Local anesthetic lidocaine-encapsulated polymyxin–chitosan nanoparticles delivery for wound healing: in vitro and in vivo tissue regeneration

In relieving local pains, lidocaine, one of ester-type local anesthetics, has been used. To develop the lidocaine membranes of enhanced local anesthetic effects, we have designed to establish the composition of wound dressings based on lidocaine chloride (LCH) (anesthetic drug)-loaded chitosan (CS)/polymyxin B sulfate (PMB). The LCH membranes (LCH-CS/PMB) was fabricated by the LCH oxide solutions within the CS/PMB matrix. The influences of different experimental limitations on CS/PMB membrane formations were examined. The double membrane particle sizes were evaluated by scanning electron microscopy (HR-SEM). Additionally, antibacterial efficacy was developed for gram-positive and negative microorganisms. Moreover, we examined in vivo healing of skin wounds formed in mouse models over 16 days. In contrast to the untreated wounds, rapid healing was perceived in the LCH-CS/PMB-treated wound with less damaging. These findings indicate that LCH-CS/PMB-based bandaging materials could be a potential innovative biomaterial for tissue repair and regeneration for wound healing applications in an animal model.     

Thioctamer: a novel thioctic acid–glatiramer acetate nanoconjugate expedites wound healing in diabetic rats

The current work aims to design thioctic acid (TA) and glatiramer acetate (GA) nanoconjugate (thioctamer) loaded hydrogel formula as well as evaluation of thioctamer preclinical efficacy in expediting wound healing in a rat model of the diabetic wound. Thioctamer was prepared by conjugation of GA and TA in a 1:1 molar ratio. Particle size, zeta potential, and thermodynamic stability of the prepared thioctamer were assessed. Thioctamer was loaded in hydroxypropyl methylcellulose-based hydrogel and in vitro release study was investigated. The ability of thioctamer to enhance the process of wound healing in diabetic rats was investigated by assessing wound contraction and immunohistochemical assessment of the inflammation markers IL-6 and TNF-α. The results demonstrated that thioctamer showed particle size of 137 ± 21.4 nm, polydispersity index (PDI) of 0.235, and positive zeta potential value of 7.43 ± 4.95 mV. On day 10 of making a skin excision, diabetic rat wounds administered thioctamer preparation showed almost complete healing (95.6 ± 8.6%). Meanwhile, % of wound contraction in animals treated with TA or GA groups exhibited values amounting to 56.5 ± 5.8% and 62.6 ± 7.1%, respectively. Histological investigation showed that the highest healing rate was noted in the thioctamer group animals, as the surface of the wound was nearly fully protected by regenerated epithelium with keratinization, with few inflammatory cells noticed. Thioctamer significantly (p<.05) inhibited IL-6 and TNF-α expression as compared with sections obtained from the negative control, TA, GA, or positive control group animals on day 14. The evidence of the ability of thioctamer to significantly expedite wound healing in the diabetic rats is presented.     

Management of recurrent aphthous ulcers exploiting polymer-based Muco-adhesive sponges: in-vitro and in-vivo evaluation

Recurrent aphthous ulcer (RAU) is a well-known painful, inflammatory disease with uncertain etiology for which local symptomatic therapy is only available. The aim of this study was to formulate and characterize muco-adhesive sponges containing a mixture of tenoxicam and miconazole nitrate to manage pain, inflammation and avoid candida infection that may accompany RAU due to poor oral hygiene. Two polymers at different concentrations were used to prepare sponges applying simple freeze-drying. Medicated chitosan (2%) sponges (mC2) showed acceptable physical appearance, surface pH (6.3 ± 0.042), porosity (25.7% ± 1.8), swelling index (5.7 ± 0.11), in-vivo and ex-vivo muco-adhesion time (115 min.±0.813 and 155 min.±1.537, respectively), ex-vivo muco-adhesion force (0.09 N ± 0.002) and scanning electron microscope (SEM) images. For concurrent clear-cut determination of tenoxicam and miconazole nitrate from mC2, a new UPLC method was developed and validated. mC2 sponges exhibited superior in-vitro drug release profiles where ∼100% of tenoxicam released within 5 min for fast pain relief with a more prolonged miconazole nitrate release. Furthermore, in-vivo animal study revealed that mC2 caused a significant decrease in the acetic acid-induced ulcer size in rats after 6 days of treatment (p < .0001) compared to negative and positive controls. Additionally, histopathological examination showed faster healing with complete restoration of the normal oral histology in rats. The present study concludes that chitosan sponge loaded with a combination of tenoxicam and miconazole nitrate could improve healing of RAU cases.     

