Use of milrinone in critically ill children

Background

Optimal dose adjustment of milrinone in critically ill children is challenging because of conflicting information about the association between dose and outcomes in this age group.

Objectives

To describe the use of milrinone in critically ill children and to explore associations between milrinone dosing and clinical outcomes, specifically effectiveness and adverse events.

Methods

This retrospective cohort study was performed in a consecutive sample of children admitted to a university-affiliated critical care unit (January to June 2004). The relations between milrinone dosing and its effectiveness (based on prevention of low cardiac output syndrome, defined as a difference in oxygen saturation between arterial and mixed venous blood of at least 30% or an increase in serum lactate > 2 mmol/L) and its adverse effects (thrombocytopenia, arrhythmia) were evaluated by logistic regression.

Results

A total of 197 children from 213 admissions (ranging in age from newborn to 18 years) were included in the study. Milrinone was initiated with a median loading dose of 99.2 μg/kg (range 22.1–162.2 μg/kg). The initial loading dose was higher if given in the operating room rather than the Critical Care Unit (median 99.7 versus 51.0 μg/kg; p < 0.001). Subsequent loading doses, for patients who received them, were lower (median 49 μg/kg). Milrinone was infused at a median rate of 0.64 μg/kg per minute (range 0.13–2.08 μg/kg per minute) for a median of 43.1 h. There was no relation between serum creatinine level and the maintenance dose of milrinone (r ² ≤ 0.0335). Low cardiac output syndrome was relatively frequent (166 [77.9%] of the 213 admissions). There was a trend for occurrence of this syndrome in patients with greater average milrinone dose rate (odds ratio [OR] 8.21, 95% confidence interval [CI] 0.98–69.15, p = 0.053) and with longer duration of milrinone therapy (OR 1.01, 95% CI 1.01–1.02, p < 0.05). Adverse events were relatively frequent (thrombocytopenia for 27 admissions [12.7%], arrhythmia for 82 admissions [38.5%]) but were not significantly associated with milrinone dosing.

Conclusions

A retrospective evaluation of milrinone use in critically ill children revealed variable utilization and frequent occurrence of both low cardiac output syndrome and adverse events. Further prospective research is needed to understand the impact of individual pharmacokinetic differences on pharmacodynamic responses, to guide optimal dose adjustment, improve outcomes, and minimize toxic effects.     

Does Etomidate Increase Vasopressor Requirements in Patients Needing Mechanical Ventilation?

Background:

Single-dose etomidate is used as an induction agent for rapid-sequence intubation and is associated with transient adrenal insufficiency. There is ongoing debate as to the clinical consequences of this transient adrenal insufficiency for critically ill patients.

Objective:

To determine if the use of etomidate is associated with higher requirements for a vasopressor, relative to other induction agents, at a single time point (24 h after administration of the induction agent) in patients needing mechanical ventilation.

Methods:

In this retrospective observational study utilizing electronic health records, a convenience sample of 50 patients who had undergone intubation in the emergency department with etomidate were matched (1:1) with patients who had received other induction agents. Matching was based on primary admitting diagnosis relating to the cause of shock, APACHE II (Acute Physiology and Chronic Health Evaluation II) score, age, and sex. All patients were subsequently admitted to critical care areas for management. As a surrogate marker of hemodynamic instability, the vasopressor dose was recorded 24 h after intubation. Vasopressor doses were converted to norepinephrine equivalents for comparison.

Results:

The mean dose of vasopressors, in norepinephrine equivalents, was 4 μg/min⁻¹ for patients receiving etomidate and 3 μg/min⁻¹ for the control group (mean difference 0.7 μg min⁻¹, 95% confidence interval [CI] −1.9 to 3.2 μg min⁻¹, p = 0.61). Twelve of the patients in the etomidate group and 16 of those in the control group required the use of vasopressors at 24 h following intubation (odds ratio 2.3, 95% CI 0.53 to 13.99, p = 0.34).

Conclusions:

Single-dose etomidate does not adversely affect hemodynamic stability, as measured by the dose of vasopressors required at 24 h after administration.     

