Protective effect of ISO-1 with inhibition of RIPK3 up-regulation and neutrophilic accumulation on acetaminophen-induced liver injury in mice.

Overdose use of acetaminophen (APAP) often occurs a severe liver injury, and its liver injury is lethal in some cases. Macrophage migration inhibitory factor (MIF) is expressed in a variety of cells and has multifunctional roles. However, the role of MIF in APAP-induced liver injury has not been fully investigated. In this study, we investigated whether treatment with (S,R)-3-(4-hydroxyphenil)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1), a MIF inhibitor, protected mice from acute APAP-induced liver injury. Acute liver injury was induced by injection of APAP (300 mg/kg body weight). Mice were treated with a single injection of ISO-1(15 mg/kg body weight) 1 h (h) before APAP administration. Histological, biochemical and molecular analyses were performed in liver of mice 12 h after APAP administration. ISO-1 remarkably improved the histological findings of APAP-induced liver injury in mice. The increases in serum levels of alanine aminotransferase (ALT), and macrophage inflammatory protein-2 (MIP-2) by APAP were inhibited by ISO-1. In addition, ISO-1 reduced the increased number of the myeloperoxidase-staining cells and that of TUNEL-positive staining cells in the liver of mice with APAP-induced liver injury. Up-regulation of hepatic receptor interacting protein kinase (RIPK)3 and heat shock protein70 by APAP was suppressed in the liver of mice given ISO-1. These results provide the additional evidence that inhibition of MIF activity may be clinically effective for treatment of acute APAP-induced liver injury.

The delivery of mRNA to colon inflammatory lesions by lipid-nano-particles containing environmentally-sensitive lipid-like materials with oleic acid scaffolds.

An mRNA gene therapy represents a potentially promising therapeutic for curing inflammatory diseases. The transient nature of the gene expression of mRNA would be expected to be beneficial for avoiding undesired side effects. Since the mRNA is a vulnerable molecule, a development of a carrier that can deliver the mRNA to the cytoplasm has a high priority. We report herein on the development of a system for delivering mRNA to the inflammatory lesion in a dextran sulfate sodium (DSS)-induced colitis model. We modulated molecular structures of an ionizable lipid, an SS-cleavable and pH-activated lipid-like material (ssPalm). Among the fatty acids investigated, oleic acid scaffolds (ssPalmO) appeared to be more biocompatible than either myristic acid or linoleic acid scaffolds with the colitis model. The structural modification of the hydrophilic head groups from linear tertiary amines to piperazine rings (ssPalmO-Paz4-C2) resulted in a more than 10-fold higher increasing in the transgene activity in inflammatory colon. The most notable observation is that the transgene activity in the inflammatory colon is significantly higher than that in liver, the major clearance organ of lipid nanoparticles. Collectively, the ssPalmO-Paz4-C2 represents a promising material for the delivery of an mRNA to inflammatory lesions.

Protective effect of chlorogenic acid on the inflammatory damage of pancreas and lung in mice with l-arginine-induced pancreatitis.

AIMS: Pancreatitis is characterized by inflammatory disease with severe tissue injury in pancreas, and the incidence of pancreatitis has been recently increasing. Although several treatments of acute pancreatitis have been developed, some patients have been resistant to current therapy. Chlorogenic acid (CGA) is one of the polyphenols, and is known to have an anti-inflammatory effect. In this study, we investigated the effects of CGA on experimental pancreatitis in mice. MATERIALS AND METHODS: Pancreatitis was induced by twice injection of l-arginine (5g/kg body weight). Mice were intraperitoneally injected with CGA (20mg/kg or 40mg/kg) 1h before administration of l-arginine. KEY FINDINGS: Administration of 40mg/kg of CGA decreased the histological severity of pancreatitis and pancreatitis-associated lung injury. Moreover, administration of CGA inhibited the levels of pancreatic enzyme activity. Interestingly, CGA reduced the serum and pancreatic levels of macrophage migration inhibitory factor (MIF) in mice with l-arginine-induced pancreatitis. SIGNIFICANCE: Our results suggest that CGA has an anti-inflammatory effect on l-arginine-induced pancreatitis and pancreatitis-associated lung injury.

