid was higher (p = .045), and intubation time was longer (p = .018) in the low Hct group than in the high Hct group, although there was no difference in hospital mortality between the groups. The AUC of the nadir Hct during CPB as a prognostic indicator of prolonged postoperative ventilator support was 0.8, with the highest accuracy at 16.7% (sensitivity 88%, specificity 76.9%). In all cohorts, female sex was an independent risk factor for a lower nadir Hct value of <21% during CPB. CONCLUSION: A lower nadir Hct value of <21% during CPB may be associated with postoperative renal dysfunction and prolonged ventilator support in patients with ATAAD.

Eicosapentaenoic acid suppresses cisplatin-induced muscle atrophy by attenuating the up-regulated gene expression of ubiquitin.

Previously it was shown that cisplatin causes muscle atrophy. Under this condition, cisplatin increased the expression of atorogenes, such as muscle ring finger 1 and atrogin-1 (also known as muscle atrophy F-box protein), in mouse skeletal muscle. It was reported recently that ubiquitin (Ub) and ubiquitinated protein levels in skeletal muscle were also up-regulated in cisplatin-induced muscle atrophy, and cisplatin-induced ubiquitinated proteins were degraded by the 26S proteasome pathway. Eicosapentaenoic acid (EPA) is effective against skeletal muscle atrophy in mice. However, it is unclear how EPA suppresses the Ub-proteasome pathway. In this study, the effect of EPA on cisplatin-induced muscle atrophy in mice was examined. Mice were intraperitoneally injected with cisplatin or vehicle control once daily for 4 d. EPA or its vehicle was orally administered 30 min before cisplatin administration. Cisplatin systemic administration induced decrease in muscle mass, myofiber diameter, and increase in Ub genes and ubiquitinated proteins in mouse skeletal muscle were recovered by co-treatment with EPA. However, weight loss and up-regulated atrogenes induced by cisplatin were not changed by co-treatment with EPA in skeletal muscle. In this study, EPA attenuated cisplatin-induced muscle atrophy via down-regulation of up-regulated Ub gene expression. Although further clinical studies are needed, EPA administration can be effective in the development of muscle atrophy in cisplatin-treated patients.

Efficacy of point-of-care thromboelastography 6s to evaluate platelet function in a patient with pseudothrombocytopenia undergoing cardiopulmonary bypass: a case report.

BACKGROUND: Pseudothrombocytopenia is a phenomenon caused by in vitro platelet aggregation induced by anticoagulants contained in blood sampling tubes. Thromboelastography (TEG) 6s is a common point-of-care viscoelastic test to assess intraoperative coagulation status, which may reduce the need for blood transfusions. The reliability and usefulness of TEG6s for patients with pseudothrombocytopenia has not been established. CASE PRESENTATION: We present a patient with pseudothrombocytopenia, who underwent suprarenal abdominal aortic aneurysm repair under cardiopulmonary bypass. At the beginning of surgery, TEG6s with citrated blood sampling showed that the results were unaffected by the disease. After completion of the aortic repair and the administration of protamine, maximum amplitude of TEG6s and adequate hemostasis were achieved without platelet transfusions, while standard laboratory tests showed thrombocytopenia. CONCLUSIONS: In the present patient who underwent cardiopulmonary bypass, TEG6s may have contributed to a reliable and useful assessment of coagulation and hemostatic status avoiding unnecessary platelet transfusions.

Down-regulation of miR-140-3p is a cause of the interlukin-13-induced up-regulation of RhoA protein in bronchial smooth muscle cells.

The current study aimed to determine the role of a microRNA (miRNA), miR-140-3p, in the control of RhoA expression in bronchial smooth muscle cells (BSMCs). In cultured human BSMCs, incubation with interleukin-13 (IL-13) caused an up-regulation of RhoA protein concurrently with a down-regulation of miR-140-3p. Transfection of the cells with a miR-140-3p inhibitor caused an increase in basal RhoA protein level. Although a mimic of miR-140-3p had little effect on the basal RhoA level, its treatment inhibited the IL-13-induced up-regulation of RhoA. These findings suggest that RhoA expression is negatively regulated by miR-140-3p, and that the negative regulation is inhibited by IL-13 to cause an up-regulation of RhoA protein in BSMCs.

Effect of PACAP on sweat secretion by immortalized human sweat gland cells.

The process of sweating plays an important role in the human body, including thermoregulation and maintenance of the environment and health of the skin. It is known that the conditions of hyperhidrosis and anhidrosis are caused by abnormalities in sweat secretion and can result in severe skin conditions such as pruritus and erythema, which significantly reduce the patient's quality of life. However, there are many aspects of the signaling mechanisms in the process of sweating that have not been clarified, and no effective therapies or therapeutic agents have yet been discovered. Previously, it was reported that pituitary adenylate cyclase-activating polypeptide (PACAP) promotes sweating, but details of the underlying mechanism has not been clarified. We used immortalized human eccrine gland cells (NCL-SG3 cell) to investigate how sweat secretion is induced by PACAP. Intracellular Ca2+ levels were increased in these cells following their exposure to physiological concentrations of PACAP. Intracellular Ca2+ was not elevated when cells were concomitantly treated with PA-8, a specific PAC1-R antagonist, suggesting that PAC1-R is involved in the elevation of intracellular Ca2+ levels in response to PACAP treatment. Furthermore, immunocytochemistry experiments showed that aquaporin-5 was translocated from the cytoplasm to the cell membrane by PACAP. These results suggest that PACAP acts on eccrine sweat glands to promote sweat secretion by translocation of aquaporin-5 to the cell membrane in response to increased levels of intracellular Ca2+. These findings also provide a solid basis for future research initiatives to develop new therapies to treat sweating disorders.