Lactoferrin inhibits apoptosis through insulin-like growth factor I in primary rat osteoblasts

Aim： Excessive apoptosis of osteoblasts is the major cause of low bone mass, and bovine lactoferrin （bLF）, an iron-binding glycoprotein, might protect osteoblastic cells from apoptosis induced by serum withdrawal. The aim of this study was to elucidate the mechanisms underlying the anti-apoptotic action of bLF in rat osteoblasts in vitro. Methods： Primary rat osteoblasts were incubated in the presence of varying concentrations of bLF for 24 h. The expression of insulin-like growth factor I （IGF-I） and IGF-I receptor （IGF-IR） was measured uisng RT-PCR and Western blotting. Cell apoptosis was examined with flow cytometry. siRNAs targeting IGF-I was used in this study.

Results： Treatment of bLF （0.1–1000 μg/mL） dose-dependently increased the expression of IGF-I and IGF-IR in the osteoblasts. Treatment with bLF （10, 100 μg/mL） markedly inhibited the osteoblast apoptosis （with the rate of total apoptosis of 70% at 10 μg/mL）, but the high concentration of bLF （1000 μg/mL） significantly promoted the osteoblast apoptosis. Knockdown of the IGF-I gene in osteoblasts with siRNA markedly increased the osteoblast apoptosis.

Conclusion： Lactoferrin （10 and 100 μg/mL） effectively inhibits apoptosis of primary rat osteoblasts by upregulating IGF-I expression.     

Dexmedetomidine blocks thermal hyperalgesia and spinal glial activation in rat model of monoarthritis

Aim:

To investigate the effect of systemic administration dexmedetomidine, a selective alpha 2 adrenergic receptor (α₂AR) agonist, on thermal hyperalgesia and spinal glial activation evoked by monoarthritis (MA).

Methods:

MA was induced by an intra-articular injection of complete Freund''s adjuvant (CFA). Thermal hyperalgesia was measured by Hargreaves'' test. The spinal glial activation status was analyzed by GFAP (an astrocytic marker) and Iba-1 (a microglial marker) immunohistochemistry or immunoblotting.

Results:

Unilateral intra-articular injection of CFA produced a robust glial activation of astrocytes and microglia in the spinal cord, which was associated with the development and maintenance of thermal hyperalgesia. Intraperitoneal (ip) injection of dexmedetomidine (2.5 and 10 μg/kg) was repeatedly given once daily for 5 days with the first injection 60 min before intra-articular CFA. At the dose of 10 μg/kg, dexmedetomidine significantly attenuated MA-induced ipsilateral hyperalgesia from day 2 to day 5. MA-induced up-regulation of GFAP expression on both sides of the spinal dorsal horn was significantly suppressed by day 5 post-MA following dexmedetomidine application, whereas MA-induced Iba-1 up-regulation was only partially suppressed.

Conclusion:

Systemic dexmedetomidine inhibits the activation of spinal glia, which is possibly associated with its antihyperalgesia in monoarthritic rats.     

Gemcitabine combined with gum mastic causes potent growth inhibition and apoptosis of pancreatic cancer cells

Aim:

To investigate the antiproliferative and apoptotic effects of gemcitabine combined with gum mastic and the underlying mechanisms in human pancreatic cancer cell lines.

Methods:

Cell proliferation and apoptosis were examined using the methyl thiazolyl tetrazolium (MTT) assay and propidium iodine staining, respectively. The expression of Bcl-2, Bax, NF-κB p65 subunit, and IκBα protein was measured using Western blotting.

Results:

Gemcitabine 0.01−100 μg/mL inhibited cell proliferation and induced apoptosis in both pancreatic cancer BxPC-3 and COLO 357 cells. Gum mastic 40 μg/mL significantly potentiated the antiproliferative and apoptotic effects of gemcitabine 10 μg/mL after 72-h treatment. When cells were treated with gemcitabine in combination with gum mastic, the IκBα level was increased, whereas NF-κB activation was blocked; the expression of Bax protein was substantially increased, but Bcl-2 protein was down-regulated.

Conclusion:

Gemcitabine combined with gum mastic causes potent apoptosis in pancreatic cancer cells. The combination may be an effective therapeutic strategy for pancreatic cancer.     

Anti-tumor activity of CrTX in human lung adenocarcinoma cell line A549

Aim:

To assess the cytotoxic effect of crotoxin (CrTX), a potent neurotoxin extracted from the venom of the pit viper Crotalus durissus terrificus, in human lung adenocarcinoma A549 cells and investigated the underlying mechanisms.