Improvement of Triglyceride Levels through the Intake of Enriched-ß-Conglycinin Soybean (Nanahomare) Revealed in a Randomized, Double-Blind, Placebo-Controlled Study.

Soybean is recognized as a beneficial food with various functional components, such as ß-conglycinin, which improves lipid metabolism. We evaluated the effects of the ß-conglycinin-rich soybean Nanahomare on triglyceride (TG) levels. In this randomized, double-blind, placebo-controlled study, we divided 134 adult subjects into test and placebo groups that consumed processed food containing enriched-ß-conglycinin soybean or low-ß-conglycinin soybean. Hematological tests and body composition measurements were performed at weeks 0 (baseline), 4, 8, and 12 of the study period. TG levels significantly decreased in the test group compared with the placebo group at weeks 4 (change from baseline to week 4, placebo: 0.27 ± 44.13 mg/dL, test: -20.31 ± 43.74 mg/dL, p = 0.035) and 12 (change from baseline to week 12, placebo: -0.14 ± 65.83 mg/dL, test: -21.30 ± 46.21 mg/dL, p = 0.041). In addition, among subjects whose baseline TG levels were ≥100 mg/dL, the levels significantly improved in the test group at weeks 4 (p = 0.010) and 12 (p = 0.030), whereas the levels were not different between the test and placebo groups among those whose baseline levels were <100 mg/dL. These results suggest that the ingestion of enriched-ß-conglycinin soybean improves serum TG levels.

The effects of herring-roe lyophilized powder on lipid metabolism.

Herring-roe, which contains large amounts of docosahexaenoic acid and eicosapentaenoic acid, has anti-dyslipidemia effects. Here, we evaluated the effects of herring-roe on lipid metabolism in 33 adult subjects in a randomized, double-blind, placebo-controlled study. We divided the subjects into a test group that ingested herring-roe lyophilized powder (herring-roe powder) and a placebo group that ingested non-herring-roe powder, with each member of each group ingesting 15 g daily for 8 weeks. Hematological tests and body composition measurements were performed before and after 4, 6, and 8 weeks of the study period. Although no significant differences in low density lipoprotein were observed, high density lipoprotein was found to be increased in subjects who ingested herring-roe powder. In addition, the level of free fatty acid was significantly improved in the herring-roe powder group. These results suggest that ingestion of herring-roe could influence lipid metabolism.

Effects of yogurt containing Lactobacillus plantarum HOKKAIDO on immune function and stress markers.

Lactobacillus plantarum HOKKAIDO (HOKKAIDO strain) was isolated from well-pickled vegetables in Hokkaido, Japan. We report a randomized, double-blind, placebo-controlled study evaluating the effects of L. plantarum HOKKAIDO on immune function and stress markers in 171 adult subjects. Subjects were divided into three groups: the L. plantarum HOKKAIDO yogurt group, the placebo-1 group who ingested yogurt without the HOKKAIDO strain, and the placebo-2 group who ingested a yogurt-like dessert without the HOKKAIDO strain. Hematological tests and body composition measurements were performed before and after 4 and 8 weeks of blinded ingestion. Although no significant differences in natural killer cell activity were observed, it was found that neutrophil ratio significantly decreased and lymphocytes tended to increase in the HOKKAIDO strain yogurt group compared with the yogurt-like dessert group. In addition, the neutrophil-to-lymphocyte ratio, a stress marker, tended to improve in the HOKKAIDO strain yogurt group compared with the yogurt-like dessert group. These results suggest that the ingestion of HOKKAIDO strain yogurt tends to improve immune activity and decrease stress markers.

Effect of particle size on their accumulation in an inflammatory lesion in a dextran sulfate sodium (DSS)-induced colitis model.

