Oral Administration of Mulberry (Morus alba L.) Leaf Powder Prevents the Development of Hepatocellular Carcinoma in Stelic Animal Model (STAM) Mice.

BACKGROUND/AIM: We examined the inhibitory effect of mulberry leaf (ML) (Morus alba L.) administration on the development of hepatocellular carcinoma (HCC) in stelic animal model (STAM) mice. This STAM mouse model of nonalcoholic steatohepatitis (NASH) closely resembles the progression from NASH to HCC in human clinical practice. MATERIALS AND METHODS: Streptozotocin (STZ, 200 µg) was administered to C57L/6J mice that were fed a high-fat diet (HFD; STAM mice) with 1% ML ad libitum. After sacrificing, the liver and blood were collected. Biochemical tests of plasma and histologic examination of the liver were performed. RESULTS: Pathologic examination of all (6/6) liver samples of the STAM mice showed HCC. On the contrary, in STAM mice that received ML, fat deposition and adenoma were observed in 6/6 and 2/6 of the liver samples, respectively, but there was no HCC. CONCLUSION: Administration of ML in STAM mice inhibited the progression from nonalcoholic hepatitis (NASH) to HCC. ML may be effective in preventing the development of HCC.

Effects of Kumaizasa (Sasa senanensis) Leaf Extract on Innate Immune Regulation in HEK293 Cells and Macrophages.

BACKGROUND/AIM: We investigated the effect of Kumaizasa leaf extract (KLE) on innate immunity using the HEK293 and RAW 264.7 cell lines. MATERIALS AND METHODS: KLE, lipopolysaccharides (LPS), or KLE with LPS were added to RAW 264.7 cells. The TNF-α and IL-1ß mRNA expression was then quantified. The expression of MAPKs, NFĸB, TNF-α and IL-1ß proteins was also quantified. In addition, KLE was added to HEK293 cells and the IL-8 concentration was measured. RESULTS: In RAW 264.7 cells, KLE increased the levels of TNF-α and IL-1ß mRNA. By contrast, when KLE and LPS were added to RAW 264.7 cells, the increase in TNF-α and IL-1ß mRNA was ameliorated. Similarly, the expression of JNK and ERK proteins was reduced. The addition of KLE to HEK293 cells induced IL-8 production. CONCLUSION: Based on these results, a KLE-mediated mechanism may regulate immunity by suppressing the expression of JNK and ERK, which are involved in inflammatory signal transduction.

Sanguisorba officinalis L. derived from herbal medicine prevents intestinal inflammation by inducing autophagy in macrophages.

Disturbed activation of autophagy is implicated in the pathogenesis of inflammatory bowel disease. Accordingly, several autophagy-related genes have been identified as Crohn's disease susceptibility genes. We screened the autophagy activators from a library including 3,922 natural extracts using a high-throughput assay system. The extracts identified as autophagy activators were administered to mice with 2% dextran sodium sulfate (DSS). Among the autophagy inducers, Sanguisorba officinalis L. (SO) suppressed DSS-induced colitis. To identify the mechanism by which SO ameliorates colitis, epithelial cell and innate myeloid cells-specific Atg7-deficient mice (Villin-cre; Atg7f/f and LysM-cre; Atg7f/f mice, respectively) were analyzed. SO-mediated inhibition of colitis was observed in Villin-cre; Atg7f/f mice. However, SO and a mixture of its components including catechin acid, ellagic acid, gallic acid, and ziyuglycoside II (Mix4) did not suppressed colitis in LysM-cre; Atg7f/f mice. In large intestinal macrophages (Mφ) of Atg7f/f mice, SO and Mix4 upregulated the expression of marker genes of anti-inflammatory Mφ including Arg1, Cd206, and Relma. However, these alterations were not induced in LysM-cre; Atg7f/f mice. These findings indicate that SO and its active components ameliorate DSS-induced colitis by providing intestinal Mφ with anti-inflammatory profiles via promotion of Atg7-dependent autophagy.

Global Liver Gene Expression Analysis on a Murine Hepatic Steatosis Model Treated with Mulberry (Morus alba L.) Leaf Powder.

