Clean-DM1, a Korean Polyherbal Formula, Improves High Fat Diet-Induced Diabetic Symptoms in Mice by Regulating IRS/PI3K/AKT and AMPK Expressions in Pancreas and Liver Tissues.

OBJECTIVE: To investigate the effects of Clean-DM1 (C-DM1), a polyherbal formulation of Radix Scrophulariae, Radix Astragali, Rhizoma Atractylodis, and Radix Salviae Miltiorrhizae, on high-fat diet (HFD)-induced diabetes mice. METHODS: The information about active components of C-DM1 extract and molecular mechanism was obtained from network pharmacology analysis. Main compounds of C-DM1 extract by high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis were conducted for quality control. For in vivo study, mice were induced diabetes by HFD for 12 weeks. The mice in the normal group (Nor) were maintained with a regular diet and treated with saline by gavage. The HFD model mice were randomly divided into 3 groups, including a HFD diabetic model group, a C-DM1 extract-administered group (C-DM1, 500 mg/kg), and metformin-administered groups (Met, 500 mg/kg), 8 mice in each group. Food intake, body weight (BW), and fasting blood glucose (FBG) levels were recorded weekly for 4 weeks. After 4 weeks of treatment, alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood glucose, low-density lipoprotein cholesterol (LDL-C) were determined using an automated clinical chemistry analyzer, and homeostatic model for assessing insulin resistance (HOMA-IR) levels and oral glucose tolerance test (OGTT) were detected. The histopathological changes of liver and pancreatic tissues were observed by hematoxylin-eosin staining. Insulin receptor substrate (IRS)/phosphatidylinositol 3 kinase (PI3K)/ protein kinase B (AKT) and adenosine 5'-monophosphate-activated protein kinase (AMPK) expressions in liver and pancreas tissues were detected by Western blot analysis. RESULTS: HPLC-MS identified dihydroisotanshinone, dihydroisotanshinone I, cryptotanshinone, harpagoside, and atractyloside A in C-DM1 extract. The administration of C-DM1 extract significantly decreased body weight, calorie intake, and the levels of blood glucose and insulin in the diabetic mice (P<0.05 or P<0.01). The C-DM1 extract administration improved the impaired glucose tolerance and insulin resistance in the diabetic mice and significantly decreased the levels of LDL-C, ALT and AST (P<0.01). The C-DM1 extract inhibited the histopathological changes of fatty liver and hyperplasia of pancreatic islets in the diabetic mice. The C-DM1 extract significantly increased the phosphorylation of IRS, AKT, and AMPK and the expression of PI3K in pancreas and liver tissues (P<0.05 or P<0.01), which was consistent with the analysis results of network pharmacology. CONCLUSION: C-DM1 extract improved diabetes symptoms in long-term HFD-induced mice by regulation of IRS/PI3K/AKT and AMPK expressions in pancreas and liver tissues, suggesting that C-DM1 formulation may help prevent the progression of T2DM.

Monotropein Improves Dexamethasone-Induced Muscle Atrophy via the AKT/mTOR/FOXO3a Signaling Pathways.

The present study aimed to investigate the effects of monotropein (MON) on improving dexamethasone (DEX)-induced muscle atrophy in mice and C2C12 mouse skeletal muscle cells. The body weights, grip strengths, and muscle weights of mice were assessed. The histological change in the gastrocnemius tissues was also observed through H&E staining. The expression of myosin heavy chain (MyHC), muscle ring finger 1 (MuRF1), and muscle atrophy F-box (Atrogin1) and the phosphorylation of AKT, mTOR, and FOXO3a in the muscle tissues of mice and C2C12 myotubes were analyzed using Western blotting. MON improved muscle atrophy in mice and C2C12 myotubes by regulating catabolic states via the AKT/mTOR/FOXO3a signaling pathways, and enhanced muscle function by the increases of muscle mass and strength in mice. This suggests that MON could be used for the prevention and treatment of muscle atrophy in patients.