Methods:

A549 cells were treated with gradient concentrations of CrTX, and the cell cycle and apoptosis were analyzed using a flow cytometric assay. The changes of cellular effectors p53, caspase-3 and cleaved caspase-3, total P38MAPK and pP38MAPK were investigated using Western blot assays. A549 xenograft model was used to examine the inhibition of CrTX on tumor growth in vivo.

Results:

Treatment of A549 cells with CrTX (25–200 μg/mL) for 48 h significantly inhibited the cell growth in a dose-dependent manner (IC₅₀=78 μg/mL). Treatment with CrTX (25 μg/mL) for 24 h caused G1 arrest and induced cell apoptosis. CrTX (25 μg/mL) significantly increased the expression of wt p53, cleaved caspase-3 and phospho-P38MAPK. Pretreatment with the specific P38MAPK inhibitor SB203580 (5 μmol/L) significantly reduced CrTX-induced apoptosis and cleaved caspase-3 level, but G₁ arrest remained unchanged and highly expressed p53 sustained. Intraperitoneal injection of CrTX (10 μg/kg, twice a week for 4 weeks) significantly inhibited A549 tumor xenograft growth, and decreased MVD and VEGF levels.

Conclusion:

CrTX produced significant anti-tumor effects by inducing cell apoptosis probably due to activation of P38MAPK and caspase-3, and by cell cycle arrest mediated by increased wt p53 expression. In addition, CrTX displayed anti-angiogenic effects in vivo.     

Bleomycin induces upregulation of lysyl oxidase in cultured human fetal lung fibroblasts

Aim:

To investigate the mechanism of bleomycin (BLM)-induced pulmonary fibrosis.

Methods:

Cultured human fetal lung fibroblast (HLF) cells were exposed to bleomycin (BLM) at 0–30 μg/mL for 24 h. Western blot analysis was used to detect lysyl oxidase (LO) protein expression. Real-time RT-PCR was used to detect LO mRNA level. LO catalytic activity was measured using diaminopentane as a substrate and Amplex red as a hydrogen peroxide probe. Copper (Cu) concentration was detected by flame atomic absorption spectrophotometry.

Results:

Exposure of HLF cells to BLM at 10 μg/mL and 30 μg/mL increased LO catalytic activity to 130% and 158% of the control in the conditioned media. The expression of LO mRNA was increased to 5.5-fold of the control in HLF cells exposure to BLM at 3 μg/mL. BLM at 3 μg/mL also increased the expression of 46 kDa preproLO, 50 kDa proLO and 32 kDa mature LO to 219%, 130%, and 135% of the control, respectively. The Cu concentrations in conditioned media of cultured HLF cells exposed to BLM (10 and 30 μg/mL) were increased significantly to 1.48 and 2.46-fold of the control, respectively.

Conclusion:

Bleomycin induces upregulation of LO in cultured human fetal lung fibroblasts, which may be the mechanism of bleomycin-induced pulmonary fibrosis.     

Involvement of cholinergic system in suppression of formalin-induced inflammatory pain by cobratoxin

Aim:

To investigate the analgesic effect of cobratoxin (CTX), a long-chain α-neurotoxin from Thailand cobra venom, in a rat model of formalin-induced inflammatory pain.

Methods:

Inflammatory pain was induced in SD rats via injecting 5% formalin (50 μL) into the plantar surface of their right hind paw. CTX and other agents were ip administered before formalin injection. The time that the animals spent for licking the injected paw was counted every 5 min for 1 h.

Results:

CTX (25, 34, and 45 μg/kg) exhibited a dose-dependent analgesic effect during the phase 1 (0–15 min) and phase 2 (20–60 min) response induced by formalin. Pretreatment with naloxone (0.5 or 2.5 mg/kg) did not block the analgesic effect of CTX. Pretreatment with atropine at 5 mg/kg, but not at 2.5 mg/kg, antagonized the analgesic effect of CTX. Treatment with the nonselective nAChR antagonist mecamylamine (3 mg/kg) inhibited the analgesic effects of CTX in Phase 1 and Phase 2 responses, while with the selective α7-nAChR antagonist methyllycaconitine (3 mg/kg) antagonized the effect of CTX only in the Phase 1 response. Treatment with the α7-nAChR agonist PNU282987 (3 mg/kg) significantly reduced the formalin-induced phase 2 pain response, but only slightly reduced the Phase 1 pain response.