BACKGROUND/AIM: Mulberry (Morus alba L.) leaves (ML) contain many functional components, such as 1-deoxynojirimycin, flavonoids (rutin, quercetin, kaempferol). It is well known that 1-deoxynojirimycin functions to suppress increases in blood glucose level by α-glucosidase inhibitory activity. Thus, the molecular mechanism underlying the protective and therapeutic effects of ML supplementation was investigated on a mouse model of high-calorie diet (Western diet: WD)-induced hepatic steatosis (HS). MATERIALS AND METHODS: The C57BL/6J mouse was used for the HS model. The mice were divided into three groups: control (normal diet: ND), WD, and WD + 1% ML groups. The WD group was fed a high-calorie (high carbohydrate and high fat) diet for 12 weeks to develop HS. At week 12, all mice were sacrificed, blood was collected for biochemical tests, and the liver was obtained for histological examination and RNA sequencing (RNA-Seq). RESULTS: Liver weight, plasma triglycerides (TG), alanine aminotransferase (ALT), and alanine aminotransferase (AST) levels of both ML groups were significantly lower than those of the WD group. On histological examination of the liver, the area of fatty deposits was found to be suppressed by ML administration. In the gene expression analysis of the liver of WD- versus ML-fed mice by RNA-Seq, 722/45,706 genes exhibited a significant change in expression (corrected p-value<0.05). Gene network analysis of these genes showed that genes related to liver inflammation were inactivated and those related to regeneration of liver were activated in the ML group. CONCLUSION: ML functions to suppress HS in WD-fed mice and regulates genes related to inflammation and regeneration of liver cells.

Transcriptome Analysis of Skin from SMP30/GNL Knockout Mice Reveals the Effect of Ascorbic Acid Deficiency on Skin and Hair.

BACKGROUND/AIM: Senescence marker protein-30/gluconolactonase knockout mice (SMP-30/GNL-KO) are a very useful model for clarifying the involvement of vitamin C (VC) in aging-related diseases. In this study, the effects of VC deficiency on skin and hair growth were investigated using SMP-30/GNL-KO mice by RNA sequencing. MATERIALS AND METHODS: SMP-30/GNL-KO mice were given water containing 1.5 g/l VC until up to 8 weeks after birth to maintain a VC concentration in their organs and plasma equivalent to that in wild-type mice. The mice were then divided into two groups: a VC(+) group, where VC was administered, and a VC(-) group, where VC was not administered. Skin samples were collected at 4 and 8 weeks after the treatment. RNA was extracted from each skin sample, followed by cDNA synthesis and RNA-seq. In addition, hair growth was compared between the VC(-) and VC(+) groups after shaving. Skin samples were collected from the shaved area for histological examination by hematoxylin & eosin (HE) staining. RESULTS: RNA-seq revealed that there were 1,736 (FDR<0.001) differentially expressed genes in the VC(-) and VC(+) groups. From the functional analysis of the differentially expressed genes in the VC(-) and VC(+) groups, predicted functionalities including cell death and cytotoxicity increased in the VC(+) group. Furthermore, it was predicted that the difference in hair growth between the VC(-) and VC(+) groups was caused by the expression of genes including keratin-related genes and the Sonic hedgehog gene. It was confirmed that hair growth was significantly promoted; hair growth from hair papilla cells was also confirmed by HE staining of the shaved backs of SMP-30/GNL-KO mice in the VC(+) group. CONCLUSION: RNA-seq of the skin from VC-deficient mice showed the effects of VC deficiency on the expression of genes involved in cell growth and the hair cycle. Visual inspection suggested that changes in the expression of the genes are involved in delaying hair growth in the VC(-) group. Further research on the relationship among VC deficiency, the hair cycle, and skin cell growth may contribute to research on hair restoration and skin aging.

Perilla leaf extract prevents atopic dermatitis induced by an extract of Dermatophagoides farinae in NC/Nga mice.