Exogenous insulin-like growth factor 1 attenuates cisplatin-induced muscle atrophy in mice.

BACKGROUND: A reduction in the skeletal muscle mass worsens the prognosis of patients with various cancers. Our previous studies indicated that cisplatin administration to mice caused muscle atrophy. This is a concern for human patients receiving cisplatin. The insulin-like growth factor 1 (IGF-1)/phosphoinositide 3-kinase (PI3K)/Akt pathway stimulates the rate of protein synthesis in skeletal muscle. Thus, IGF-I can be a central therapeutic target for preventing the loss of skeletal muscle mass in muscle atrophy, although it remains unclear whether pharmacological activation of the IGF-1/PI3K/Akt pathway attenuates muscle atrophy induced by cisplatin. In this study, we examined whether exogenous recombinant human IGF-1 attenuated cisplatin-induced muscle atrophy. METHODS: Male C57BL/6J mice (8-9 weeks old) were injected with cisplatin or saline for four consecutive days. On Day 5, quadriceps muscles were isolated. Mecasermin (recombinant human IGF-1) or the vehicle control was subcutaneously administered 30 min prior to cisplatin administration. A dietary restriction group achieving weight loss equivalent to that caused by cisplatin administration was used as a second control. C2C12 myotubes were treated with cisplatin with/without recombinant mouse IGF-1. The skeletal muscle protein synthesis/degradation pathway was analysed by histological and biochemical methods. RESULTS: Cisplatin reduced protein level of IGF-1 by about 85% compared with the vehicle group and also reduced IGF-1/PI3K/Akt signalling in skeletal muscle. Under this condition, the protein levels of muscle ring finger protein 1 (MuRF1) and atrophy gene 1 (atrogin-1) were increased in quadriceps muscles (MuRF1; 3.0 ± 0.1 folds, atrogin-1; 3.0 ± 0.3 folds, P < 0.001, respectively). The administration of a combination of cisplatin and IGF-1 significantly suppressed the cisplatin-induced downregulation of IGF-1/PI3K/Akt signalling and upregulation of MuRF1 and atrogin-1 (up to 1.6 ± 0.3 and 1.5 ± 0.4 folds, P < 0.001, respectively), resulting in diminished muscular atrophy. IGF-1 showed similar effects in cisplatin-treated C2C12 myotubes, as well as the quadriceps muscle in mice. CONCLUSIONS: The downregulation of IGF-1 expression in skeletal muscle might be one of the factors playing an important role in the development of cisplatin-induced muscular atrophy. Compensating for this downregulation with exogenous IGF-1 suggests that it could be a therapeutic target for limiting the loss of skeletal muscle mass in cisplatin-induced muscle atrophy.

Increased Gene expression of CCL2/CCR2 axis in bronchial smooth muscles of allergen-challenged mice.

PURPOSE: Augmented bronchial smooth muscle (BSM) contraction is a cause of airway hyperresponsiveness (AHR) in asthma. Increasing evidence suggest that C-C motif chemokine 2 (CCL2) modulates smooth muscle contractility by activating its binding partner C-C chemokine receptor type 2 (CCR2). In the present study, changes in the gene expression of CCL2/CCR2 axis were determined in the BSMs of a murine model of allergic asthma. MATERIALS AND METHODS: The ovalbumin (OA)-sensitized mice were repeatedly challenged with aerosolized OA to induce asthmatic reaction. Twenty-four hours after the last antigen challenge, total RNAs of the main BSM tissues and bronchoalveolar lavage fluids (BALFs) were obtained. RESULTS: Our published microarray data (GEO accession No. GSE116504) detected changes in gene expression associated with the chemokine signaling pathway (KEGG Map ID: 04062) in BSMs of mice with AHR induced by antigen exposure. Among them, quantitative RT-PCR analyses showed significant increase in mRNA expression of Ccl2 and Ccr2. Analysis of BALFs also revealed a significant increase in Ccl2 protein in the airways of the diseased animals. CONCLUSION: It is thus possible that, in association with the AHR, the CCL2/CCR2 axis is enhanced in the airways of allergic bronchial asthma.

[Non-coding RNAs and bronchial smooth muscle hyperresponsiveness in allergic bronchial asthma].

Non-coding RNAs, such as microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), play important roles in normal and diseased cell functions. A small GTPase RhoA is a key protein of bronchial smooth muscle (BSM) contraction, and an up-regulation of RhoA has been demonstrated in BSMs of experimental asthma. Our previous study also demonstrated that RhoA translation was controlled by a miRNA, miR-133a, in BSMs. In human BSM cells (hBSMCs), an up-regulation of RhoA was observed when the function of endogenous miR-133a was inhibited by its antagomir. Treatment of hBSMCs with interleukin-13 (IL-13) caused an up-regulation of RhoA and a down-regulation of miR-133a. In a murine experimental asthma, increased expression of IL-13 and RhoA and the BSM hyperresponsiveness were observed. Interestingly, the level of miR-133a was significantly decreased in BSMs of the diseased animals. These findings suggest that RhoA expression is negatively regulated by miR-133a in BSMs, and that the miR-133a down-regulation causes an up-regulation of RhoA, resulting in an augmentation of the contraction. Recent studies also revealed an inhibitory effect of lncRNA Malat1 on the miR-133a function. Thus, lncRNAs/miRNAs might be key regulators of BSM hyperresponsiveness, and provide us a new insight into the treatment of airway hyperresponsiveness in asthmatics.