Taking advantage of the enhanced permeation and retention (EPR) effect is a promising approach for delivering macromolecules or nanoparticles to tumors. Recent studies revealed that this strategy is also applicable for targeting other pathological lesions (i.e. inflammatory disease). In the present study, we report the optimal size of a nanoparticle for allowing the higher accumulation of a particle in an inflammatory lesion using a dextran sulfate sodium (DSS)-induced colitis model. As a nanoparticle platform, we utilized a SS-cleavable and pH-activated lipid-like material (ssPalm), that can be used to produce particles in a variety of sizes ranging from 50nm to 180nm while using the same lipid composition. In healthy mice, particle accumulation remained low regardless of size. In contrast, the accumulation in inflammatory colon tissue was enhanced depending on the progress of the inflammation. In this situation, the apparent uptake clearance accumulation of a mid-sized particle (113nm on average) was higher than that for smaller and larger (54nm and 183nm in average, respectively) ones. Therefore, controlling particle size is an important parameter for the extensive targeting of inflammatory lesion.

Effects of white rice containing enriched gamma-aminobutyric acid on blood pressure.

Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter with beneficial effects including antihypertension and antistress properties. In this study, we examined the effects of GABA-enriched white rice (GABA rice) on blood pressure (BP) in 39 mildly hypertensive adults in a randomized, double-blind, placebo-controlled study. The participants were divided into a test group (n = 22) who consumed rice with 11.2 mg GABA/100 g of rice and a placebo group (n = 17) who consumed rice with 2.7 mg GABA/100 g of rice. For 8 weeks, the participants took 150 g of either the GABA rice or the placebo rice. Hematological examinations were performed on both groups at 0, 4, and 8 weeks after the start of rice consumption. Home BP was self-measured two times daily, morning and evening, from 1 weeks before to 2 weeks after the intervention. Although the hospital BP and evening BP measurements of the participants showed no significant change, consumption of the GABA rice improved the morning BP compared with the placebo rice after the 1(st) week and during the 6(th) and 8(th) weeks. These results showed the possibility that the GABA rice improves morning hypertension.

Effects of the extract from roasted chicory (Cichorium intybus L.) root containing inulin-type fructans on blood glucose, lipid metabolism, and fecal properties.

The extract from roasted chicory (Cichorium intybus L.; jú jù) root (chicory root extract), which contains inulin-type fructans, has favorable effects including antihyperglycemic and antidyslipidemic effects and the improvement of bowel movement. In this study, we examined the effects of chicory root extract on blood glucose, lipid metabolism, and fecal properties in 47 healthy adult participants in a randomized, double-blind, placebo-controlled study. The participants were divided into a test group that drank chicory root extract and a placebo group that drank nonchicory root extract (ingesting 300 mL daily for 4 weeks). We performed hematological examinations and body composition measurements, and administered a visual analog scale (VAS) questionnaire for fecal properties at the baseline (Week 0) and after the intervention (Week 4) for the two groups. Although no significant differences in fasting plasma glucose or insulin were observed, hemoglobin A1c was found to decrease by ingesting chicory root extract. No intergroup differences in the levels of lipid metabolism parameters were observed. However, the level of adiponectin was significantly improved in the chicory root extract group when the baseline and postintervention values were compared. In addition, chicory root extract tends to improve the VAS score for fecal properties. These results suggest that chicory root extract could delay or prevent the early onset of diabetes mellitus and improve bowel movements.

Pumpkin Seed Oil Extracted From Cucurbita maxima Improves Urinary Disorder in Human Overactive Bladder.

The pumpkin seed oil obtained from Cucurbita pepo has been shown to be useful for the treatment of nocturia in patients with urinal disorders in several western countries. In this study, we evaluated the effect of the pumpkin seed oil from Cucurbita maxima on urinary dysfunction in human overactive bladder (OAB). Forty-five subjects were enrolled in this study. An extract of pumpkin seed oil from C. maxima (10 g of oil/day) was orally administrated for 12 weeks. After 6 and 12 weeks, urinary function was evaluated using Overactive Bladder Symptom Score (OABSS). Pumpkin seed oil from C. maxima significantly reduced the degree of OABSS in the subjects. The results from our study suggest that pumpkin seed oil extracts from C. maxima as well as from C. pepo are effective for urinary disorders such as OAB in humans.

Improvement of Blood Pressure, Glucose Metabolism, and Lipid Profile by the Intake of Powdered Asparagus ( Lú Sun) Bottom-stems and Cladophylls.