BACKGROUND: Perilla (Perilla frutescens Britton) leaf comprises many types of active components, mainly flavonoids, and acts as an anti-inflammatory agent in in vitro and in vivo atopic dermatitis (AD) models. OBJECTIVE: We investigated the effects of orally administered perilla leaf extract (PLE) on the symptoms of AD induced by Dermatophagoides farinae extract (DFE) in NC/Nga AD model mice. METHODS: The mice were allowed free intake of 0.5% PLE. Skin lesions were assessed, and blood was sampled from the caudal vein on days 0, 7, 14, 21, and 31. On day 31, all mice were sacrificed to obtain blood, skin, spleen, and intestinal tissue samples. RESULTS: The assessment scores of the skin lesions and total serum IgE levels of PLE-treated mice (PLE group) were significantly lower than DFE-treated mice (DFE group) on days 7, 14, and 21. On day 31, the serum periostin and thymus and activation-regulated chemokine (TARC) levels in the PLE group were significantly lower than those in the DFE group. Histological analysis of the skin revealed that hyperplasia of the epidermal and dermal layers and infiltration of inflammatory cells (cell infiltration in corium tissues) were suppressed by PLE. Periostin deposition was observed in the skin tissue obtained from the DFE group. Moreover, the CD4+/CD8+ ratio of splenic T cells was suppressed in the PLE group but not in the DFE group.

Immunopotentiator from Pantoea agglomerans Prevents Atopic Dermatitis Induced by Dermatophagoides farinae Extract in NC/Nga Mouse.

BACKGROUND/AIM: Pantoea agglomerans LPS (immunopotentiator from Pantoea agglomerans 1: IP-PA1) has been reported to have anti-inflammatory effects in in vitro and in vivo models. The aim of the present study was to investigate the effects of orally-administered IP-PA1 on atopic dermatitis (AD) symptoms induced by Dermatophagoides farinae body extract (DFE) in NC/Nga mice. MATERIALS AND METHODS: Using the NC/Nga AD murine model, mice were orally administered 0.1% (High) or 0.01% (Low) water-containing IP-PA1. Skin lesion assessment and blood collection from the caudal vein was performed on days 0, 7, 21 and 31. On day 31, all mice were sacrificed and blood, skin, spleen, as well as intestine samples, were obtained. RESULTS: Assessment score of the skin lesion and serum immunoglobulin E (IgE) level of both IP-PA1 groups were significantly lower than that of the DFE group on days 14 and 21. The serum periostin and thymus and activation-regulated chemokine (TARC) level of IP-PA1-Low group was significantly lower than that of the DFE group on day 31. On histological examination of the skin, hyperplasia of epidermal and dermal layers and infiltration of inflammatory cells were suppressed by IP-PA1 administration. Deposition of periostin was observed in the DFE group skin tissue. Moreover, the CD4(+)/CD8(+) ratio of splenic T-cells increased by IP-PA1 administration. CONCLUSION: IP-PA1 administration may have an inhibitory effect on AD skin lesions.

[Effect of notoginseng extracts and their components on lipopolysaccharide and galactosamine mixture-induced impaired hepatic function in mice].

The protective effects of notoginseng against hepatic damage were investigated in mice. To prepare a model animal of hepatitis, a mixture of lipopolysaccharide and galactosamine (LPS/GAlN) was administered intraperitoneally, leading to the impairment of hepatic function. Extracts of notoginseng or its components (ginsenoside Rb1 and ginsenoside Rg1) were orally administered 2 h before LPS/GalN injection. Eight hours after LPS/GalN injection, blood and liver tissue samples were collected. The levels of serum aspartate amino transferase (AST), alanine aminotransferase (ALT), tumor necrosis factor (TNF)-α, and interferon (IFN)-γ were measured using commercial assay kits. Histologic changes in the tissue samples were also observed after hematoxylin-eosin staining. LPS/GalN administration increased the serum levels of AST and ALT, and histologic changes were noted, indicating hepatic cell damage. Prior to the increase in ALT, the serum levels of TNF-α and IFN-γ were elevated after LPS/GalN injection. Pretreatment of the mice with either notoginseng extract or gensenoside Rb1 and Rg1 attenuated the LPS/GalN-induced hepatic damage markedly. The protective effects of the components against hepatic damage appeared to be less potent than those of the crude extract and prescription of notoginseng. Notoginseng may be clinically useful in patients with hepatitis.

Comparative effects of azelnidipine and amlodipine on myocardial function and mortality after ischemia/reperfusion in dogs.