Different Adaptations in AMPA Receptor Transmission in the Nucleus Accumbens after Short vs Long Access Cocaine Self-Administration Regimens

Ca²⁺-permeable AMPA receptors (CP-AMPARs) accumulate in the nucleus accumbens (NAc) after ∼1 month of withdrawal from a long-access cocaine self-administration regimen (6 h/d, 10d). This is functionally significant because CP-AMPARs mediate the ‘incubated'' cue-induced cocaine craving produced by this regimen. Our present goal was to determine if other commonly employed cocaine self-administration regimens also elicit CP-AMPAR accumulation. We compared four regimens, named according to whether sessions were short-access (ShA, 2 h) or long-access (LgA, 6 h) and the total number of sessions: LgA/10d (already shown to elicit CP-AMPAR accumulation), ShA/11d, ShA/20-24d, and LgA/20-24d. In the latter regimens, rats began with 10 days of ShA and then entered a differential phase (10–14 days) in which ShA sessions either continued or switched to LgA. Controls self-administered saline. After >40 days of withdrawal, whole-cell patch-clamp recordings were performed in NAc core medium spiny neurons to assess the contribution of CP-AMPAR transmission, based on the magnitude of synaptic suppression elicited by bath application of the selective CP-AMPAR antagonist naspm (100 μM). Naspm produced a non-significant (∼10%) attenuation of electrically evoked local excitatory postsynaptic current in the saline and ShA groups. By contrast, a significant naspm-induced synaptic attenuation (25–30%) was observed in both the LgA groups. Further analyses indicate that this emergence of CP-AMPAR transmission in the LgA groups is associated with increased baseline responsiveness of MSN to excitatory drive. Together with data on cocaine infusions in each group, our results show that CP-AMPAR accumulation and enhanced glutamate transmission is associated with longer sessions (6 h), rather than the number of sessions or cocaine infusions.     

Pollution dilemma in Asian population: CNG and wound healing

Automobile exhaust constituents contribute significantly to air pollution in urban areas and compressed natural gas (CNG) is considered one of the most promising fuel alternatives for the future. CNG-powered four-stroke engine auto-rickshaws are ubiquitous in South Asian cities as taxi and for commercial transportation. Automotive exhaust contains several toxins, which are overwhelmingly toxic to the processes of wound healing. By utilizing the in vivo mouse model of wound healing, this report analyzes the effects of CNG-powered four-stroke auto-rickshaws smoke solution (4SARSS) on different events of wound healing; dermal matrix regeneration, re-epithelialization and neovascularization. A total of 72 adult mice, divided in eight groups were exposed to 4SARSS for 12 days. A highly significant reduction (P < 0.001) in wound closure was observed among all 4SARSS treated groups, at each time point of the experiment. An immature development in both the neoepidermis and the neodermis was observed among all 4SARSS treated wounds with defective re-epithelialization, dermal matrix regeneration and maturation of collagen bundles. Abbott curve, angular spectrum, 3D surface topographies, and histological investigations of wounds explicated highly significant activation (P < 0.001) of delayed-neovascularization among 4SARSS treated wounds. All these annotations advocate excessive toxicity of emission from CNG-powered auto-rickshaws to the process of wound healing and people occupationally exposed to this toxic emissions may suffer varying degree of delayed wound healing.     

Toxicological evaluation of the effects of 2-stroke auto-rickshaw smoke solutions on wound healing

Vehicle exhaust from traffic is a widespread air pollutant. The use of 3-wheel auto-rickshaws powered by a 2-stroke engine is widespread in south Asia; exhaust from these vehicles may cause different types of toxicities resulting in different pathologies. The aim of this study was to explore the association between exposure to 2-stroke auto-rickshaw smoke solution (2SARSS) and wound healing. The in vivo model of wound healing was customized to evaluate different stages of wound healing: dermal matrix regeneration, re-epithelialization, and neovascularization. A total of 72 adult mice were divided into 8 groups and exposed to 2SARSS for 12 days. A highly significant reduction (p < 0.001) in wound closure was observed among all 2SARSS-treated groups at day 8 post-wounding. Histological examination revealed a significant delay in the outcome of re-epithelialization, dermal matrix regeneration, and maturation of collagen bundles among all 2SARSS-exposed wounds. Delayed activation of neovascularization was seen in the 2SARSS-treated groups at day 12 post-wounding. The Abbot curve, angular spectrum, and several other 3D surface parameters of reverse wound topographies revealed a highly significant reduction (p < 0.001) in angiogenesis. These results demonstrate that application of 2SARSS causes a substantial delay in the progression of angiogenesis, resulting in delayed onset of wound healing. These observations validate the damaging effects of 2SARSS on wound healing. Thus, people who are directly or indirectly exposed to this toxic exhaust are expected to have delayed wound healing, which could result in chronic wounds.     

