Synthetic Strategies for Improving Solubility: Optimization of Novel Pyrazolo[1,5-a]pyrimidine CFTR Activator That Ameliorates Dry Eye Disease.

Dry eye disease (DED) is one of the most prevalent ocular diseases but has limited treatment options. Cystic fibrosis transmembrane conductance regulator (CFTR), a major chloride channel that stimulates fluid secretion in the ocular surface, may pave the way for new therapeutic strategies for DED. Herein, we report the optimization of Cact-3, a potent CFTR activator with poor solubility, to 16d, a potent CFTR activator with suitable solubility for eye drop formulation. Notably, 16d was well distributed in target tissues including cornea and conjunctiva with minimal systemic exposure in rabbit. Topical ocular instillation of 16d significantly enhanced tear secretion and improved corneal erosion in a mouse model of DED. In addition, 16d significantly reduced mRNA expression of pro-inflammatory cytokines including IL-1ß, IL-17, and TNF-α and MMP2 in cornea and conjunctiva of DED mice.

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.

Methylglyoxal-Induced Dysfunction in Brain Endothelial Cells via the Suppression of Akt/HIF-1α Pathway and Activation of Mitophagy Associated with Increased Reactive Oxygen Species.

Methylglyoxal (MG) is a dicarbonyl compound, the level of which is increased in the blood of diabetes patients. MG is reported to be involved in the development of cerebrovascular complications in diabetes, but the exact mechanisms need to be elucidated. Here, we investigated the possible roles of oxidative stress and mitophagy in MG-induced functional damage in brain endothelial cells (ECs). Treatment of MG significantly altered metabolic stress as observed by the oxygen-consumption rate and barrier-integrity as found in impaired trans-endothelial electrical resistance in brain ECs. The accumulation of MG adducts and the disturbance of the glyoxalase system, which are major detoxification enzymes of MG, occurred concurrently. Reactive oxygen species (ROS)-triggered oxidative damage was observed with increased mitochondrial ROS production and the suppressed Akt/hypoxia-inducible factor 1 alpha (HIF-1α) pathway. Along with the disturbance of mitochondrial bioenergetic function, parkin-1-mediated mitophagy was increased by MG. Treatment of N-acetyl cysteine significantly reversed mitochondrial damage and mitophagy. Notably, MG induced dysregulation of tight junction proteins including occludin, claudin-5, and zonula occluden-1 in brain ECs. Here, we propose that diabetic metabolite MG-associated oxidative stress may contribute to mitochondrial damage and autophagy in brain ECs, resulting in the dysregulation of tight junction proteins and the impairment of permeability.

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.

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.

Reduced metabolic activity of gut microbiota by antibiotics can potentiate the antithrombotic effect of aspirin.

In this study, we investigated the effects of antibiotics on the pharmacological effects of aspirin. The antithrombotic activity of aspirin was evaluated after antibiotic treatment using tail bleeding assay. The pyrosequencing analysis and selective medium culture assay were performed to investigate the alterations in gut microbiota. In addition, the in vitro metabolism assay with fecal suspension and in vivo pharmacokinetic experiments with antibiotic treatment were conducted. Ampicillin treatment significantly prolonged the bleeding time in aspirin-dosed rats. Oral administration of ampicillin significantly reduced gut microbial aspirin-metabolizing activity by 67.0% in rats. Furthermore, systemic exposure to aspirin and its primary metabolite (M1) was significantly increased in ampicillin-treated rats. The results from the pyrosequencing and selective medium culture with rat fecal samples revealed that ampicillin treatment led to the changes of the amounts and composition profile of gut microbiota. These findings suggest that co-administration of antibiotics can modulate the metabolism and pharmacokinetics of aspirin via suppression of metabolic activity of gut microbiota, which could potentiate the therapeutic potency of aspirin.

Selective inhibition of JAK2/STAT1 signaling and iNOS expression mediates the anti-inflammatory effects of coniferyl aldehyde.