Effects of azelnidipine were examined and compared with those of amlodipine on stunned myocardium in dogs. The left anterior descending (LAD) coronary artery was ligated for 20 min and subsequently released for 60 min. A vehicle, azelnidipine (0.3 mg/kg), or amlodipine (0.3 or 1 mg/kg) was injected intravenously 20 min before LAD ligation. The heart rate increased after a depressor response in the presence of amlodipine, while it decreased despite a decrease in arterial pressures in the presence of azelnidipine. After reperfusion, the coronary flow (CF) significantly increased in the presence of azelnidipine, but did not change with amlodipine after reperfusion. A positive inotropic effect was observed after treatment with both calcium antagonists. Ischemia significantly decreased the percentage of segment shortening (%SS) in all groups. Treatment with both calcium antagonists significantly increased %SS after reperfusion, although high-energy phosphate levels did not improve in the presence of calcium antagonists 60 min after reperfusion. Mortality with azelnidipine was significantly lower than that with 0.3 mg/kg amlodipine immediately after reperfusion. In conclusion, improvement in myocardial stunning after pretreatment with azelnidipine is associated with an increase in CF after reperfusion. The negative chronotropic action may have contributed to decreased mortality due to reperfusion arrhythmias. Azelnidipine is more beneficial than amlodipine and may provide an additional advantage to patients with angina and hypertension.

Pharmacokinetic interactions between Japanese traditional Kampo medicine and modern medicine (IV). Effect of Kamisyoyosan and Tokisyakuyakusan on the pharmacokinetics of etizolam in rats.

Kamisyoyosan (KSS) and Tokisyakuyakusan (TSS) are widely used herbal formulas in Japanese traditional kampo medicine to relieve the symptoms occurred in climacteric disturbance. Since Japanese physicians frequently prescribe these formulas combined with etizolam, one of benzodiazepine anxiolytics, we evaluated the pharmacokinetic interaction between KSS or TSS and etizolam, and in vitro inhibitory effect of KSS and TSS on rat cytochrome P450 (CYP) 3A activity in rat microsomes, to obtain drug information to prevent from disadvantage or adverse effects by their combined therapy. In in vitro experiment, KSS and TSS inhibited CYP3A activity comparable to grapefruit juice. However in in vivo experiments, oral administration of KSS did not influence the plasma concentration profile of etizolam. The maximum concentration (Cmax) of etizolam was significantly reduced when TSS was co-administered at 20 times amount of human daily dosage. Since the double of human daily dose of TSS did not suppress the absorption of etizolam, TSS would not influence the pharmacokinetics of etizolam at the usual clinical dosage. Since both KSS and TSS did not influence the metabolism of etizolam, the result of in vitro experiment would not reflect to that of in vivo experiment or in clinic. The combination of etizolam with KSS or TSS at usual dosage would not cause drug interaction.

Pharmacokinetic interactions between Japanese traditional medicine (kampo) and modern medicine (III). Effect of Sho-seiryu-to on the pharmacokinetics of azelastine hydrochloride in rats.

Sho-seiryu-to (SST) is widely used herbal formula in Japanese traditional medicine (kampo) to treat allergic diseases. Since Japanese physicians frequently prescribe this formula combined with azelastine hydrochloride, one of anti-histamine and anti-allergic medicines, we evaluated the pharmacokinetic interactions between SST and azelastine hydrochloride in rats to obtain the drug information for the prevention from disadvantage or adverse effects by their combined therapy. Oral administration of SST did not influence the plasma concentration profile of azelastine after its intravaneous injection, suggesting that SST would not change the activities of metabolic enzymes such as cytochrome P450s. However, maximum concentration (C(max)) of azelastine after oral administration of azelastine hydrochloride was significantly reduced and mean residence time (MRT) was significantly lengthened when SST was orally administered at 20 times amount of human daily dosage. There was not significant difference in the area under the plasma concentration-time curve (AUC), suggesting that SST might delay the absorption of azelastine without affecting the extent of bioavailability. Since this delay was independent of ephedrine that is a main constituent of SST and that a suppressor for gastric transit, SST might form unsoluble complex with azelastine to reduce its absorption. At the double of human daily dose, SST did not made the absorption of azelastine delay. The possibility that SST reduce the absorption of azelastine hydrochloride could not be denied completely, however, it is suggested that SST would not cause pharmacokinetic interaction with azelastine hydrochloride clinically.