Asparagus ( Lú Sǔn; Asparagus officinalis L.) is a common vegetable, long used as an herbal medicine. The cladophylls and bottom-stems of asparagus have various pharmacological effects, but they are generally discarded at harvesting. The present open clinical trial was performed to examine the effects of the intake of cladophylls and bottom-stems on the improvement of metabolic syndrome characterized by hypertension, hyperglycemia, and dyslipidemia. Twenty-eight healthy volunteers ingested either cladophyll or bottom-stem powder (6 g/day) daily for 10 weeks. The cladophyll intake resulted in significant reduction in the subjects' diastolic blood pressure and fasting plasma glucose (FPG), and decreased both the left cardio-ankle vascular index score and the total cholesterol level (T-CHO). The bottom-stem intake significantly reduced the subjects' systolic and diastolic blood pressure and FPG as well as T-CHO. These results suggest the possibility that asparagus cladophylls and bottom-stems differentially improve hypertension, hyperglycemia, and dyslipidemia.

Macrophage migration inhibitory factor contributes to development of nonsteroidal anti-inflammatory drugs-induced gastric injury in mice.

Macrophage migration inhibitory factor (MIF) plays an important role in the development of inflammation. In this study, we evaluated the role of MIF in gastric injury induced by non-steroidal anti-inflammatory drugs (NSAIDs) in mice. To induce gastric injury, mice were intraperitnoneally injected with 35 mg/kg of indomethacin. The level of MIF protein was up-regulated and severe gastric injury with inflammatory infiltrate was observed in the stomach of wild-type (WT) mice treated with indomethacin. The severity of gastric injury in MIF-deficient mice was less than that in WT mice. Increase in TNF-α in gastric tissue of mice treated with indomethacin was suppressed in MIF-deficient mice. The expression of HSP70, which has a cytoprotective role, was remarkably up-regulated in the stomach of MIF-deficient mice compared with WT mice after indomethacin treatment. Our results suggest that MIF is essential for the development of gastric injury-induced by NSAIDs.

Rikkunshito ameliorates the aging-associated decrease in ghrelin receptor reactivity via phosphodiesterase III inhibition.

Aging is associated with decreased food intake, a phenomenon termed the anorexia of aging. In this study, we sought to clarify changes in peripheral and central appetite-related factors in aged mice. Furthermore, we investigated the effects of rikkunshito, a traditional Japanese medicine, on age-related anorexia. C57BL/6J mice that were 6 or 75 wk old were studied. We investigated changes in food intake, ghrelin and leptin levels, and the expression of appetite-related genes with age. In addition, we verified the effects of ghrelin, rikkunshito, phosphodiesterase 3 (PDE3), and phosphoinositide 3-kinase inhibitors on appetite. Food intake was significantly decreased in 75-wk-old mice compared with the 6-wk-old mice. In 75-wk-old mice, plasma acylated ghrelin levels under fasting conditions were lower than in 6-wk-old mice, whereas leptin levels under feeding conditions were substantially higher. The expression levels of hypothalamic preproghrelin under feeding conditions and the expression levels of neuropeptide Y and agouti-related protein under fasting conditions were lower compared with those of the 6-wk-old mice. Ghrelin supplementation (33 microg/kg) failed to increase food intake in 75-wk-old mice. Conversely, oral administration of LY294002, a phosphoinositide 3-kinase inhibitor, and cilostamide, a PDE3 inhibitor, increased food intake in 75-wk-old mice. Moreover, rikkunshito increased food intake in aged mice. The components of rikkunshito (nobiletin, isoliquiritigenin, and heptamethoxyflavone) had inhibitory effects on PDE3. These results suggest that dysregulation of ghrelin secretion and ghrelin resistance in the appetite control system occurred in aged mice and that rikkunshito ameliorated aging-associated anorexia via inhibition of PDE3.

Lack of macrophage migration inhibitory factor suppresses innate immune response in murine dextran sulfate sodium-induced colitis.

