Xue Ma

Exendin-4, a Glucagon-Like Peptide-1 Receptor Agonist, Prevents Osteopenia by Promoting Bone Formation and Suppressing Bone Resorption in Aged Ovariectomized Rats

Osteoporosis mainly affects postmenopausal women and older men. Gastrointestinal hormones released after meal ingestion, such as glucose‐dependent insulinotropic peptide (GIP) and glucagon‐like peptide (GLP)‐2, have been shown to regulate bone turnover. However, whether GLP‐1, another important gastrointestinal hormone, and its analogues also have antiosteoporotic effects, especially in aged postmenopausal situation, has not been confirmed. In the present study, we evaluated the effects of the GLP‐1 receptor agonist exendin‐4 on ovariectomy (OVX)‐induced osteoporosis in old rats. Twelve‐month‐old female Sprague‐Dawley rats were subjected to OVX, and exendin‐4 was administrated 4 weeks after the surgery and lasted for 16 weeks. Bone characters and related serum and gene biomarkers were analyzed. Sixteen weeks of treatment with exendin‐4 slowed down body weight gain by decreasing fat mass and prevented the loss of bone mass in old OVX rats. Exendin‐4 also enhanced bone strength and prevented the deterioration of trabecular microarchitecture. Moreover, exendin‐4 decreased the urinary deoxypyridinoline (DPD)/creatinine ratio and serum C‐terminal cross‐linked telopeptides of type I collagen (CTX‐I) and increased serum alkaline phosphatase (ALP), osteocalcin (OC), and N‐terminal propeptide of type 1 procollagen (P1NP) levels, key biochemical markers of bone turnover. Interestingly, gene expression results further showed that exendin‐4 not only inhibited bone resorption by increasing the osteoprotegerin (OPG)/receptor activator of NF‐κB ligand (RANKL) ratio, but also promoted bone formation by increasing the expression of OC, Col1, Runx2, and ALP, which exhibited dual regulatory effects on bone turnover as compared with previous antiosteoporotic agents. In conclusion, these findings demonstrated for the first time the antiosteoporotic effects of exendin‐4 in old OVX rats and that it might be a potential candidate for treatment of aged postmenopausal osteoporosis.

Antisense inhibition of gene expression and growth in gram-negative bacteria by cell-penetrating peptide conjugates of peptide nucleic acids targeted to rpoD gene.

Gram-negative bacteria (GNB) cause common and severe hospital- and community-acquired infections with a high incidence of multidrug resistance (MDR) and mortality. The emergence and spread of MDR-GNB strains limit therapeutic options and highlight the need to develop new therapeutic strategies. In this study, the peptide (RXR)(4)XB- and (KFF)(3)K-conjugated peptide nucleic acids (PPNAs) were developed to target rpoD, which encodes an RNA polymerase primary σ(70) that is thought to be essential for bacterial growth. Their antimicrobial activities were tested against different clinical isolates of MDR-GNB in vitro and in infection models. The (RXR)(4)XB- and (KFF)(3)K- conjugated PNAs were bactericidal against different strains of MDR-GNB in concentration-dependent and sequence-selective manner, whereas a PPNA with a scrambled base sequence had no effect on growth. Among tested PPNAs, (RXR)(4)XB conjugate PPNA06 showed more potent and broad spectrum inhibition in multidrug-resistant Escherichia coli, Salmonella enterica, Klebsiella pneumoniae, and Shigella flexneri in vitro and in vivo. The results were associated with suppression of rpoD mRNA and σ(70) expression, as well as σ(70) downstream regulated genes including ftsZ, mazF, prfB, rpoS, seqA, turfB and ygjD. The treatment of PPNA06 on mono- or multiple MDR-GBN infected human gastric mucosal epithelial cells demonstrated the complete inhibition on bacterial growth and no influence on morphology and growth of human cells. Also, PPNA06 did not show the induction of antibiotic resistance as compared with classical antibiotics in GNB. These findings firstly demonstrate that rpoD is potential target for developing antisense antibiotics, and indicate that peptide conjugates of anti-rpoD PNA are active against GNBs in vitro and in vivo. Our results offer a feasible strategy for treating MDR-GNB infections.

