Therapeutic efficacy of new botulinum toxin identified in CCUG 7968 strain.

Botulinum neurotoxin type A (BoNT/A) induces muscle atrophy by cleaving synaptosomal-associated protein 25. Thus, BoNT/A has been actively utilized for the treatment of masseter and gastrocnemius hypertrophy. In this study, INI101 toxin was newly identified from the CCUG 7968 strain, and its therapeutic efficacy was evaluated both in vitro and in vivo. The INI101 toxin showed identical genetic sequence, amino acid sequence, and protein subunit composition to BoNT/A produced from strain Hall A. Electromyography (EMG), and immunofluorescence staining demonstrated that INI101 (at 2 ~ 8 U/rat) effectively blocked the neuromuscular junction with no toxicity in a rat model. The EMG results showed INI101 toxin-induced weight loss and volume reduction of the gastrocnemius, similar to the effects of Botox® (BTX). Histological and immunofluorescence staining was consistent with this EMG result, showing that INI101 toxin caused muscle fiber reduction in the gastrocnemius. Notably, INI101 toxin diffused less into adjacent muscle tissue than BTX, indicating that INI101 toxin may reduce potential side effects due to diffusion into normal tissues. INI101 toxin isolated from the novel strain CCUG 7968 is a newly identified meaningful biopharmaceutical comparable to the conventional BoNT/A in the medical field. KEY POINTS: • Botulinum neurotoxin type A (BoNT/A, INI101) was identified from the CCUG 7968 strain. • INI101 toxin showed similar safety and therapeutic efficacy comparable to conventional BoNT/A both in vitro and in vivo. • INI101 toxin is a meaningful biopharmaceutical comparable to the conventional BoNT/A in the medical field.

Efficacy of nobiletin in improving hypercholesterolemia and nonalcoholic fatty liver disease in high-cholesterol diet-fed mice.

BACKGROUND/OBJECTIVES: Nobiletin (NOB), a citrus flavonoid, is reported to have beneficial effects on cardiovascular and metabolic health. However, there is limited research investigating the effect of long-term supplementation with low-dose NOB on high-cholesterol diet (HCD)-induced hypercholesterolemia and non-obese nonalcoholic fatty liver disease (NAFLD). Therefore, we investigated the influence of NOB on hypercholesterolemia and NAFLD in HCD-fed mice. SUBJECTS/METHODS: C57BL/6J mice were fed a normal diet (ND) or HCD (35 kcal% fat, 1.25% cholesterol, 0.5% cholic acid) with or without NOB (0.02%) for 20 weeks. RESULTS: HCD feeding markedly reduced the final body weight compared to ND feeding, with no apparent energy intake differences. NOB supplementation suppressed HCD-induced weight loss without altering energy intake. Moreover, NOB significantly decreased the total cholesterol (TC) levels and the low-density lipoprotein (LDL)/very-LDL-cholesterol to TC ratio, and increased the high-density lipoprotein-cholesterol/TC ratio in plasma, compared to those for HCD feeding alone. The plasma levels of inflammatory and atherosclerosis markers (C-reactive protein, oxidized LDL, interleukin [IL]-1ß, IL-6, and plasminogen activator inhibitor-1) were significantly lower, whereas those of anti-atherogenic adiponectin and paraoxonase were higher in the NOB-supplemented group than in the HCD control group. Furthermore, NOB significantly decreased liver weight, hepatic cholesterol and triglyceride contents, and lipid droplet accumulation by inhibiting messenger RNA expression of hepatic genes and activity levels of cholesterol synthesis-, esterification-, and fatty acid synthesis-associated enzymes, concomitantly enhancing fatty acid oxidation-related gene expression and enzyme activities. Dietary NOB supplementation may protect against hypercholesterolemia and NAFLD via regulation of hepatic lipid metabolism in HCD-fed mice; these effects are associated with the amelioration of inflammation and reductions in the levels of atherosclerosis-associated cardiovascular markers. CONCLUSIONS: The present study suggests that NOB may serve as a potential therapeutic agent for the treatment of HCD-induced hypercholesterolemia and NAFLD.

Pudendal nerve entrapment syndrome caused by ganglion cysts along the pudendal nerve.