Effects of Jowiseungki-tang on high fat diet-induced obesity in mice and functional analysis on network pharmacology and metabolomics analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional Chinese and Korean medicine, Jowiseungki-tang (JST) is a prescription for diabetes mellitus (DM) treatment. However, little scientific evidence is known of its effect in diabetic condition. AIMS: We assessed the effects of JST on high-fat diet (HFD)-induced obesity with inflammatory condition in mice and to analyze the therapeutic function of JST on network pharmacology as well as targeted metabolomics. MATERIALS AND METHODS: JST administration at 100 mg/kg and 500 mg/kg for a period of 4 weeks in HFD-induced obese mice, body weight gain, energy utility, calorie intake, and levels of glucose, insulin, total cholesterol, triglyceride, LDL-cholesterol as well as interleukin-6 were measured. Measurements of HDL-cholesterol (HDL-C) were performed and compared to those of the control group. Moreover, the therapeutic function of JST on obesity was analyzed furtherly based on network pharmacology and targeted metabolomics methods. RESULTS: Administration of JST at 100 mg/kg and 500 mg/kg for a period of 4 weeks in HFD-induced obesity mice significantly decreased the body weight gain, energy utility, calorie intake, and levels of insulin, total cholesterol, LDL-cholesterol, triglyceride, and interleukin-6. However, HDL-cholesterol (HDL-C) levels showed marked elevation relative to control groups. JST administration strongly inhibited expressions of inducible nitric oxide synthase, inflammatory proteins, and cyclooxygenase-2 in the pancreas, stomach, and liver tissues, and reduced hepatic steatosis and pancreatic hyperplasia. In network pharmacological analysis, the putative functional targets of JST are underlie on modulation of cofactor-, coenzyme-, and fatty acid-bonding, insulin resistance, and inflammatory response, fine-tuned the phosphatase binding and signal pathway activation, such as mitogen activated protein kinases, phosphatidylinositol 3-kinases/protein kinase B, protein kinase C, and receptor of glycation end products as well-advanced glycation end products. According to the metabolomics analysis, the contents and energy metabolites, and medium and long chain fatty acids was significantly changed in mice pancreases. CONCLUSIONS: JST is a valuable prescription for treatment of patients with DM in traditional clinics through inhibition of obesity, inflammatory condition and metabolism.

Effects of Root Extract of Morinda officinalis in Mice with High-Fat-Diet/Streptozotocin-Induced Diabetes and C2C12 Myoblast Differentiation.

Type 2 diabetes is the most common type of diabetes and causes a decline in muscle quality. In this study, we investigated the effects of the root extract of Morinda officinalis (MORE) on skeletal muscle damage in mice with high-fat-diet (HFD)/streptozotocin (STZ)-induced diabetes and the expression of myogenic and biogenesis regulatory proteins in C2C12 myoblast differentiation. An in vivo model comprised C57BL/6N mice fed HFD for 8 weeks, followed by a single injection of STZ at 120 mg/kg. MORE was administered at 100 and 200 mg/kg once daily (p.o.) for 4 weeks. The changes in body weight, calorie intake, and serum levels of glucose, insulin, total cholesterol (TCHO), HDL-cholesterol (HDL-C), LDL-cholesterol (LDL-C), aspartate transaminase (AST), and alanine aminotransferase (ALT) were investigated in diabetic mice. The histological changes in the gastrocnemius muscle were observed by H&E staining, and then the myofiber size was measured. The expression of the myogenic (MHC, myogenin, and MyoD) and biogenesis (PGC-1α, SIRT1, NRF1, and TFAM) regulatory proteins was examined in the muscle tissues and differentiated C2C12 myoblasts by Western blot, respectively. The administration of MORE at 200 mg/kg in mice with HFD/STZ-induced diabetes significantly reduced weight gains, calorie intake, insulin resistance, and serum levels of glucose, TCHO, LDL-C, AST, and ALT. MORE administration at 100 and 200 mg/kg significantly increased serum insulin and HDL-C levels in diabetic mice. In addition, MORE significantly increased the expression of MHC, myogenin, MyoD, PGC-1α, SIRT1, NRF1, and TFAM in muscle tissues as well as increased the myofiber size in diabetic mice. In C2C12 myoblast differentiation, MORE treatment at 0.5, 1, and 2 mg/mL significantly increased the expression of myogenic and biogenesis regulatory proteins in a dose-dependent manner. MORE improves diabetes symptoms in mice with HFD/STZ-induced diabetes by improving muscle function. This suggests that MORE could be used to prevent or treat diabetes along with muscle disorders.

An Aluminosilicate Zeolite Containing Rings of Tetrahedral Atoms with All Odd Numbers from Five to Eleven.

Herein we report the synthesis, structure solution, and catalytic properties of PST-31, which has an unprecedented framework topology. This high-silica (Si/Al=16) zeolite was synthesized using a pyrazolium-based dication with a tetramethylene linker as an organic structure-directing agent (OSDA) in hydroxide media. The PST-31 structure is built from new building layers containing four-, five-, six-, and seven-membered rings, which are connected by single four-membered rings in the interlayer region to form a two-dimensional pore system. Its channels consist of [4.56 .6.9.11] and [5.6.7.9.10.11] cavities and are thus delimited by nine-, ten-, and eleven-membered rings. The OSDA cations in as-synthesized PST-31 were determined to reside without disorder in the large [42 .514 .64 .72 .94 ] cavities composed of smaller [4.56 .6.9.11] and [5.6.7.9.10.11] ones, leading to a symmetry coincidence between the OSDA and the surrounding zeolite cavity. The proton form of PST-31 was found to be selective for the cracking of n-hexane to light olefins.