Novel Anion Liposome-Encapsulated Antisense Oligonucleotide Restores Susceptibility of Methicillin-Resistant Staphylococcus aureus and Rescues Mice from Lethal Sepsis by Targeting mecA

ABSTRACT β-Lactam resistance in methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) is caused by the production of an additional low-affinity penicillin-binding protein 2a, which is encoded by the mecA gene. The disruption of mecA may inhibit mecA expression and thereafter lead to the restoration of MRSA susceptibility to β-lactams. In this study, we developed a novel anionic liposome for encapsulating and delivering the complexes of a specific anti-mecA phosphorothioate oligodeoxynucleotide (PS-ODN833) and polycation polyethylenimine (PEI). The efficiencies of liposome encapsulation of the complexes were around 79.7% ± 2.7%. The liposomes showed sustained release of PS-ODN833 at 37°C but very low levels of release at 4°C and room temperature. The addition of the encapsulated anti-mecA PS-ODN833-PEI complex to cultures of MRSA strains caused 45, 76, 82, and 93% reductions in mecA expression, accompanied by the inhibition of MRSA growth on Mueller-Hinton agar containing oxacillin (6 μg/ml) in a concentration-dependent manner. The encapsulated-PS-ODN833 treatment also reduced the MICs of five of the most commonly used antibiotics for MRSA clinical isolates to values within the sensitivity range and rescued mice from MRSA-caused septic death by downregulating mecA. The survival rates of septic mice increased from 0% for the control group to 53% for the PS-ODN833-treated group. The results were associated with reductions of bacterial titers in the blood of surviving mice. The findings of the present study indicate that an antisense oligodeoxynucleotide targeted to mecA can significantly restore the susceptibility of MRSA to existing β-lactam antibiotics, providing an apparently novel strategy for treating MRSA infections.

Arrhythmogenic Effect of Sympathetic Histamine in Mouse Hearts Subjected to Acute Ischemia

The role of histamine as a newly recognized sympathetic neurotransmitter has been presented previously, and its postsynaptic effects greatly depended on the activities of sympathetic nerves. Cardiac sympathetic nerves become overactivated under acute myocardial ischemic conditions and release neurotransmitters in large amounts, inducing ventricular arrhythmia. Therefore, it is proposed that cardiac sympathetic histamine, in addition to norepinephrine, may have a significant arrhythmogenic effect. To test this hypothesis, we observed the release of cardiac sympathetic histamine and associated ventricular arrhythmogenesis that was induced by acute ischemia in isolated mouse hearts. Mast cell-deficient mice (MCDM) and histidine decarboxylase knockout (HDC−/−) mice were used to exclude the potential involvement of mast cells. Electrical field stimulation and acute ischemia-reperfusion evoked chemical sympathectomy-sensitive histamine release from the hearts of both MCDM and wild-type (WT) mice but not from HDC−/− mice. The release of histamine from the hearts of MCDM and WT mice was associated with the development of acute ischemia-induced ventricular tachycardia and ventricular fibrillation. The incidence and duration of induced ventricular arrhythmias were found to decrease in the presence of the selective histamine H2 receptor antagonist famotidine. Additionally, the released histamine facilitated the arrhythmogenic effect of simultaneously released norepinephrine. We conclude that, under acute ischemic conditions, cardiac sympathetic histamine released by overactive sympathetic nerve terminals plays a certain arrhythmogenic role via H2 receptors. These findings provided novel insight into the pathophysiological roles of sympathetic histamine, which may be a new therapeutic target for acute ischemia-induced arrhythmias.