OBJECTIVE: Macrophage migration inhibitory factor (MIF) plays an important role in the development of inflammatory diseases. Recent studies have indicated an association of MIF with gastrointestinal inflammation including colitis, but the mechanism by which MIF exacerbates gut inflammation has not been fully clarified. In this study, in order further to clarify the role of MIF in intestinal inflammation, we investigated the association of MIF with innate immunity in experimental colitis using MIF-deficient mice. MATERIAL AND METHODS: Colitis was induced by treating mice with 3% dextran sulfate sodium (DSS) solution for 7 days. The expressions of chemokines in the colon were determined by reverse transcriptase-polymerase chain reaction (RT-PCR). Myeloperoxidase activity in the colon was measured and immunohistochemistry for F4/80 was analyzed. RESULTS: DSS treatment increased the level of myeloperoxidase activity and infiltration of F4/80-stained cells in the colon, and up-regulated the mRNA expressions in macrophage inflammatory protein (MIP)-1alpha, MIP-2, macrophage chemotaxic protein (MCP)-1, and interferon inducible protein (IP)-10 in wild-type mice. In contrast, this increase and up-regulation were not observed in the colon of MIF-deficient mice treated with DSS. CONCLUSION: Our findings indicate that a lack of MIF suppresses the innate immune response in DSS-induced colitis.

Rikkunshito, an herbal medicine, suppresses cisplatin-induced anorexia in rats via 5-HT2 receptor antagonism.

BACKGROUND & AIMS: Chemotherapy with an anticancer agent generally causes gastrointestinal tract disorders such as vomiting and anorexia, but the mechanism remains unclear. Rikkunshito, a kampo preparation, is known to alleviate such adverse reactions. In this study, we attempted to clarify the mechanism. METHODS: We investigated the decreases of plasma acylated-ghrelin level and food intake caused by cisplatin, serotonin (5-HT), 5-HT agonists, and vagotomy as well as the decrease-suppressing effects of rikkunshito and 5-HT antagonists. In addition, binding affinities of rikkunshito components were determined in receptor-binding assays using 5-HT2B and 5-HT2C receptors. RESULTS: Cisplatin, 5-HT, BW723C86 (5-HT2B-receptor agonist), and m-chlorophenylpiperazine HCl (5-HT2C agonist) markedly decreased plasma acylated-ghrelin levels, although 5-HT3 and 5-HT4 agonists had no effect. In contrast, 5-HT2B and 5-HT2C antagonists suppressed the cisplatin-induced decrease of plasma acylated-ghrelin level and food intake. Administration of rat ghrelin improved the cisplatin-induced decrease in food intake. Vagotomy decreased the plasma acylated-ghrelin level, which was decreased further by cisplatin. Rikkunshito suppressed such cisplatin-induced decreases of plasma acylated-ghrelin level and food intake. The suppressive effect of rikkunshito was blocked by a ghrelin antagonist. Components of rikkunshito, 3,3',4',5,6,7,8-heptamethoxyflavone, hesperidin, and iso-liquiritigenin showed a 5-HT2B-antagonistic effect in vitro, and oral administration of rikkunshito suppressed the cisplatin-induced decrease in the plasma acylated-ghrelin level. CONCLUSIONS: The cisplatin-induced decreases of the plasma acylated-ghrelin level and food intake are mediated by 5-HT2B/2C receptors and suppressed by flavonoids in rikkunshito.

Genotyping of human papillomaviruses by a novel one-step typing method with multiplex PCR and clinical applications.

We describe here a rapid, high-throughput genotyping procedure that allows the simultaneous detection of 16 high- and low-risk genital human papillomavirus (HPV) types by multiplex PCR in a single reaction tube. Multiplex PCR is based on the amplification of HPV DNA by sets of HPV genotype-specific primers, and the genotypes of HPV are visually identified by the sizes of amplicons after they are separated by capillary electrophoresis. The procedure does not include a hybridization step with HPV-specific probes and is rapid and labor-saving. We detected all 16 HPV genotypes (types 16, 58, 52, 51, 56, 31, 18, 39, 66, 59, 6, 33, 30, 35, 45, and 11) with a high sensitivity and a high degree of reproducibility. By using this newly developed method, we conducted a pilot study to examine the correlation between the prevalence and genotype distributions of HPV and the cytological group classifications for 547 cervical samples. Compared with the group of samples considered normal (14.7%), there was a significant increase in the prevalence of HPV in women with atypical squamous cells of unknown significance (61.3%), low-grade intraepithelial lesions (75.8%), and high-grade intraepithelial lesions (HSILs) (82.2%). The prevalence and distribution of type 58 were correlated with cytological malignancies, with the highest prevalence in women with HSILs. In conclusion, the novel multiplex PCR method described appears to be highly suitable not only for the screening of cervical cancer precursor lesions but also for the characterization of genotype distributions in large-scale epidemiological studies and HPV vaccination trials.