Conclusion:

The results suggest that CTX exerts an antinociceptive effect in formalin-induced inflammatory pain, which appears to be mediated by mAChR and α7-nAChR.     

Mercury Exposure Among Artisanal Gold Miners in Madre de Dios, Peru: A Cross-sectional Study

Introduction

Exposure to mercury, a toxic metal, occurs primarily from inhaling mercury vapors or consuming methylmercury-contaminated fish. One third of all anthropogenic mercury emissions worldwide are from artisanal gold mining, which uses mercury to extract gold. Although recent reports suggest that the Madre de Dios region in Peru (with >30,000 artisanal miners) has extensive mercury contamination, residents had never been assessed for mercury exposure. Thus, our objective was to quantify mercury exposure among residents of an artisanal mining town in Madre de Dios and to assess risk factors for exposure.

Methods

We conducted a cross-sectional assessment of 103 residents of an artisanal gold mining town in July 2010. Each participant provided a urine and blood sample and completed a questionnaire assessing potential exposures and health outcomes. We calculated geometric mean (GM) urine total mercury and blood methylmercury concentrations and compared log-transformed concentrations between subgroups using linear regression.

Results

One third (34.0 %) of participants were gold miners. All participants had detectable urine total mercury (GM, 5.5 μg/g creatinine; range, 0.7–151 μg/g creatinine) and 91 % had detectable blood methylmercury (GM, 2.7 μg/L; range, 0.6–10 μg/L); 13 participants (13 %) reported having kidney dysfunction or a neurological disorder. Urine total mercury concentrations were higher among people who heated gold–mercury amalgams compared with people who never heated amalgams (p < 0.05); methylmercury concentrations were higher among fish consumers compared with nonfish consumers (p < 0.05).

Conclusion

Our findings suggest that mercury exposure may be widespread in Huaypetue.     

Nerve growth factor induces cord formation of mesenchymal stem cell by promoting proliferation and activating the PI3K/Akt signaling pathway

Aim:

To investigate whether nerve growth factor (NGF) induced angiogenesis of bone marrow mesenchymal stem cells (MSCs) and the underlying mechanisms.

Methods:

Bone marrow MSCs were isolated from femors or tibias of Sprague-Dawley rat, and cultured. The cells were purified after 3 to 5 passages, seeded on Matrigel-coated 24-well plates and treated with NGF. Tube formation was observed 24 h later. Tropomyosin-related kinase A (TrkA) and p75NTR gene expression was examined using PCR analysis and flow cytometry. Growth curves were determined via cell counting. Expression of VEGF and pAkt/Akt were analyzed with Western blot.

Results:

NGF (25, 50, 100 and 200 μg/L) promoted tube formation of MSCs. The tubular length reached the maximum of a 2.24-fold increase, when the cells were treated with NGF (50 μg/L). NGF (50 μg/L) significantly enhanced Akt phosphorylation. Pretreatment with the specific PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (10 μmol/L) blocked NGF-stimulated Akt phosphorylation, tube formation and angiogenesis. NGF (25–200 μg/L) did not affect the expression of TrkA and vascular endothelial growth factor (VEGF), but significantly suppressed the expression of p75NTR. NGF (50 μg/L) markedly increased the proliferation of MSCs.

Conclusion:

NGF promoted proliferation of MSCs and activated the PI3K/Akt signaling pathway, which may be responsible for NGF induction of MSC angiogenesis.     

Management of Lead Encephalopathy with DMSA After Exposure to Lead-Contaminated Moonshine

Background

Lead encephalopathy is a severe manifestation of lead poisoning that can present with altered mental status and seizures and has been associated with illicit moonshine consumption. Lead encephalopathy has traditionally been treated using dimercaprol (British anti-Lewisite, BAL) and calcium disodium ethylenediaminetetraacetic acid (CaNa₂EDTA).