Bioadhesive hydrogel comprising bilirubin/β-cyclodextrin inclusion complexes promote diabetic wound healing

ContextChronic non-healing diabetic wound therapy is an important clinical challenge. Manipulating the release of bioactive factors from an adhesive hydrogel is an effective approach to repair chronic wounds. As an endogenous antioxidant, bilirubin (BR) has been shown to promote wound healing. Nonetheless, its application is limited by its low water solubility and oxidative degradation.ObjectiveThis study developed a bilirubin-based formulation for diabetic wound healing.Materials and methodsBilirubin was incorporated into β-CD-based inclusion complex (BR/β-CD) which was then loaded into a bioadhesive hydrogel matrix (BR/β-CD/SGP). Scratch wound assays were performed to examine the in vitro pro-healing activity of BR/β-CD/SGP (25 μg/mL of BR). Wounds of diabetic or non-diabetic rats were covered with BR or BR/β-CD/SGP hydrogels (1 mg/mL of BR) and changed every day for a period of 7 or 21 days. Histological assays were conducted to evaluate the in vivo effect of BR/β-CD/SGP.ResultsCompared to untreated (18.7%) and BR (55.2%) groups, wound closure was more pronounced (65.0%) in BR/β-CD/SGP group. In diabetic rats, the wound length in BR/β-CD/SGP group was smaller throughout the experimental period than untreated groups. Moreover, BR/β-CD/SGP decreased TNF-α levels to 7.7% on day 3, and elevated collagen deposition and VEGF expression to 11.9- and 8.2-fold on day 14. The therapeutic effects of BR/β-CD/SGP were much better than those of the BR group. Similar observations were made in the non-diabetic model.Discussion and conclusionBR/β-CD/SGP promotes wound healing and tissue remodelling in both diabetic and non-diabetic rats, indicating an ideal wound-dressing agent.     

Naoxintong accelerates diabetic wound healing by attenuating inflammatory response

ContextNaoxintong (NXT), a prescribed traditional Chinese medicine, widely used in cerebrovascular and cardiovascular diseases, could be effective in diabetic wounds.ObjectiveThis study evaluates the wound healing activity of NXT by employing an excisional wound splinting model.Materials and methodsNXT was dissolved in saline and given daily by gavage. Wounds were induced at the dorsum of non-diabetic (db/+) and diabetic (db/db) mice and treated with saline or 700 mg/kg/d NXT for 16 days. Wound closure was measured every four days. Extracellular matrix (ECM) remodelling, collagen deposition, leukocyte infiltration and expression of Col-3, CK14, CXCL1, CXCL2, MPO, Ly6G, CD68, CCR7, CD206, p-JAK1, p-STAT3 and p-STAT6 was analysed.ResultsNXT significantly accelerated rate of wound closure increased from 70% to 84%, accompanied by up-regulation of collagen deposition and ECM at days 16 post-injury. Moreover, NXT alleviated neutrophil infiltration, accompanied by down-regulation of CXCL1 and CXCL2 mRNA expression. In addition, NXT markedly augmented neutrophil efferocytosis. In diabetic wounds, the levels of M1 marker gene (CCR7) increased, while M2 marker gene (CD206) decreased, demonstrating a pro-inflammatory shift. Application of NXT increased M2 macrophage phenotype in db/db mice. Mechanistically, NXT treatment increased expression level of p-STAT3 and p-STAT6 at days 3 post-injury, indicating NXT mediated macrophages towards M2 phenotype and alleviated inflammation in diabetic wounds by activation of STAT3 and STAT6.ConclusionsOur study provides evidence that NXT accelerates diabetic wound healing by attenuating inflammatory response, which provides an important basis for use of NXT in the treatment of chronic diabetic wound healing.     