A Novel Approach Based on Metabolomics Coupled With Intestinal Flora Analysis and Network Pharmacology to Explain the Mechanisms of Action of Bekhogainsam Decoction in the Improvement of Symptoms of Streptozotocin-Induced Diabetic Nephropathy in Mice.

Bekhogainsam decoction (BHID), a representative prescription for the treatment of diabetes mellitus (DM) and diabetic complications in both traditional Korean and Chinese medicine, was examined for its ability to ameliorate diabetic nephropathy (DN), and its mechanism of action was evaluated by metabolomics, gut microbiota, and network pharmacology. In this study, male specific pathogen-free C57BL/6 mice were intraperitoneally injected with streptozotocin (STZ, 100 mg/kg) once per day for 3 days consecutively, and were then orally administered BHID at 100 and 500 mg/kg, and metformin at 250 mg/kg once per day for 4 weeks. Our results showed that the administration of BHID to mice with STZ-induced DN prevented physiological and serological changes, structural damage, and kidney dysfunction. Based on a metabolomics test with serum, the profoundly altered metabolites in the BHID treatment group were identified. Thirty-six BHID-related proteins and four signaling pathways, including valine, leucine, and isoleucine biosynthesis, nicotinate and nicotinamide metabolism, tryptophan metabolism, and alanine, aspartate, and glutamate metabolism pathways, were explored. Principal coordinates analysis (PCoA) of the gut microbiota revealed that BHID treatment significantly affected the flora composition. In addition, the network pharmacology analysis revealed that BHID acted through phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) and MAPK-related protein targets. Our findings on the anti-DN effects of BHID and its mechanism of action, from the perspective of systems biology, have provided scientific evidence to support the clinical treatment of patients with diabetes, and implied that BHID has the potential to prevent the progression of DN.

Effects of a rhizome aqueous extract of Dioscorea batatas and its bioactive compound, allantoin in high fat diet and streptozotocin-induced diabetic mice and the regulation of liver, pancreas and skeletal muscle dysfunction.

ETHNOPHARMACOLOGICAL RELEVANCE: Dysfunction of glucose metabolism is associated with the occurrence of metabolic syndromes, including type 2 diabetes mellitus (T2DM). In this study, we investigated the anti-diabetic effects of yam aqueous extract and allantoin in high-fat-diet (HFD) and streptozotocin (STZ)-induced diabetic mice and the mechanism of action on the dysfunction of the liver, pancreas, and skeletal muscle. MATERIALS AND METHODS: Male C57BL/6 mice were induced into a diabetic condition by HFD for 16 weeks and a single injection of STZ (120 mg/kg) and then orally administered yam aqueous extract (500 and 1000 mg/kg) or allantoin (20 and 50 mg/kg) once daily for 4 weeks. The changes in physiological parameters, serological parameters, and morphology of tissues were investigated. The expression levels of antioxidant enzymes, biogenetic proteins, and myogenetic proteins were determined in the liver, pancreas and skeletal muscle tissues of mice. RESULTS: The administration of yam aqueous extract and allantoin at high doses in HFD/STZ-induced diabetic mice compared with the control group significantly decreased the increase in body weight, caloric intake, and water intake. Yam aqueous extract and allantoin significantly decreased high glucose and leptin, total cholesterol, triglyceride, low-density lipoprotein-cholesterol, aspartate transaminase, alanine aminotransferase levels and increased insulin and albumin levels in the plasma of mice. Yam aqueous extract and allantoin inhibited the structural damage of the liver with regard to fat accumulation, the pancreas with atrophy of Langerhans' islets, and skeletal muscle with regard to atrophy and significantly increased the expression of antioxidant enzymes and mitochondria-mediated biogenetic factors in the liver, pancreas, and muscle tissues. In addition, Yam aqueous extract and allantoin significantly increased the expression of myogenetic proteins in skeletal muscle tissues. CONCLUSION: Our results indicated that Yam aqueous extract and allantoin improve diabetic symptoms through the regulation of oxidation and glucose imbalance in liver, pancreas, and skeletal muscle tissues in mice. These findings suggest that Yam aqueous extract and allantoin can be used as antidiabetic factors in supplementary foods and medications for T2DM patients.