Case Report

We describe a patient with lead encephalopathy related to lead-contaminated moonshine consumption, who was treated using dimercaptosuccinic acid (DMSA) due to a national shortage of CaNa₂EDTA. A 66-year-old woman presented to a hospital with headache, irritability, and altered mental status. On hospital day 16, she was found to have a whole blood lead concentration of 148.2 μg/dL and a 24-h urine lead concentration of 232 μg/day. Due to a national shortage of CaNa₂EDTA, the patient was given one dose of BAL and then started on DMSA via nasogastric tube. She dramatically improved over 4 days and was subsequently transitioned to oral DMSA and outpatient treatment. One day prior to discharge, her whole blood lead concentration was 47.2 μg/dL and her mental status was normal. DMSA was used in lieu of CaNa₂EDTA to treat the patient with lead encephalopathy. The patient subsequently experienced clinical improvement and declining whole blood level concentrations.

Conclusion

Further prospective studies are needed to compare the efficacy of DMSA versus CaNa₂EDTA in patients with lead encephalopathy.     

Diclofenac readily penetrates the cerebrospinal fluid in children

AIMS

The primary aim was to study the cerebrospinal fluid (CSF) penetration of intravenous diclofenac in children. The secondary aim was to evaluate the plasma diclofenac concentration at the onset of wound pain after inguinal surgery in children.

METHODS

A total of 31 children (24 boys) aged 3 months to 12 years received a single intravenous injection of diclofenac 1 mg kg⁻¹. Paired CSF and blood samples were obtained 5 min to 22 h (median 69 min) later. In children having inguinal surgery a second blood sample was obtained at the time that the children felt wound pain for the first time after surgery. Diclofenac concentrations in CSF, plasma and protein free plasma were measured by gas chromatography with mass spectrometric detection.

RESULTS

In the 28 CSF samples obtained at 5 min to 3 h 43 min after injection, diclofenac concentrations ranged between 0.5 and 4.7 μg l⁻¹. At 5.5 h the CSF concentration was 0.1 μg l⁻¹, and no diclofenac was detected in the two CSF samples obtained at 22 h. The median of plasma diclofenac concentration at the time when pain returned after inguinal surgery was 104 μg l⁻¹ (range 70–272 μg l⁻¹). No serious or unexpected adverse effects were reported.

CONCLUSIONS

Diclofenac penetrates the CSF rapidly, and a sufficient concentration to inhibit cyclooxygenase enzymes is sustained for up to 4 h.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT

• Diclofenac, a nonselective nonsteroidal anti-inflammatory drug,, exerts analgesic action both in the peripheral tissues and in the central nervous system by inhibiting cyclooxygenase enzymes COX-1/2, but central nervous system penetration of diclofenac has not been evaluated in humans.

WHAT THIS STUDY ADDS

• Diclofenac penetrates the cerebrospinal fluid rapidly, and after a single intravenous dose of 1 mg kg⁻¹, sufficient concentrations to inhibit COX-1/2 are sustained for up to 4 h.

     

Effect of erythromycin and rifampicin on monoethylglycinexylidide test

Background:

The dynamic liver function test based on the hepatic conversion of lidocaine to monoethylglycinexylidide (MEGX) provides a direct measure of the actual functional state of the liver. Cytochrome P450 (CYP) 3A4 has been proposed as the main CYP isoform responsible for MEGX formation. The concomitant use of either CYP3A4 inducer rifampicin or CYP3A4 inhibitor erythromycin may influence the results of MEGX test. Hence, the objective of this study was to evaluate the effect of a CYP3A4 inhibitor erythromycin and inducer rifampicin on the MEGX test.

Materials and Methods:

The study included 20 healthy male volunteers whose routine laboratory tests were normal. As per study protocol, MEGX test was carried out in all the participants after an overnight fast. All the participants were given 1 mg/kg lidocaine dose intravenously and MEGX concentration at 30 and 60 min after IV dose was measured using HPLC. These MEGX values served as control values. Ten subjects received 600 mg/day erythromycin orally for six days while remaining ten participants received 600 mg/day rifampicin orally for six days. On the sixth day, MEGX test was carried out two hours after the last dose.

Result:

Rifampicin increased the mean plasma concentration of MEGX₃₀ from 93.94 ± 26.31 to 98.54 ± 24.94 μg/ml (P = 0.085) and MEGX₆₀ from 134.34 ± 35.42 to 136.36 ± 33.14 μg/ml (P = 0.051). Erythromycin lowered the serum concentration of MEGX₃₀ from 101.37 ± 39.39 to 96.67 ± 36.09 μg/ml (P = 0.128) and MEGX₆₀ from 142.52 ± 42.65 to 138.98 ± 40.23 μg/ml (P = 0.159).