Development of endothelin receptors in perinatal rabbit pulmonary resistance arteries

1. Contractile responses to endothelin-1 (ET-1) and sarafotoxin S6c (S6c) were studied in pulmonary resistance arteries (∼320 μm i.d.) from fetal, 0–24 h, 4 day and 7 day rabbits. The effects of the ET_A-selective antagonist {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317, the selective ET_B receptor antagonist BQ-788 and the non-selective ET_A/ET_B receptor antagonist SB 209670, on these responses, were determined. Acetylcholine-induced vasodilation and noradrenaline-evoked contractions were also examined.
2. ET-1 potency was in the following order (pEC₅₀ values): fetal (8.7) = 0–24 h (8.8) = 4 day (8.6) > 7 day (8.0). The order of potency for S6c was 7 days (11.1) = 4 days (10.8) >0–24 h (9.7) > fetal (8.6). Hence, S6c and ET-1 were equipotent in the fetus but S6c was increasingly more potent than ET-1 with increasing age, being some 1000 times more potent by 7 days. By 7 days, responses to ET-1 were also resistant to both {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 and BQ-788. {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 inhibited responses to ET-1 in vessels from 0–24 h and 4 day, but not fetal, rabbits (pK_b: 6.4 in 4 day rabbits). BQ-788 inhibited responses to ET-1 at all age points except for 7 days (pK_b: 6.7 at 0–24 h; 6.2 at 4 days). BQ-788 inhibited responses to S6c at all age points (pK_b: 8.5 at 4 days). SB 209670 inhibited responses to ET-1 and S6c at 0–24 h and 4 days (pK_b for ET-1: 8.3 and 8.0 respectively; pK_b for S6c: 9.2 and 10.2 respectively).
3. Acetylcholine (1 μM) induced vasodilation at all age points (inhibited by 100 μM L-N^ω-nitroarginine methylester) although the degree of vasodilation was significantly reduced (∼75%) at 0–24 h. Noradrenaline induced contraction at all age points except 7 days and its response was significantly enhanced at 0–24 h.
4. Over the first week of life, the potency of S6c increases whilst that to ET-1 decreases suggesting differential development of responses to ET-1 and S6c and heterogeneity of ET_A- or `ET_B-like'' receptor-mediated responses. There is no synergism between ET_A and ET_B receptors at birth but this is established by 7 days. Immediately after birth rabbit Pulmonary Resistance Arteries are hyperresponsive to ET-1 and noradrenaline but exhibit impaired nitric-oxide dependent vasodilation.

     

Wound Healing Potential of Formulated Extract from Hibiscus Sabdariffa Calyx

Wound healing agents support the natural healing process, reduce trauma and likelihood of secondary infections and hasten wound closure. The wound healing activities of water in oil cream of the methanol extract of Hibiscus sabdariffa L. (Malvaceae) was evaluated in rats with superficial skin excision wounds. Antibacterial activities against Pseudomonas aeroginosa, Staphylococcus aureus and Echerichia coli were determined. The total flavonoid content, antioxidant properties and thin layer chromatographic fingerprints of the extract were also evaluated. The extract demonstrated antioxidant properties with a total flavonoid content of 12.30±0.09 mg/g. Six reproducible spots were obtained using methanol:water (95:5) as the mobile phase. The extract showed no antimicrobial activity on the selected microorganisms, which are known to infect and retard wound healing. Creams containing H. sabdariffa extract showed significant (P<0.05) and concentration dependent wound healing activities. There was also evidence of synergism with creams containing a combination of gentamicin and H. sabdariffa extract. This study, thus, provides evidence of the wound healing potentials of the formulated extract of the calyces of H. sabdariffa and synergism when co-formulated with gentamicin.     

Enhanced healing efficacy of an optimized gabapentin-melittin nanoconjugate gel-loaded formulation in excised wounds of diabetic rats