Pudendal nerve entrapment (PNE) syndrome refers to the condition in which the pudendal nerve is entrapped or compressed. Reported cases of PNE associated with ganglion cysts are rare. Deep gluteal syndrome (DGS) is defined as compression of the sciatic or pudendal nerve due to a non-discogenic pelvic lesion. We report a case of PNE caused by compression from ganglion cysts and treated with steroid injection; we discuss this case in the context of DGS. A 77-year-old woman presented with a 3-month history of tingling and burning sensations in the left buttock and perineal area. Ultrasonography showed ganglion cystic lesions at the subgluteal space. Magnetic resonance imaging revealed cystic lesions along the pudendal nerve from below the piriformis to the Alcock's canal and a full-thickness tear of the proximal hamstring tendon. Aspiration of the cysts did not yield any material. We then injected steroid into the cysts, which resolved her symptoms. Steroid injection into a ganglion cyst should be considered as a treatment option for PNE caused by ganglion cysts.

The Electrophysiologic and Ultrasonographic Change after Carpal Tunnel Release in a Patient with Hereditary Neuropathy with Liability to Pressure Palsy.

Hereditary neuropathy with liability to pressure palsy (HNPP) often manifests via peripheral nerve entrapment including median nerve compression at the carpal tunnel. However, there are few reports on surgical interventions for focal compression of the median nerve at the wrist in patients with HNPP. We report a case of a patient with HNPP who improved clinically, electrophysiologically, and ultrasonographically after carpal tunnel release (CTR). A 56-year-old woman presented with an 18-month history of pain in both thumbs. Nerve conduction study (NCS) revealed bilateral median neuropathy at the wrist. Ultrasonography demonstrated a markedly increased cross-sectional area (CSA) of both median nerves. Gene analysis revealed a deletion of the PMP22 gene. She received bilateral CTR. Follow-up NCS at one year demonstrated the improvement of motor and sensory conduction; follow-up ultrasonography revealed significantly reduced CSA. Our case suggests that surgical decompression can be applicable to well-selected patients with HNPP.

Supplementation of the Flavonoid Myricitrin Attenuates the Adverse Metabolic Effects of Long-Term Consumption of a High-Fat Diet in Mice.

The flavonoid myricitrin exhibits various pharmacological and physiological effects. However, studies on the effects of myricitrin on obesity are limited. We hypothesized that dietary myricitrin would attenuate the adiposity and metabolic dysfunction that occur in obesity. To test this hypothesis, mice were randomly fed a high-fat diet (HFD) or HFD supplemented with myricitrin for 16 weeks. Myricitrin significantly reduced white adipose tissue (WAT) mass, adipocyte size, and plasma leptin levels, and also attenuated dyslipidemia. These changes appeared to result from increased energy expenditure and activation of the carnitine acyltransferase (CPT) and ß-oxidation in WAT. Expressions of the proinflammatory genes NF-κB, TLR2, MCP1, and TNF-α were also lower in the WAT of myricitrin-supplemented mice. Moreover, myricitrin markedly reduced hepatic triglyceride accumulation and plasma aspartate transaminase levels by increasing CPT activity and reducing fatty acid synthase activity in the liver. Myricitrin-supplemented mice also showed improved glucose tolerance, insulin sensitivity, and decreased hyperinsulinemia, along with decreased levels of circulating resistin. In conclusion, long-term consumption of a myricitrin-supplemented diet may effectively protect against HFD-induced obesity and related metabolic disorders.

Honokiol Improves Insulin Resistance, Hepatic Steatosis, and Inflammation in Type 2 Diabetic db/db Mice.

This study focuses on the effect of honokiol (HON) on glucose homeostasis, insulin resistance, dyslipidemia, hepatic steatosis, and inflammation in type 2 diabetic db/db mice. Male C57BL/KsJ-db/db mice were fed a normal diet with or without HON (0.02%, w/w) or pioglitazone (PIO, anti-diabetic agent, 0.01%, w/w) for 5 weeks. Blood biomarker, tissue morphology and enzymatic and genetic parameters were determined. PIO significantly decreased food intake, fasting blood glucose, and glycosylated hemoglobin (HbA1c) levels, but markedly increased body weight, adipose tissue weight, and plasma leptin levels. HON did not significantly affect food intake, body weight, or levels of plasma leptin and blood glucose. However, HON led to significant decreases in adipose tissue weight, plasma insulin, blood HbA1c and HOMA-IR levels and improved glucose tolerance. The anti-diabetic and anti-adiposity effects of HON were partially related to the inhibition of gluconeogenic enzymes and their mRNA expression in the liver; and the inhibition of lipogenic enzymes in adipose tissue, respectively. Unlike PIO, HON did not affect dyslipidemia, but ameliorated hepatic steatosis by inhibiting hepatic lipogenic enzymes activity. Moreover, HON exhibited anti-inflammatory effects similar to PIO. These results suggest that HON can protect against type 2 diabetes by improving insulin resistance, glucose and lipid metabolism, and inflammation.