Conclusion:

It can be concluded from this study that the MEGX test is not affected by concomitant administration of CYP3A4 modifiers rifampicin and erythromycin.     

Ganoderma lucidum polysaccharides reduce methotrexate-induced small intestinal damage in mice via induction of epithelial cell proliferation and migration

Aim:

To study the effects of Ganoderma lucidum polysaccharides (Gl-PS) on methotrexate (MTX)-induced small intestinal damage in mice and the underlying mechanisms.

Methods:

BALB/c mice were used for in vivo study. The mice were administered with Gl-PS (50, 100, or 200 mg/kg, ig) for 10 d, and injected with MTX (50 mg/kg, ip) on d 7 and 8 to induce intestinal damage, and then sacrificed on d 11 for morphological study and tissue malondialdehyde (MDA) and superoxide dismutase (SOD) measurements. Before sacrificing, blood samples were collected to analyze immunoglobulin A (IgA). Rat intestinal IEC-6 cells were used for in vitro study. Cell proliferation and migration were assessed using MTT method and an in vitro wounding model, respectively. Transforming growth factor β (TGFβ) protein expression was determined using ELISA assay. Ornithine decarboxylase (ODC) and c-Myc mRNA expression profiles were determined using RT-PCR.

Results:

MTX treatment caused severe mucosal damage, significantly increased small intestine MDA levels, and decreased SOD and serum IgA levels in BALB/c mice. Gl-PS (100 and 200 mg/kg) markedly reversed the MTX effects. In IEC-6 cells, Gl-PS (0.1, 1, and 10 μg/mL) significantly stimulated the cell proliferation. Furthermore, Gl-PS (10 μg/mL) significantly stimulated the cell migration. In addition, Gl-PS (10 and 20 μg/mL) significantly increased the expression of ODC and c-Myc mRNAs. However, Gl-PS (up to 20 μg/mL) had no effect on the expression of TGFβ protein.

Conclusion:

The results suggest that Gl-PS protects small intestine against MTX-induced injury via induction of epithelial cell proliferation and migration.     

Effects of ATPM-ET, a novel κ agonist with partial μ activity, on physical dependence and behavior sensitization in mice

Aim:

To investigate the effects of ATPM-ET [(−)-3-N-Ethylaminothiazolo [5,4-b]-N-cyclopropylmethylmorphinan hydrochloride] on physical dependence and behavioral sensitization to morphine in mice.

Methods:

The pharmacological profile of ATPM-ET was characterized using competitive binding and GTPγS binding assays. We then examined the antinociceptive effects of ATPM-ET in the hot plate test. Morphine dependence assay and behavioral sensitization assay were used to determine the effect of ATPM-ET on physical dependence and behavior sensitization to morphine in mice.

Results:

The binding assay indicated that ATPM-ET ATPM-ET exhibited a high affinity to both κ- and μ-opioid receptors with K_i values of 0.15 nmol/L and 4.7 nmol/L, respectively, indicating it was a full κ-opioid receptor agonist and a partial μ-opioid receptor agonist. In the hot plate test, ATPM-ET produced a dose-dependent antinociceptive effect, with an ED₅₀ value of 2.68 (2.34–3.07) mg/kg. Administration of ATPM-ET (1 and 2 mg/kg, sc) prior to naloxone (3.0 mg/kg, sc) injection significantly inhibited withdrawal jumping of mice. In addition, ATPM-ET (1 and 2 mg/kg, sc) also showed a trend toward decreasing morphine withdrawal-induced weight loss. ATPM-ET (1.5 and 3 mg/kg, sc) 15 min before the morphine challenge significantly inhibited the morphine-induced behavior sensitization (P<0.05).

Conclusion:

ATPM-ET may have potential as a therapeutic agent for the treatment of drug abuse.     

Telmisartan attenuates isoproterenol-induced cardiac remodeling in rats via regulation of cardiac adiponectin expression

Aim:

To investigate whether telmisartan (Telm) pretreatment attenuates isoproterenol (Iso)-induced postinfarction remodeling (PIR) in rats, and whether the effect of Telm is associated with cardiac expression of adiponectin.