The present study aimed to design and optimize, a nanoconjugate of gabapentin (GPN)-melittin (MLT) and to evaluate its healing activity in rat diabetic wounds. To explore the wound healing potency of GPN-MLT nanoconjugate, an in vivo study was carried out. Diabetic rats were subjected to excision wounds and received daily topical treatment with conventional formulations of GPN, MLT, GPN-MLT nanoconjugate and a marketed formula. The outcome of the in vivo study showed an expedited wound contraction in GPN-MLT-treated animals. This was confirmed histologically. The nanoconjugate formula exhibited antioxidant activities as evidenced by preventing malondialdehyde (MDA) accumulation and superoxide dismutase (SOD) and glutathione peroxidase (GPx) enzymatic exhaustion. Further, the nanoconjugate showed superior anti-inflammatory activity as it inhibited the expression of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). This is in addition to enhancement of proliferation as indicated by increased expression of transforming growth factor-β (TGF- β), vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor receptor-β (PDGFRB). Also, nanoconjugate enhanced hydroxyproline concentration and mRNA expression of collagen type 1 alpha 1 (Col 1A1). In conclusion, a GPN-MLT nanoconjugate was optimized with respect to particle size. Analysis of pharmacokinetic attributes showed the mean particle size of optimized nanoconjugate as 156.9 nm. The nanoconjugate exhibited potent wound healing activities in diabetic rats. This, at least partly, involve enhanced antioxidant, anti-inflammatory, proliferative and pro-collagen activities. This may help to develop novel formulae that could accelerate wound healing in diabetes.     

Gelatin hydrogel/contact lens composites as rutin delivery systems for promoting corneal wound healing

Corneal wound healing is a highly regulated biological process that is of importance for reducing the risk of blinding corneal infections and inflammations. Traditional eye drop was the main approach for promoting corneal wound healing. However, its low bioavailability required a high therapeutic concentration, which can lead to ocular or even systemic side effects. To develop a safe and effective method for treating corneal injury, we fabricated rutin-encapsulated gelatin hydrogel/contact lens composites by dual crosslinking reactions including in situ free radical polymerization and carboxymethyl cellulose/N-hydroxysulfosuccinimide crosslinking. In vitro drug release results evidenced that rutin in the composites could be sustainedly released for up to 14 days. In addition, biocompatibility assay indicated nontoxicity of the composites. Finally, the effect of rutin-encapsulated composites on the healing of the corneal injury in rabbits was investigated. The injury was basically cured in corneas using rutin-encapsulated composites (healing rate, 98.3% ± 0.7%) at 48 h post-operation, while the damage was still present in corneas using the composite (healing rate, 87.0% ± 4.5%). Further proteomics analysis revealed that corneal wound healing may be promoted by the ERK/MAPK and PI3K/AKT signal pathways. These results inform a potential intervention strategy to facilitate corneal wound healing in humans.     

Cardamonin suppressed the migration,invasion, epithelial mesenchymal transition (EMT) and lung metastasis of colorectal cancer cells by down-regulating ADRB2 expression

ContextCardamonin (CDN) can suppress cell growth in colorectal cancer (CRC), a common digestive malignancy.ObjectiveWe explored the effect and mechanism of CDN on metastatic CRC.Materials and methodsTwo cell lines (HT29 and HCT116) were initially treated with CDN at different concentrations (5, 10 and 20 μmol/L) or 50 μmol/L propranolol (positive control) for 24 or 48 h. Then, the two cell lines were separately transfected with siADRB2 and ADRB2 overexpression plasmids, and further treated with 10 μmol/L CDN for 24 h. The cell viability, migration and invasion were determined by cell counting kit-8 (CCK-8), wound healing and transwell assays, respectively. The levels of ADRB2, matrix metalloprotease (MMP)-2, MMP-9, E-cadherin and N-cadherin were measured by Western blotting or/and RT-qPCR. A CRC metastasis model was established to evaluate the antimetastatic potential of CDN (25 mg/kg).ResultsADRB2 (3.2-fold change; p < 0.001) was highly expressed in CRC tissues. CDN at 10 μmol/L suppressed viability (69% and 70%), migration (33% and 66%), invasion (43% and 72%) and ADRB2 expression (2.2- and 2.84-fold change) in HT29 and HCT116 cells (p < 0.001). CDN at 10 μmol/L inhibited MMP-2, MMP-9 and N-cadherin expression but promoted E-cadherin expression in CRC cells (p < 0.001). Importantly, the effect of CDN on CRC cells was impaired by ADRB2 overexpression, but further enhanced by ADRB2 down-regulation (p < 0.01). Additionally, ADRB2 overexpression reversed the inhibitory effect of CDN on metastatic lung nodules (p < 0.05). Discussion and conclusions: CDN is a potential candidate for the treatment of metastatic CRC in clinical practice.     