Urgent needs still exist for selective control of excessive inflammation. Despite the therapeutic potential of natural compounds against inflammation-associated chronic conditions, lack of specific molecular targets renders these bioactive compounds difficult for further development. Here we examined the bioactivity of coniferyl aldehyde (CA), a natural phenolic compound found in several dietary substances and medicinal plants, elucidating its efficacy both in vivo and in vitro with underlying molecular mechanisms. IFN-γ/TNF-α-stimulated human keratinocytes and lipopolysaccharide (LPS)-stimulated murine macrophages were used to examine the effect of CA in vitro and to elucidate the underlying mechanisms. In vivo models of phorbol 12-myristate 13-acetate (TPA)-induced ear edema and carrageenan (CRG)-induced paw edema were employed to investigate the topical and systemic anti-inflammatory effects of CA, respectively. CA significantly reduced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in LPS-stimulated macrophages. While nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPKs) pathways, the representative cellular pathways for iNOS induction, were not affected by CA, phosphorylation of Janus kinase 2 (JAK2) and signal Transducers and Activators of Transcription 1 (STAT1) and subsequent nuclear translocation of p-STAT1 were significantly decreased by CA. The effect of CA on JAK2-STAT1-iNOS axis was also observed in human keratinocytes stimulated with IFN-γ/TNF-α. Topical application of CA to mice produced significant protection against TPA-induced ear edema along with suppressed epidermal hyperproliferation and leucocyte infiltration. Systemic administration of CA significantly reduced CRG-induced paw edema in rats, where CRG-induced iNOS expression and STAT1 phosphorylation were decreased by CA. In summary, CA has significant anti-inflammatory properties both in vitro and in vivo, mediated by significant selective inhibition of JAK2-STAT1-iNOS signaling. CA is an attractive novel candidate for treating inflammatory diseases associated with excessive production of NO.

A newly synthesized macakurzin C-derivative attenuates acute and chronic skin inflammation: The Nrf2/heme oxygenase signaling as a potential target.

Impaired immune responses in skin play a pivotal role in the development and progression of chemical-associated inflammatory skin disorders. In this study, we synthesized new flavonoid derivatives from macakurzin C, and identified in vitro and in vivo efficacy of a potent anti-inflammatory flavonoid, Compound 14 (CPD 14), with its underlying mechanisms. In lipopolysaccharide (LPS)-stimulated murine macrophages and IFN-γ/TNF-α-stimulated human keratinocytes, CPD 14 significantly inhibited the release of inflammatory mediators including nitric oxide (NO), prostaglandins, and cytokines (IC50 for NO inhibition in macrophages: 4.61µM). Attenuated NF-κB signaling and activated Nrf2/HO-1 pathway were responsible for the anti-inflammatory effects of CPD 14. The in vivo relevance was examined in phorbol 12-myristate 13-acetate (TPA)-induced acute skin inflammation and oxazolone-induced atopic dermatitis models. Topically applied CPD 14 significantly protected both irritation- and sensitization-associated skin inflammation by suppressing the expression of inflammatory mediators. In summary, we demonstrated that a newly synthesized flavonoid, CPD 14, has potent inhibitory effects on skin inflammation, suggesting it is a potential therapeutic candidate to treat skin disorders associated with excessive inflammation.

Potent Anti-inflammatory and Analgesic Actions of the Chloroform Extract of Dendropanax morbifera Mediated by the Nrf2/HO-1 Pathway.

Dendropanax morbifera LEVEILLE (DP) has been used in traditional Korean medicines to treat a variety of inflammatory diseases. Although the in vitro anti-inflammatory potential of this plant is understood, its in vivo efficacy and underlying molecular mechanism of anti-inflammatory effects are largely unknown. We elucidated the anti-inflammatory and analgesic activities and the underlying molecular mechanisms of DP using in vitro and in vivo models. Lipopolysaccharide (LPS)-stimulated murine macrophages were used to analyze the in vitro anti-inflammatory potential of DP extract and to elucidate the underlying mechanisms. In vivo animal models of phorbol 12-myristate 13-acetate (TPA)-induced ear edema and acetic acid-induced writhing response tests were used to analyze the in vivo anti-inflammatory effects and anti-nociceptive effects of DP extract, respectively. Methanolic extract of DP (DPME) significantly inhibited the release of nitric oxide (NO) and prostaglandin E2 (PGE2) in LPS-activated macrophages. Among the five sub-fractions, the chloroform fraction (DP-C) showed the most potent suppressive effects against pro-inflammatory mediators and cytokines in LPS-stimulated macrophages. These effects were attributed to inhibition of nuclear factor-κB (NF-κB) nuclear translocation and c-Jun N terminal kinase (JNK) 1/2 phosphorylation and to activation of NF-E2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) signaling. DP-C exhibited strong protective in vivo effects in TPA-induced ear edema mouse model and acetic acid-induced writhing response test. Our data suggest that DP-C has potent anti-inflammatory and analgesic activities and may be a promising treatment against a variety of inflammatory diseases.