Methods:

PIR was induced in male Wistar rats with two consecutive injections of Iso (80 mg/kg, sc) at an interval of 24 h. Primary culture of ventricular myocytes from neonatal rats was prepared. Iso-induced cardiomyocyte injury was assessed based on cell growth and lactate dehydrogenase (LDH) activity. Cardiac adiponectin expression was measured using qRT-PCR and immunoblot analysis.

Results:

In the rats with PIR, Telm (10 mg·kg⁻¹·d⁻¹, po for 65 d) suppressed Iso-induced increases in gravimetric parameters, cardiomyocyte diameter and collagen volume fraction, but had no effect on Iso-induced myocardial hypertrophy and interstitial fibrosis. The protective effect of Telm was associated with enhanced protein expression of cardiac adiponectin. In cultured cardiomyocytes, Telm (5–20 μmol/L) inhibited the cell death and LDH release induced by Iso (10 μmol/L), and reversed Iso-induced reduction in adiponectin protein expression. In cardiomyocytes exposed to Iso (20 μmol/L), GW9662 (30 μmol/L), a selective antagonist of PPAR-γ, blocked the effects of Telm pretreatment on adiponectin protein expression, as well as the protective effects of Telm on Iso-induced cell injury.

Conclusion:

Telm attenuates Iso-induced cardiac remodeling and cell injury, which is associated with induction of cardiac adiponectin expression.     

Atorvastatin prevents amyloid-13 peptide oligomer- induced synaptotoxicity and memory dysfunction in rats through a p38 MAPK-dependent pathway

Aim： To investigate whether atorvastatin treatment could prevent Aβ1-42 oligomer （AβO）-induced synaptotoxicity and memory dysfunction in rats, and to elucidate the mechanisms involved in the neuroprotective actions of atorvastatin.
Methods： SD rats were injected with AβOs （5 nmol, icv）. The rats were administrated with atorvastatin （10 mg·kg^-1·d^-1, po） for 2 consecutive weeks （the first dose was given 5 d before AβOs injection）. The memory impairments were evaluated with Morris water maze task. The expression of inflammatory cytokines in the hippocampus was determined using ELISA assays. The levels of PSD-95 and p38MAPK proteins in rat hippocampus were evaluated using Western blot analysis. For in vitro experiments, cultured rat hippocampal neurons were treated with AβOs （50 nmol/L） for 48 h. The expression of MAP-2 and synaptophysin in the neurons was detected with immunofluorescence.
Results： The AβO-treated rats displayed severe memory impairments in Morris water maze tests, and markedly reduced levels of synaptic proteins synaptophysin and PSD-95, increased levels of inflammatory cytokines （IL-1β, IL-6 and TNF-α） and p38MAPK activation in the hippocampus. All these effects were prevented or substantially attenuated by atorvastatin administration. Pretreatment of cultured hippocampal neurons with atorvastatin （1 and 5 μmol/L） concentration-dependently attenuated the AβO-induced synaptotoxicity, including the loss of dendritic marker MAP-2, and synaptic proteins synaptophysin and PSD-95. Pretreatment of the cultured hippocampal neurons with the p38MAPK inhibitor SB203580 （5 μmol/L） blocked the AβO-induced loss of synaptophysin and PSD-95.
Conclusion： Atorvastatin prevents AβO-induced synaptotoxicity and memory dysfunction through a p38MAPK-dependent pathway.     

Huperzine A derivative M3 protects PC12 cells against sodium nitroprusside-induced apoptosis

Aim:

To investigate the effects of M3, a derivative of huperzine A, on the apoptosis induced by sodium nitroprusside (SNP) in PC12 cells.

Methods:

Cell viability was detected using MTT method. Apoptosis was examined with annexin V/prodium iodide (PI) stain. The levels of reactive oxygen species (ROS) were measured using fluorophotometric quantitation. The amount of malonaldehyde (MDA) was determined with MDA detection kits. The expression of caspase-3 and Hsp70 were analyzed using Western blotting.

Results:

Exposure of PC12 cells to SNP (200 μmol/L) for 24 h decreased the cell viability to 69.0% of that in the control group. Pretreatment with M3 (10 μmol/L) or huperzine A (10 μmol/L) significantly protected the cells against SNP-induced injury and apoptosis; the ratio of apoptotic bodies in PC12 cells was decreased from 27.3% to 15.0%. Pretreatment with M3 (10 μmol/L) significantly decreased ROS and MDA levels, and increased the expression of Hsp70 in the cells. Quercetin (10 μmol/L) blocked the protective effect of M3, while did not influence on that of huperzine A.