Stimulation of locus coeruleus neurons by non-I1/I2-type imidazoline receptors: an in vivo and in vitro electrophysiological study

1. Imidazoline binding sites have been reported to be present in the locus coeruleus (LC). To investigate the role of these sites in the control of LC neuron activity, we studied the effect of imidazolines using in vivo and in vitro single-unit extracellular recording techniques.
2. In anaesthetized rats, local (27 pmoles) and systemic (1 mg kg⁻¹, i.v.) administrations of 2-(2-benzofuranyl)-2-imidazoline (2-BFI), a selective I-imidazoline receptor ligand, increased the firing rate of LC cells (maximal increase: 22±5%, P<0.001 and 16±7%, P<0.001 respectively). Chronic pretreatment with the irreversible monoamine oxidase inhibitor clorgyline (3 mg kg⁻¹, i.p., every 12 h for 14 days) abolished this effect.
3. In rat midpontine brain slices containing the LC, bath application (1 mM) of the imidazolines 2-BFI, 2-(4,5-dihydroimidaz-2-yl)-quinoline (BU224), idazoxan, efaroxan, phentolamine and (2-2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline (RX821002) reversibly stimulated LC cells. The maximal effect was ∼90% except for RX821002 and efaroxan which induced smaller maximal effects (∼58% and ∼35% respectively). Simultaneous application of idazoxan and 2BFI did not lead to additive effects.
4. Bath application of the α₂-adrenoceptor antagonists, yohimbine (1–10 μM) and N-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) (10 μM), failed to modify LC activity. The irreversible blockade of α₂-adrenoceptors with EEDQ (10 μM) did not alter the effect of idazoxan or that of efaroxan. Previous application of clorgyline (10 μM) did not modify the excitatory effect of 2-BFI or efaroxan.
5. Changes in the pH of the bathing solution (6.84–7.84) did not influence the effect caused by idazoxan. Bath application of 2-BFI (1 mM) reversed the inhibition induced by diazoxide (300 μM), an ATP-sensitive K⁺ channel opener, whereas application of glibenclamide (3 μM), an ATP-sensitive K⁺ channel blocker, partially blocked the effect of 2-BFI.
6. This study shows that imidazoline compounds stimulate the firing rate of LC neurons. This effect is not mediated by α₂-adrenoceptors nor by I₁ or I₂-imidazoline receptors but involves a different subtype of imidazoline receptor. Our results indicate that this receptor is located extracellularly and modulates ATP-sensitive K⁺ channels.

     

Accelerated wound healing and anti-inflammatory effects of physically cross linked polyvinyl alcohol–chitosan hydrogel containing honey bee venom in diabetic rats

Diabetes is one of the leading causes of impaired wound healing. The objective of this study was to develop a bee venom-loaded wound dressing with an enhanced healing and anti-inflammatory effects to be examined in diabetic rats. Different preparations of polyvinyl alcohol (PVA), chitosan (Chit) hydrogel matrix-based wound dressing containing bee venom (BV) were developed using freeze–thawing method. The mechanical properties such as gel fraction, swelling ratio, tensile strength, percentage of elongation and surface pH were determined. The pharmacological activities including wound healing and anti-inflammatory effects in addition to primary skin irritation and microbial penetration tests were evaluated. Moreover, hydroxyproline, glutathione and IL-6 levels were measured in the wound tissues of diabetic rats. The bee venom-loaded wound dressing composed of 10 % PVA, 0.6 % Chit and 4 % BV was more swellable, flexible and elastic than other formulations. Pharmacologically, the bee venom-loaded wound dressing that has the same pervious composition showed accelerated healing of wounds made in diabetic rats compared to the control. Moreover, this bee venom-loaded wound dressing exhibited anti-inflammatory effect that is comparable to that of diclofenac gel, the standard anti-inflammatory drug. Simultaneously, wound tissues covered with this preparation displayed higher hydroxyproline and glutathione levels and lower IL-6 levels compared to control. Thus, the bee venom-loaded hydrogel composed of 10 % PVA, 0.6 % Chit and 4 % BV is a promising wound dressing with excellent forming and enhanced wound healing as well as anti-inflammatory activities.     