Activation of GLP-1 Receptor Promotes Bone Marrow Stromal Cell Osteogenic Differentiation through β-Catenin

Summary Glucagon-like peptide 1 (GLP-1) plays an important role in regulating bone remodeling, and GLP-1 receptor agonist shows a positive relationship with osteoblast activity. However, GLP-1 receptor is not found in osteoblast, and the mechanism of GLP-1 receptor agonist on regulating bone remodeling is unclear. Here, we show that the GLP-1 receptor agonist exendin-4 (Ex-4) promoted bone formation and increased bone mass and quality in a rat unloading-induced bone loss model. These functions were accompanied by an increase in osteoblast number and serum bone formation markers, while the adipocyte number was decreased. Furthermore, GLP-1 receptor was detected in bone marrow stromal cells (BMSCs), but not in osteoblast. Activation of GLP-1 receptor by Ex-4 promoted the osteogenic differentiation and inhibited BMSC adipogenic differentiation through regulating PKA/β-catenin and PKA/PI3K/AKT/GSK3β signaling. These findings reveal that GLP-1 receptor regulates BMSC osteogenic differentiation and provide a molecular basis for therapeutic potential of GLP-1 against osteoporosis.

Wide distribution and subcellular localization of histamine in sympathetic nervous systems of different species

Previous studies have demonstrated that histamine (HA) acts as a neurotransmitter in the cardiac sympathetic nervous system of the guinea pig. The aim of the current study was to examine whether HA widely exists in the sympathetic nervous systems of other species and the subcellular localization of HA in sympathetic terminals. An immunofluorescence histochemical multiple-staining technique and anterograde tracing method were employed to visualize the colocalization of HA and norepinephrine (NE) in sympathetic ganglion and nerve fibers in different species. Pre-embedding immunoelectron microscopy was used to observe the subcellular distribution of HA in sympathetic nerve terminals. Under the confocal microscope, coexistence of NE and HA was displayed in the superior cervical ganglion and celiac ganglion neurons of the mouse and dog as well as in the vas deferens, mesenteric artery axon, and varicosities of the mouse and guinea pig. Furthermore, colocalization of NE and HA in cardiac sympathetic axons and varicosities was labeled by biotinylated dextranamine injected into the superior cervical ganglion of the guinea pig. By electron microscopy, HA-like high-density immunoreactive products were seen in the small vesicles of the guinea pig vas deferens. These results provide direct cellular and subcellular morphological evidence for the colocalization of HA and NE in sympathetic ganglion and nerve fibers, and support that HA is classified as a neurotransmitter in sympathetic neurons.

oprM as a new target for reversion of multidrug resistance in Pseudomonas aeruginosa by antisense phosphorothioate oligodeoxynucleotides

Multidrug-resistant Pseudomonas aeruginosa (MDR-PA) is one of the leading Gram-negative organisms associated with nosocomial infections. The increasing frequency of MDR-PA has represented a huge challenge in conventional antibacterial therapy. The loss of effectiveness of commonly used antibiotics calls for the immediate need to develop an alternative strategy for combating MDR-PA infections. The multiantibiotic resistance of MDR-PA is largely attributable to the production of multidrug efflux pumps, MexAB-OprM. OprM forms the antibiotic-ejecting duct and plays a crucial role in exporting incoming chemotherapeutic agents across the membranes. Disruption of the OprM expression may inhibit the function of multidrug efflux pumps and lead to restoration of MDR-PA susceptibility to antibiotics. In this study, we developed a novel anion liposome for encapsulating and delivering specific anti-oprM phosphorothioate oligodeoxynucleotide (PS-ODN617) and polycation polyethylenimine (PEI) complexes. The additions of the encapsulated anti-oprM PS-ODN617/PEI to MDR-PA isolates caused a significant reduction of oprM expression and inhibition of MDR-PA growth in the presence of piperacillin in a concentration-dependent manner. The encapsulated PS-ODN617 treatment also reduced minimal inhibitory concentrations of five most commonly used antibiotics to the sensitive margin values on MDR-PA clinical isolates, respectively. The results of present study firstly indicate that PS-ODN targeted to oprM can significantly restore the susceptibility of MDR-PA to existing antibiotics, which appears to be a novel strategy for treating MDR-PA infections.