A case report of the therapeutic effect of cryofiltration in a patient with glucocorticoid-resistant ulcerative colitis.

Ulcerative colitis (UC) is characterized by chronic inflammation of the colon and its cause and pathogenesis have not been fully clarified. Although UC is treated with various drugs, including 5-amino-salycilate and glucocorticoids, some patients are resistant to them. It was recently reported that apheresis, such as leukocytapheresis and granulocytapheresis, improves intestinal inflammation in refractory cases of UC. On the other hand, cryofiltration, in which plasma apheresis is used to remove immunoglobulin and immune complexes, has been used for the treatment of autoimmune diseases. We herein report a case of glucocorticoid-resistant UC successfully treated with cryofiltration. Interestingly, the level of interleukin-10 (IL-10) in the patient's serum was markedly increased after eight sessions of cryofiltration. This suggests that cryofiltration suppresses intestinal inflammation, in part via up-regulation of IL-10.

Polaprezinc protects human colon cells from oxidative injury induced by hydrogen peroxide: relevant to cytoprotective heat shock proteins.

AIM: To investigate the effect of polaprezinc on cellular damage induced by hydrogen peroxide (H(2)O(2)) in human colon CaCo2 cells. METHODS: CaCo2 cells were treated with polaprezinc (10-100 micromol/L) for 6 h. After polaprezinc treatment, the cells were incubated with H(2)O(2) (20 micromol/L) for 1 h. Cell viability was measured by MTT assay. Western blot analysis for heat shock protein (HSP) 27 and HSP72 in the cells was performed. Moreover, cells were pretreated with quercetin (200 micromol/L), an inhibitor of HSP synthesis, 2 h before polaprezinc treatment, and cell viability and the expression of HSP27 and 72 were assessed in these cells. RESULTS: Polaprezinc significantly protected CaCo2 cells from cell damage induced by H(2)O(2), and up-regulated the expressions of HSP27 and HSP72 in the cells (10, 30 and 100 micromol/L of polaprezinc; 35.0% +/- 7.7%, 58.3% +/- 14.6% and 64.2% +/- 8.2%, respectively. P < 0.01 versus polaprezinc-nontreated cells; 6.0% +/- 4.4%). Quercetin inhibited the up-regulation of HSP27 and HSP72 by polaprezinc and diminished the protective effect of polaprezinc against H(2)O(2)-caused injury in the cells. CONCLUSION: Polaprezinc is a useful therapeutic agent for treatment of colitis and its effects depend on the function of cytoprotective HSP in colon.

Resistance to experimental colitis depends on cytoprotective heat shock proteins in macrophage migration inhibitory factor null mice.

Macrophage migration inhibitory factor plays an important role in inflammatory diseases. We investigated the role of macrophage migration inhibitory factor (MIF) in the development of dextran sulfate sodium (DSS)-induced colitis using MIF null ((-/-)) mice. MIF(-/-) mice given 3% DSS showed no clinical and histological feature of colitis in contrast to wild-type (WT) mice. Lack of MIF suppressed the up-regulation of TNF-alpha and IFN-gamma as Th1-derived cytokines, and increased the level of IL-4 as Th2-derived cytokine in the colon tissues. Moreover, we found that the expressions of heat shock protein (HSP)40 and HSP70 were markedly up-regulated in the colon of MIF(-/-) mice in response to DSS compared with WT mice. Additionally, quercetin, an inhibitor of HSP synthesis, inhibited the up-regulation of HSP40 and 70 expressions and developed DSS-induced colitis in MIF(-/-) mice. Our findings in this study provide more information in the role of MIF in colitis.