Morphine-induced early delays in wound closure: Involvement of sensory neuropeptides and modification of neurokinin receptor expression

Dose-limiting side effects of centrally acting opioid drugs have led to the use of topical opioids to reduce the pain associated with chronic cutaneous wounds. However, previous studies indicate that topical morphine application impairs wound healing. This study was designed to elucidate the mechanisms by which morphine delays wound closure. Rats were depleted of sensory neuropeptides by treatment with capsaicin, and full-thickness 4-mm diameter wounds were excised from the intrascapular region. Wounds were treated topically twice daily with 5 mM morphine sulfate, 1 mM substance P, 1 mM neurokinin A, or 5 mM morphine combined with 1 mM substance P or neurokinin A and wound areas assessed. During closure, wound tissue was taken 1, 3, 5, and 8 days post-wounding from control and morphine-treated rats and immunostained for neurokinin receptors and markers for macrophages, myofibroblasts, and vasculature. Results obtained from capsaicin-treated animals demonstrated a significant delay in the early stages of wound contraction that was reversed by neuropeptide application. Treatment of capsaicin-treated rats with topical morphine did not further delay wound closure, suggesting that topical opioids impair wound closure via the inhibition of peripheral neuropeptide release into the healing wound. Morphine application altered neurokinin-1 and neurokinin-2 receptor expression in inflammatory and parenchymal cells essential for wound healing in a cell-specific manner, demonstrating a direct effect of morphine on neurokinin receptor regulation within an array of cells involved in wound healing. These data provide evidence indicating a potentially detrimental effect of topical morphine application on the dynamic wound healing process.     

Gastroprotective effects of water extract of domesticated Amauroderma rugosum against several gastric ulcer models in rats

ContextAmauroderma rugosum (Blume & T. Nees) Torrend (Ganodermataceae) is an edible mushroom with medicinal properties. However, the effects of A. rugosum on gastric ulcer remain unclear.ObjectiveTo investigate the gastroprotective efficacy of water extract of A. rugosum (WEA) on gastric ulcer.Materials and methodsSprague-Dawley rats were randomly grouped as control, model, lansoprazole and 200, 100 and 50 mg/kg of WEA. After pre-treatment for seven days, ethanol- and indomethacin-induced gastric ulcer models were established. The gastric ulcer and histopathology were investigated. Enzyme-linked immunosorbent assay (ELISA), quantitative polymerase chain reaction (Q-PCR) and Western blot assays were conducted to explore the potential anti-inflammatory effect and mechanism of WEA. Additionally, the pyloric ligation model was used to explore the influence of WEA on gastric acid and mucus.ResultsPre-treatment with WEA (200, 100 and 50 mg/kg) effectively reduced ulcerous area in both ethanol-induced (71%, 88% and 71%) and indomethacin-induced (77%, 65% and 86%) gastric ulcer model. The gastric levels of tumour necrosis factor-alpha (TNF-α) (34% and 50 mg/kg), interleukin-6 (IL-6) (32% and 100 mg/kg) and interleukin-1β (IL-1β) (36%, 45% and 41%) were reduced significantly (p < 0.05) by WEA. Serum nitric oxide was decreased significantly (p < 0.05) at 200 and 50 mg/kg and PGE2 concentration was increased remarkably (p < 0.05) at 100 mg/kg. Gene expression of inflammasome Nlrp3, and the nuclear translocation of nuclear factor-κB (NF-κB) P65 were significantly decreased by WEA pre-treatment. However, the pH of gastric acid and secretion of mucus did not show any significant change.ConclusionsThe gastroprotective effect of WEA on gastric damage is attributed to anti-inflammation through the inhibition on NF-κB P65 nuclear migration and Nlrp3 gene expression.     

Effects of Ca2+ channel blocker neurotoxins on transmitter release and presynaptic currents at the mouse neuromuscular junction