Topical administration of EGF suppresses immune response and protects skin barrier in DNCB-induced atopic dermatitis in NC/Nga mice.

Atopic dermatitis (AD) is a common inflammatory skin disease characterized by a complex, heterogeneous pathogenesis including skin barrier dysfunction, immunology, and pruritus. Although epidermal growth factor (EGF) is essential for epithelial homeostasis and wound healing, the effect of EGF on AD remains to be explored. To develop a new therapy for AD, the anti-AD potential of EGF was investigated by inducing AD-like skin lesions in NC/Nga mice using 2,4-dinitrochlorobenzene (DNCB). EGF was administrated to NC/Nga mice to evaluate its therapeutic effect on DNCB-induced AD. EGF treatment improved dermatitis score, ear thickness, epidermal hyperplasia, serum total immunoglobulin E level, and transepidermal water loss in NC/Nga mice with DNCB-induced AD. In addition, levels of skin barrier-related proteins such as filaggrin, involucrin, loricrin, occludin, and zonula occludens-1 (ZO-1) were increased by EGF treatment. These beneficial effects of EGF on AD may be mediated by EGF regulation of Th1/Th2-mediated cytokines, mast cell hyperplasia, and protease activated receptor-2 (PAR-2) and thymic stromal lymphopoietin (TSLP), which are triggers of AD. Taken together, our findings suggest that EGF may potentially protect against AD lesional skin via regulation of skin barrier function and immune response.

Long-term dietary supplementation with low-dose nobiletin ameliorates hepatic steatosis, insulin resistance, and inflammation without altering fat mass in diet-induced obesity.

SCOPE: We evaluated the long-term effect of low-dose nobiletin (NOB), a polymethoxylated flavone, on diet-induced obesity and related metabolic disturbances. METHODS AND RESULTS: C57BL/6J mice were fed a high-fat diet (HFD, 45 kcal% fat) with or without NOB (0.02%, w/w) for 16 weeks. NOB did not alter food intake or body weight. Despite increases in fatty acid oxidation-related genes expression and enzymes activity in adipose tissue, NOB did not affect adipose tissue weight due to simultaneous increases in lipogenic genes expression and fatty acid synthase activity. However, NOB significantly decreased not only pro-inflammatory genes expression in adipose tissue but also proinflammatory cytokine levels in plasma. NOB-supplemented mice also showed improved glucose tolerance and insulin resistance, along with decreased levels of plasma insulin, free fatty acids, total cholesterol, non-HDL-cholesterol, and apolipoprotein B. In addition, NOB caused significant decreases in hepatic lipid droplet accumulation and triglyceride content by activating hepatic fatty acid oxidation-related enzymes. Hepatic proinflammatory TNF-α mRNA expression, collagen accumulation, and plasma levels of aminotransferases, liver damage indicators, were also significantly lower in NOB-supplemented mice. CONCLUSION: These findings suggest that long-term supplementation with low-dose NOB can protect against HFD-induced inflammation, insulin resistance, dyslipidemia, and nonalcoholic fatty liver disease, without ameliorating adiposity.

Applicability of citronella oil (Cymbopogon winteratus) for the prevention of mosquito-borne diseases in the rural area of Tikapur, far-western Nepal.

INTRODUCTION: Mosquito-borne diseases are a serious global problem, particularly in tropical and sub-tropical countries such as Nepal. Citronella oil is a natural mosquito repellent as well as a local fragrance in Nepal, which is accessible at very low cost because citronella plants are widely cultivated in rural areas of the Terai belt in Nepal. This study was conducted using a real-life randomized controlled pilot trial to confirm the effectiveness and applicability of locally-produced citronella oil as a mosquito repellent for the prevention of mosquito-borne diseases in Nepal. METHODS: A repellency activity test was performed with 100% citronella oil (Cymbopogon winteratus) from April to May 2013 in the Tikapur Municipality of the Kailali district, Nepal. The test was divided into two trials: an indoor exposure (IE) test (N=101) and an outdoor exposure (OE) test (N=140) from 5.00 pm to 7.00 pm. Each trial contained an experimental citronella oil-applied group and a non-applied (control) group. The outcome measures were the protective effect of citronella oil against mosquitoes, the number of mosquito bites, the repellency percentage, the smell satisfaction and the irritation level. RESULTS: Experimental group had a significant protective effect against mosquito bites in IE (96.5%, n=57) and OE (95.7%, n=70) tests compared to the control group in IE (29.5%, n=44) and OE (28.6%, n=70) tests (experimental vs control groups, p<0.001). The repellency percentage for the OE test was 96.7%. In the smell satisfaction test (n=127), most of the participants responded with high satisfaction: 'good' (67.7%), 'very good' (16.5%), 'bad' (13.4%) and 'very bad' (2.4%). IE and OE tests showed similar satisfaction levels in each category. In the irritation level test (n=127), 87.4% and 12.6% responded with no irritation and slight irritation, respectively. There were no reports of moderate or severe irritation. CONCLUSIONS: The topical application of citronella oil can be employed as an easily-available, affordable and effective alternative mosquito repellent to prevent mosquito-borne diseases in rural areas such as Tikapur, Nepal.