Conclusion:

M3 protects PC12 cells against SNP-induced apoptosis, possible due to ROS scavenging and Hsp70 induction.     

Interleukin-17A is involved in development of spontaneous pulmonary emphysema caused by Toll-like receptor 4 mutation

Aim:

To explore the pathogenic role of Th17 cells and interleukin-17A (IL-17A)-associated signaling pathways in spontaneous pulmonary emphysema induced by a Toll-like receptor 4 mutant (TLR4^mut).

Methods:

Lungs were obtained from wild-type (WT) or TLR4mut mice that were treated with or without recombinant mouse IL-17A (1 μg·kg⁻¹·d⁻¹, ip) from the age of 3 weeks to 3 months. Pulmonary emphysema was determined using histology, immunochemistry, and biochemical analysis. T cell polarization was determined with flow cytometry, the levels of cytokines were measured using ELISA, and the levels of IL-17A-associated signaling molecules were detected using Western blot.

Results:

Compared to WT mice, 3 month-old TLR4^mut mice were characterized by significantly reduced infiltration of Th17 cells into lungs (2.49%±1.13 % νs 5.26%±1.39%), and significantly reduced expression levels of IL-17A (3.66±0.99 pg/μg νs 10.67±1.65 pg/μg), IL-23 (12.43±1.28 pg/μg νs 28.71±2.57 pg/μg) and IL-6 (51.82±5.45 pg/μg νs 92.73±10.91 pg/μg) in bronchoalveolar lavage fluid. In addition, p38 MAPK phosphorylation and AP-1 expression were decreased to 27%±9% and 51%±8%, respectively, of that in WT mice. Treatment of TLR4^mut mice with IL-17A increased the infiltration of Th17 cells into lungs and expression levels of IL-17A, IL-6, and IL-23 in bronchoalveolar lavage fluid, attenuated MDA and apoptosis, and improved emphysema accompanied with increased phosphorylation of p38 MAPK and expression of AP-1.

Conclusion:

Th17 cells, in particular the cytokine IL-17A, play a crucial role in the pathogenesis of TLR4^mut-induced spontaneous pulmonary emphysema. Both of them are potential targets for therapeutic strategies for pulmonary emphysema.     

Enhancement of cytotoxicity of antimicrobial peptide magainin II in tumor cells by bombesin-targeted delivery

Aim:

To investigate whether the conjugation of magainin II (MG2), an antimicrobial peptides (AMPs), to the tumor-homing peptide bombesin could enhance its cytotoxicity in tumor cells.

Methods:

A magainin II-bombesin conjugate (MG2B) was constructed by attaching magainin II (MG2) to bombesin at its N-terminus. The peptides were synthesized using Fmoc-chemistry. The in vitro cytotoxicity of the peptide in cancer cells was quantitatively determined using the CCK-8 cell counting kit. Moreover, the in vivo antitumor effect of the peptide was determined in tumor xenograft models.

Results:

The IC₅₀ of MG2B for cancer cells (10–15 μmol/L) was at least 10 times lower than the IC₅₀ of unconjugated MG2 (125 μmol/L). Moreover, the binding affinity of MG2B for cancer cells was higher than that of unconjugated MG2. In contrast, conjugation to a bombesin analog lacking the receptor-binding domain failed to increase the cytotoxicity of MG2, suggesting that bombesin conjugation enhances the cytotoxicity of MG2 in cancer cells through improved binding. Indeed, MG2B selectively induced cell death in cancer cells in vitro with the IC₅₀ ranging from 10 to 15 μmol/L, which was about 6–10 times lower than the IC₅₀ for normal cells. MG2B (20 mg/kg per day, intratumorally injected for 5 d) also exhibited antitumor effects in mice bearing MCF-7 tumor grafts. The mean weights of tumor grafts in MG2B- and PBS-treated mice were 0.21±0.05 g and 0.59±0.12 g, respectively.

Conclusion:

The results suggest that conjugation of AMPs to bombesin might be an alternative approach for targeted cancer therapy.