1. The effects of the voltage-dependent calcium channel (VDCC) blockers ω-agatoxin IVA (ω-AgaIVA), ω-conotoxin GVIA (ω-CgTx), ω-conotoxin MVIIC (ω-MVIIC) and ω-conotoxin MVIID (ω-MVIID) were evaluated on transmitter release in the mouse diaphragm preparation. The effects of ω-AgaIVA and ω-MVIIC were also evaluated on the perineurial calcium and calcium-dependent potassium currents, I_Ca and I_K(Ca), respectively, in the mouse levator auris preparation.
2. The P- and Q-type VDCC blocker ω-AgaIVA (100 nM) and P- Q- and N-type channel blockers ω-MVIIC (1 μM) and ω-MVIID (3 μM) strongly reduced transmitter release (>80–90% blockade) whereas the selective N-type channel blocker ω-CgTx (5 μM) was ineffective.
3. The process of release was much more sensitive to ω-MVIIC (IC₅₀=39 nM) than to ω-MVIID (IC₅₀=1.4 μM). After almost completely blocking transmitter release (quantal content ∼0.3% of its control value) with 3 μM ω-MVIIC, elevating the external [Ca²⁺] from 2 to 10 mM induced an increase of ∼20 fold on the quantal content of the endplate potential (e.p.p.) (from 0.2±0.04 to 4.8±1.4).
4. Nerve-evoked transmitter release in a low Ca²⁺-high Mg²⁺ medium (low release probability, quantal content = 2±0.1) had the same sensitivity to ω-AgaIVA (IC₅₀=16.8 nM) as that in normal saline solutions. In addition, K⁺-evoked transmitter release was also highly sensitive to the action of this toxin (IC₅₀=11.5 nM; 100 nM >95% blockade). The action of ω-AgaIVA on transmitter release could be reversed by toxin washout if the experiments were carried out at 31–33°C. Conversely, the effect of ω-AgaIVA persisted even after two hours of toxin washout at room temperature.
5. Both the calcium and calcium-dependent potassium presynaptic currents, I_Ca and I_K(Ca), respectively, were highly sensitive to low concentrations (10–30 nM) of ω-AgaIVA. The I_Ca and the I_K(Ca) were also strongly reduced by 1 μM ω-MVIIC. The most marked difference between the action of these two toxins was the long incubation times required to achieve maximal effects with ω-MVIIC.
6. In summary these results provide more evidence that synaptic transmission at the mammalian neuromuscular junction is mediated by Ca²⁺ entry through P- and/or Q-type calcium channels.

     

Silymarin improved 6-OHDA-induced motor impairment in hemi-parkisonian rats: behavioral and molecular study

Background

Neuroinflammation and oxidative stress has been shown to be associated with the development of Parkinson disease (PD). In the present study, we investigated the effect of intraperitoneal (i.p.) administration of silymarin, on 6-OHDA-induced motor-impairment, brain lipid per-oxidation and cerebrospinal fluid (CSF) levels of inflammatory cytokine in the rats.

Results

The results showed that silymarin is able to improve motor coordination significantly (p < 0.001) in a dose dependent manner. There was a significant (p < 0.001) increase in MDA levels of 6-OHDA-lesioned rats whereas; in silymarin (100, 200 and 300 mg/kg, i.p. for 5 days) pre-treated hemi-parkinsonian rats MDA levels was decreased markedly (p < 0.001). Furthermore the CSF levels of IL-1β was decreased (p < 0.001) in silymarin (100, 200 and 300 mg/kg) pre-treated rats up to the range of normal non-parkinsonian animals.

Conclusion

We found that pre-treatment with silymarin could improve 6-OHDA-induced motor imbalance by attenuating brain lipid per-oxidation as well as CSF level of IL-1β as a pro-inflammatory cytokine. We suggest a potential prophylactic effect for silymarin in PD. However, further clinical trial studies should be carried out to prove this hypothesis.     

Inhibition of ornithine decarboxylase potentiates nitric oxide production in LPS-activated J774 cells

1. We have examined whether modulation of the polyamine biosynthetic pathway, through inhibition by α-difluoromethylornithine (DFMO) of the rate limiting enzyme, ornithine decarboxylase (ODC), modulates NO synthesis in J774 macrophages.
2. DFMO potentiated LPS-stimulated nitrite production in both a concentration- and time-dependent manner, increasing nitrite levels by 48±5% at 10 mM. This effect was observed in cells pre-treated with DFMO for 24 h prior to stimulation with LPS. Addition of DFMO 12 h after LPS failed to potentiate LPS-induced nitrite production.
3. Supplementation of the culture medium with horse serum (10%) in place of foetal calf serum (10%) caused no significant change in either LPS-induced nitrite production or in the ability of DFMO (10  mM) to potentiate LPS-induced NO synthesis.
4. Metabolism of L-[³H]arginine to L-[³H]citrulline by partially purified inducible nitric oxide synthase (iNOS) was not significantly altered by either DFMO (1–10 mM) or by putrescine (0.001–1 mM), spermidine (0.001–1 mM) or spermine (0.001–1 mM). iNOS activity was also unaffected by 1 mM EGTA but was markedly attenuated (70±0.07%) by L-NMMA (100 μM).
5. Pre-incubation of cells with DFMO (10 mM; 24 h) prior to activation with LPS resulted in enhanced (∼2 fold) iNOS protein expression.
6. These results show that DFMO potentiates LPS-induced nitrite production in the murine macrophage cell line J774. Since the only known mechanism of action of DFMO is inhibition of ODC, and thus polyamine biosynthesis, we conclude that expression of iNOS can be critically regulated by endogenous polyamines.