Design and Application of Rolling Circle Amplification for a Tumor-Specific Drug Carrier.

It is challenging to design rolling circle amplification (RCA) for tumor-selective delivery of drugs. Here, we devise a doxorubicin nanocarrier composed of RCA products, cholesterol-DNA, and folate-DNA conjugates. RCA products, designed to contain tandem repeats of short hairpin DNA, employ the repeated sequences complementary to both DNA conjugates, and thus RCA products/cholesterol-DNA/folate-DNA complexes, generated via sequential base pairing processes, acquire the amphiphilic properties that facilitate self-assembly into the highly condensed nanoparticles (RCA nanoparticles). Doxorubicin-loaded RCA nanoparticles, especially with high cargo capacity, release drugs to the environment with the aid of acidity and show selective cytotoxicity to cancer cells. Particularly, the condensed structures enable RCA nanoparticles to be resistant to nucleases in the blood. These results show that RCA nanoparticles have great potential as a doxorubicin carrier for targeted cancer therapy, and furthermore, our strategy provides an alternative tool to exploit RCA techniques on drug delivery systems.

High-fat diet decreases energy expenditure and expression of genes controlling lipid metabolism, mitochondrial function and skeletal system development in the adipose tissue, along with increased expression of extracellular matrix remodelling- and inflammation-related genes.

The aim of the present study was to identify the genes differentially expressed in the visceral adipose tissue in a well-characterised mouse model of high-fat diet (HFD)-induced obesity. Male C57BL/6J mice (n 20) were fed either HFD (189 % of energy from fat) or low-fat diet (LFD, 42 % of energy from fat) for 16 weeks. HFD-fed mice exhibited obesity, insulin resistance, dyslipidaemia and adipose collagen accumulation, along with higher levels of plasma leptin, resistin and plasminogen activator inhibitor type 1, although there were no significant differences in plasma cytokine levels. Energy intake was similar in the two diet groups owing to lower food intake in the HFD group; however, energy expenditure was also lower in the HFD group than in the LFD group. Microarray analysis revealed that genes related to lipolysis, fatty acid metabolism, mitochondrial energy transduction, oxidation-reduction, insulin sensitivity and skeletal system development were down-regulated in HFD-fed mice, and genes associated with extracellular matrix (ECM) components, ECM remodelling and inflammation were up-regulated. The top ten up- or down-regulated genes include Acsm3, mt-Nd6, Fam13a, Cyp2e1, Rgs1 and Gpnmb, whose roles in the deterioration of obesity-associated adipose tissue are poorly understood. In conclusion, the genes identified here provide new therapeutic opportunities for prevention and treatment of diet-induced obesity.

Inhibition of prothrombin kringle-2-induced inflammation by minocycline protects dopaminergic neurons in the substantia nigra in vivo.

Prothrombin kringle-2 (pKr-2), a domain of prothrombin, can cause the degeneration of mesencephalic dopaminergic neurons through microglial activation. However, the chemical products that inhibit pKr-2-induced inflammatory activities in the brain are still not well known. The present study investigated whether minocycline, a semisynthetic tetracycline derivative, could inhibit pKr-2-induced microglial activation and prevent the loss of nigral dopaminergic (DA) neurons in vivo. To address this question, rats were administered a unilateral injection of pKr-2 in the substantia nigra in the presence or absence of minocycline. Our results show that pKr-2 induces the production of proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß), and inducible nitric oxide synthase from the activated microglia. In parallel, 7 days after pKr-2 injection, tyrosine hydroxylase immunocytochemical analysis and western blot analysis showed a significant loss of nigral DA neurons. This neurotoxicity was antagonized by minocycline and the observed neuroprotective effects were associated with the ability of minocycline to suppress the expression of tumor necrosis factor-α, interleukin-1ß, and nitric oxide synthase. These results suggest that minocycline may be promising as a potential therapeutic agent for the prevention of DA neuronal degeneration associated with pKr-2-induced microglial activation.

Omija fruit extract improves endurance and energy metabolism by upregulating PGC-1α expression in the skeletal muscle of exercised rats.