Heme oxygenase 1-mediated novel anti-inflammatory activities of Salvia plebeia and its active components.

ETHNOPHARMACOLOGICAL RELEVANCE: Salvia plebeia R. Br. (SP) has been widely used as a traditional folk medicine for the treatment of infectious diseases and pain. An anti-inflammatory potential of SP has remains largely unknown. AIM OF THE STUDY: We tried to elucidate the principle mechanism and the active ingredients underlying the anti-inflammatory activities of SP. MATERIALS AND METHODS: We investigated the protective activities of SP methanolic extract (SPME) and seven representative ingredients against inflammation. Quantitative analysis using HPLC-DAD-ESI/MS was conducted to determine the relative amounts of these seven active ingredients in SPME. Both in vitro murine macrophages and in vivo mouse models were employed to elucidate SP- and active ingredient-mediated anti-inflammatory effects. RESULTS: SPME significantly reduced inflammatory processes both in vivo in a TPA-induced ear edema model and in vitro in lipopolysaccharide (LPS)-activated macrophages. SPME decreased the release of nitric oxide (NO) and prostaglandin E2 (PGE2) and expression of inducible nitric oxide synthase (iNOS). Seven active components (luteoloside (C1), nepitrin (C2), homoplantagenin (C3), luteolin (C4), nepetin (C5), hispidulin (C6), and eupatorin (C7)) of SPME were analyzed and their relative concentrations were determined, demonstrating that C2, C3, C5 and C6 were present in higher amounts than were C1, C4, and C7. These major compounds inhibited NO and PGE2 production, and iNOS and COX-II protein expression through heme oxygenase-1 (HO-1) induction via activation of nuclear factor erythroid 2-related factor2 (Nrf2). CONCLUSION: Our data demonstrate that SPME possesses potent in vitro and in vivo anti-inflammatory activities. Nepetin and hispidulin, and their glycosides are the major active compounds in SPME, and their effects are mediated by Nrf2/HO-1 signaling. Taken together, we propose that SPME and its active ingredients may serve as novel therapeutic candidates for diseases associated with excessive inflammation.

Protective activity of Dendropanax morbifera against cisplatin-induced acute kidney injury.

BACKGROUND/AIMS: Drug-induced acute kidney injury (AKI) has been a severe threat to hospitalized patients, raising the urgent needs to develop strategies to reduce AKI. We investigated the protective activity of Dendropanax morbifera (DP), a medicinal plant which has been widely used to treat infectious and pain diseases, on acute kidney injury (AKI) using cisplatin-induced nephropathic models. METHODS: Both in vitro renal tubular cells (NRK-52E) and in vivo rat models were used to demonstrate the nephroprotective effect of DP. RESULTS: Methanolic extract from DP significantly reduced cisplatin-induced toxicity in renal tubular cells. Through successive liquid extraction, the extract of DP was separated into n-hexane, CHCl3, EtOAc, n-BuOH, and H2O fractions. Among these, the CHCl3 fraction (DPCF) was found to be most potent. The protective activity of DPCF was found to be mediated through anti-oxidant, mitochondrial protective, and anti-apoptotic activities. In in vivo rat models of AKI, treatment with DPCF significantly reversed the cisplatin-induced increase in blood urea nitrogen and serum creatinine and histopathologic damage, recovered the level of anti-oxidant enzymes, and inhibited renal apoptosis. CONCLUSION: We demonstrated that DP extracts decreased cisplatin-induced renal toxicity, indicating its potential to ameliorate drug-associated acute kidney damage.

High glucose condition induces autophagy in endothelial progenitor cells contributing to angiogenic impairment.

Cardiovascular complications are the major causes of death in patients with diabetes mellitus. Several studies have demonstrated that endothelial progenitor cells (EPCs), adult stem cells contributing to the regeneration of vascular endothelium, are dysfunctional under diabetic condition resulting in impaired peripheral circulation and delayed wound healing. In this study, we investigated the cellular alteration of EPCs under high glucose condition, to elucidate the mechanisms underlying diabetes-associated EPC dysfunction. EPCs were isolated from bone marrow and cultured in normal glucose (5.5 mM)- or high glucose (HG; 30 mM)-containing medium. High glucose treated-EPCs showed decreased ability to form tubule-like networks in Matrigel compared to EPCs under normal glucose, which matched well to the clinical observation of diabetic EPC dysfunction. Conversion of LC3-I to LC3-II was increased in EPCs under HG condition, showing that HG induced autophagy in EPCs. Flow cytometric analysis revealed generation of oxidative stress and disruption of mitochondrial permeability in HG exposed EPCs. Increased mitochondrial oxidative stress was also observed by mitochondria-specific superoxide indicator, MitoSOX(TM). Taken together, we demonstrated that autophagy and mitochondrial impairment were induced in EPCs under high glucose condition, giving a new insight into the mechanism underlying dysfunction of diabetic EPCs. We hope that our finding can contribute to the development of a new treatment option for cardiovascular complications in diabetic patients.