Poly‐GLP‐1, a novel long‐lasting glucagon‐like peptide‐1 polymer, ameliorates hyperglycaemia by improving insulin sensitivity and increasing pancreatic beta‐cell proliferation

Aim: The clinical value of glucagon‐like peptide‐1 (GLP‐1) is restricted because of its short half‐life. To overcome this limitation, a new polymer of GLP‐1 was developed by prodrug strategy, termed Poly‐GLP‐1, and its pharmacological properties were investigated.

An Orally Active Allosteric GLP-1 Receptor Agonist Is Neuroprotective in Cellular and Rodent Models of Stroke.

Diabetes is a major risk factor for the development of stroke. Glucagon-like peptide-1 receptor (GLP-1R) agonists have been in clinical use for the treatment of diabetes and also been reported to be neuroprotective in ischemic stroke. The quinoxaline 6,7-dichloro-2-methylsulfonyl-3-N-tert- butylaminoquinoxaline (DMB) is an agonist and allosteric modulator of the GLP-1R with the potential to increase the affinity of GLP-1 for its receptor. The aim of this study was to evaluate the neuroprotective effects of DMB on transient focal cerebral ischemia. In cultured cortical neurons, DMB activated the GLP-1R, leading to increased intracellular cAMP levels with an EC50 value about 100 fold that of exendin-4. Pretreatment of neurons with DMB protected against necrotic and apoptotic cell death was induced by oxygen-glucose deprivation (OGD). The neuroprotective effects of DMB were blocked by GLP-1R knockdown with shRNA but not by GLP-1R antagonism. In C57BL/6 mice, DMB was orally administered 30 min prior to middle cerebral artery occlusion (MCAO) surgery. DMB markedly reduced the cerebral infarct size and neurological deficits caused by MCAO and reperfusion. The neuroprotective effects were mediated by activation of the GLP-1R through the cAMP-PKA-CREB signaling pathway. DMB exhibited anti-apoptotic effects by modulating Bcl-2 family members. These results provide evidence that DMB, a small molecular GLP-1R agonist, attenuates transient focal cerebral ischemia injury and inhibits neuronal apoptosis induced by MCAO. Taken together, these data suggest that DMB is a potential neuroprotective agent against cerebral ischemia.

Antisense Growth Inhibition of Methicillin-Resistant Staphylococcus aureus by Locked Nucleic Acid Conjugated with Cell-Penetrating Peptide as a Novel FtsZ Inhibitor

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) infections are becoming increasingly difficult to treat, owing to acquired antibiotic resistance. The emergence and spread of MRSA limit therapeutic options and require new therapeutic strategies, including novel MRSA-active antibiotics. Filamentous temperature-sensitive protein Z (FtsZ) is a highly conserved bacterial tubulin homologue that is essential for controlling the bacterial cell division process in different species of S. aureus. We conjugated a locked nucleic acid (LNA) that targeted ftsZ mRNA with the peptide (KFF)3K, to generate peptide-LNA (PLNA). The present study aimed to investigate whether PLNA could be used as a novel antibacterial agent. PLNA787, the most active agent synthesized, exhibited promising inhibitory effects on four pathogenic S. aureus strains in vitro. PLNA787 inhibited bacterial growth and resolved lethal Mu50 infections in epithelial cell cultures. PLNA787 also improved the survival rates of Mu50-infected mice and was associated with reductions of bacterial titers in several tissue types. The inhibitory effects on ftsZ mRNA and FtsZ protein expression and inhibition of the bacterial cell division process are considered to be the major mechanisms of PLNA. PLNA787 demonstrated activity against MRSA infections in vitro and in vivo. Our findings suggest that ftsZ mRNA is a promising new target for developing novel antisense antibiotics.