This study was conducted to examine the effects of omija fruit extract on the endurance and energy metabolism of rats trained under a progressive loaded exercise program. Thirty male Sprague-Dawley rats (6 weeks old) were divided into three groups according to exercise and experimental diets for 6 weeks: the sedentary control (SC) group (n=10), the exercise control (EC) group (n=10), and the exercise group supplemented with 0.6% w/w omija extract (OM; n=10). The omija supplement significantly extended the running endurance time compared with the EC group. The soleus muscle weight was significantly higher in the OM group compared with the EC group and the plasma lactate and ammonia levels were significantly lower in the OM group than in the EC group. Plasma glucose, free fatty acid, and gastrocnemius muscle glycogen concentrations were also significantly lower in the OM group compared with the EC group. The lactate dehydrogenase activity was significantly higher in the gastrocnemius muscle of the OM group compared with the EC group. Expression of the peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) mRNA in the gastrocnemius muscle and soleus muscle were significantly elevated in the OM group and the soleus muscle peroxisome proliferator-activated receptor α (PPARα), carnitine palmitoyltransferase 1b (CPT1b), and ß-hydroxyacyl coenzyme A dehydrogenase (ß-HAD) mRNA expressions showed the same tendency. Taken together, these results show that supplementation of omija fruit extract enhances endurance and energy metabolism by upregulating the PGC-1α expression in the skeletal muscle.

Garcinia Cambogia attenuates diet-induced adiposity but exacerbates hepatic collagen accumulation and inflammation.

AIM: To investigate long-term effects of Garcinia Cambogia (GC), weight-loss supplement, on adiposity and non-alcoholic fatty liver disease in obese mice. METHODS: Obesity-prone C57BL/6J mice were fed a high-fat diet (HFD, 45 kcal% fat) with or without GC (1%, w/w) for 16 wk. The HFD contained 45 kcal% fat, 20 kcal% protein and 35 kcal% carbohydrate. They were given free access to food and distilled water, and food consumption and body weight were measured daily and weekly, respectively. Data were expressed as the mean ± SE. Statistical analyses were performed using the statistical package for the social science software program. Student's t test was used to assess the differences between the groups. Statistical significance was considered at P < 0.05. RESULTS: There were no significant changes in body weight and food intake between the groups. However, the supplementation of GC significantly lowered visceral fat accumulation and adipocyte size via inhibition of fatty acid synthase activity and its mRNA expression in visceral adipose tissue, along with enhanced enzymatic activity and gene expression involved in adipose fatty acid ß-oxidation. Moreover, GC supplementation resulted in significant reductions in glucose intolerance and the plasma resistin level in the HFD-fed mice. However, we first demonstrated that it increased hepatic collagen accumulation, lipid peroxidation and mRNA levels of genes related to oxidative stress (superoxide dismutase and glutathione peroxidase) and inflammatory responses (tumor necrosis factor-α and monocyte chemoattractant protein-1) as well as plasma alanine transaminase and aspartate transaminase levels, although HFD-induced hepatic steatosis was not altered. CONCLUSION: GC protects against HFD-induced obesity by modulating adipose fatty acid synthesis and ß-oxidation but induces hepatic fibrosis, inflammation and oxidative stress.

Long-term supplementation of honokiol and magnolol ameliorates body fat accumulation, insulin resistance, and adipose inflammation in high-fat fed mice.

SCOPE: This study investigated the effect of honokiol (HON) and magnolol (MAG), phenolic compounds in Magnolia plants, on adiposity and adiposity-related metabolic disturbances in mice fed high-fat diet (HFD), and the potential underlying mechanisms focusing on the lipid metabolism and inflammatory response. METHOD AND RESULTS: C57BL/6J mice were fed HFD (45 kcal% fat) with or without HON (0.02%, w/w) or MAG (0.02%, w/w) for 16 wk. Despite no changes in body weight, food intake, and hepatic fat accumulation, HON and MAG significantly lowered the weight of white adipose tissue (WAT) as well as adipocyte size and protected against insulin resistance induced by HFD. These effects were associated with increases in energy expenditure and adipose fatty acid oxidation and decreases in fatty acid synthase activity and expression of genes related to fatty acid synthesis, desaturation, and uptake, as well as adipocyte differentiation in WAT. Moreover, HON and MAG significantly lowered the expression of proinflammatory genes in WAT and elevated the plasma IL-10 level. Particularly, HON significantly decreased the plasma resistin level and increased the plasma adiponectin level compared to the control group. CONCLUSION: HON and MAG have potential as novel agents for amelioration of adiposity and associated insulin resistance and inflammation.