Modulation of mitochondrial function and autophagy mediates carnosine neuroprotection against ischemic brain damage.

BACKGROUND AND PURPOSE: Despite the rapidly increasing global burden of ischemic stroke, no therapeutic options for neuroprotection against stroke currently exist. Recent studies have shown that autophagy plays a key role in ischemic neuronal death, and treatments that target autophagy may represent a novel strategy in neuroprotection. We investigated whether autophagy is regulated by carnosine, an endogenous pleiotropic dipeptide that has robust neuroprotective activity against ischemic brain damage. METHODS: We examined the effect of carnosine on mitochondrial dysfunction and autophagic processes in rat focal ischemia and in neuronal cultures. RESULTS: Autophagic pathways such as reduction of phosphorylated mammalian target of rapamycin (mTOR)/p70S6K and the conversion of microtubule-associated protein 1 light chain 3 (LC3)-I to LC3-II were enhanced in the ischemic brain. However, treatment with carnosine significantly attenuated autophagic signaling in the ischemic brain, with improvement of brain mitochondrial function and mitophagy signaling. The protective effect of carnosine against autophagy was also confirmed in primary cortical neurons. CONCLUSIONS: Taken together, our data suggest that the neuroprotective effect of carnosine is at least partially mediated by mitochondrial protection and attenuation of deleterious autophagic processes. Our findings shed new light on the mechanistic pathways that this exciting neuroprotective agent influences.

Dysfunction of endothelial progenitor cells under diabetic conditions and its underlying mechanisms.

Cardiovascular complications have been major concerns in the treatment of diabetes, and up to 80% of all deaths in diabetic patients are linked to cardiovascular problems. Impaired angiogenesis is one of the most serious symptoms associated with diabetes, resulting in delayed wound healing and lower limb amputation. Endothelial progenitor cells (EPCs), a subpopulation of adult stem cells, are recruited from bone marrow to the injured vessel to promote endothelial regeneration and neovascularization, playing an important role in angiogenesis. Interestingly, several clinical studies have showed that the number of recruited EPCs is reduced and their function is decreased under diabetic conditions, implying that diabetic EPC dysfunction may contribute to defective angiogenesis and resultant cardiovascular complications in diabetes. To recover the functional abilities of diabetic EPCs and to address possible application of EPC cell therapy to diabetic patients, some studies provided explanations for diabetic EPC dysfunction including increased oxidative stress, involvement of the inflammatory response, alteration in the nitric oxide pathway and reduced signals for EPC recruitment. This review discusses clinical evidence of impairment of EPC functions under diabetic conditions and the suggested mechanisms for diabetic EPC dysfunction.

Unusual causes of tubo-ovarian abscess: CT and MR imaging findings.

Actinomycosis, tuberculosis, and xanthogranulomatous inflammation are rare but specific causes of tubo-ovarian abscess (TOA). TOAs with these causes are frequently misdiagnosed as ovarian malignancies due to their unusual appearances at computed tomography (CT) and magnetic resonance (MR) imaging. Tubo-ovarian actinomycosis frequently has a predominantly solid appearance. A linear, solid, well-enhancing lesion extending directly from the mass is a characteristic CT and MR imaging finding. Small rim-enhancing lesions in the solid part of the mass are also suggestive of actinomycosis. Tuberculous TOAs usually mimic peritoneal carcinomatosis from ovarian cancers. The granulomatous and fibrotic nature of this infection may be reflected in the CT and MR imaging appearances, which can help in differentiation. TOAs from xanthogranulomatous inflammation demonstrate more nonspecific imaging findings than actinomycotic or tuberculous TOAs, although multiple xanthogranulomas in the mass may be seen on MR images. Knowledge of these characteristics can help one make the correct diagnosis and treat the patients appropriately.