Jowiseungki decoction affects diabetic nephropathy in mice through renal injury inhibition as evidenced by network pharmacology and gut microbiota analyses.

BACKGROUND: Jowiseungki decoction (JSD) is a prescription commonly used for the treatment of diabetic complications or diabetic nephropathy (DN) in traditional medicine clinics. However, the underlying therapeutic mechanisms of JSD are still unclear. METHODS: Streptozotocin (STZ)-induced DN mice were administered 100 and 500 mg/kg JSD for 4 weeks, and the therapeutic mechanisms and targets of JSD were analyzed by network pharmacology and gut microbiota analyses. RESULTS: JSD significantly decreased the increase in food and water intake, urine volume, fasting blood glucose, serum glucose and triglyceride levels, and urinary albumin excretion. JSD administration significantly increased the decrease in insulin secretion and creatinine clearance and reduced the structural damage to the kidney tissues. Moreover, JSD administration significantly inhibited the expression of protein kinase C-alpha (PKC-α), transforming growth factor beta-1 (TGF-ß1), α-smooth muscle actin (α-SMA), nuclear factor-κB (NF-κB), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) in the kidney tissues of DN mice, while it significantly increased the phosphorylation of insulin receptor substrate 1 (IRS-1), phosphatidylinositol-3-kinase (PI3K), and protein kinase B (Akt). In the network pharmacological analysis, JSD obviously influenced phosphatase binding, protein serine/threonine kinase, and mitogen-activated protein kinase (MAPK)-related signaling pathways. Our data suggest that JSD can improve symptoms in STZ-induced DN mice through the inhibition of kidney dysfunction, in particular, by regulating the PKCα/PI3K/Akt and NF-κB/α-SMA signaling pathways. Gut microbiota analysis can help to discover the pharmaco-mechanisms of the influence of JSD on bacterial diversity and flora structures in DN. CONCLUSION: JSD can improve the symptoms of DN, and the underlying mechanism of this effect is renal protection through the inhibition of fibrosis and inflammation. JSD can also change bacterial diversity and community structures in DN.

Effects of spicatoside A isolated from the tuberous roots of Liriope platyphylla on ovalbumin-induced asthma in mice.

The tuberous roots of Liriope platyphylla (Liriopis Tuber; LT) is traditionally used in Korean Medicine for treating colds, cough, and sputum production. In this study, we investigated the effect of spicatoside A isolated from LT methanol extract on ovalbumin (OVA)-sensitized/challenged asthmatic mice. For induction of allergic asthma, BALB/c mice were sensitized with OVA by an intraperitoneal injection at three times a week, and then challenged into the nasal cavities using a nebulizer. Spicatoside A at dose of 1mg/kg body weight was treated in mice with an oral administration once daily for a week during OVA challenge. The concentrations of OVA-specific IgE, IL-4, IL-5 and IL-13 were measured in the sera or bronchoalveolar lavage fluids (BALF) of mice by enzyme-linked immunosorbent assay (ELISA). The numbers of total cells, macrophages, lymphocytes, neutrophils and eosinophils were counted in BALFs using Diff-Quik staining, and histopathological changes of lung tissues were observed by hematoxylin and eosin (H&E), Periodic acid Schiff (PAS) and Masson's trichrome staining. The purity of spicatoside A was 98.1% with a white powder (yield: 465.6mg). The treatment of spicatoside A in asthmatic mice significantly decreased the production of allergic mediator, OVA-specific IgE and Th2 cytokines, IL-4, IL-5 and IL-13 in sera and BALF. The numbers of inflammatory cells such as macrophages, lymphocytes, neutrophils and eosinophils in BALF of asthmatic mice were significantly reduced by the treatment of spicatoside A. Furthermore, the treatment of spicatoside A in asthmatic mice inhibited the structural damages of lung tissues with thickened bronchiolar epithelium and infiltration of inflammatory cells, the accumulation of mucus by the goblet cells hyperplasia and collagen in the bronchioles. These results suggest that spicatoside A of LT has a preventive effect on allergic asthma through the inhibition of lung inflammation and allergic response.

Regulatory effects of the fruit extract of Lycium chinense and its active compound, betaine, on muscle differentiation and mitochondrial biogenesis in C2C12 cells.

Our study was conducted to investigate the effects of the fruits of Lycium chinense Mill. (Lycii Fructus, LF) and its bioactive compound, betaine, on muscle differentiation and mitochondrial biogenesis in C2C12 cells. LF extract and betaine was analyzed by high-performance liquid chromatography (HPLC). The expression of myosin heavy chain (MyHC) and peroxisome proliferator-activated receptor gamma coactivator1-alpha (PGC-1α), sirtuin-1(Sirt-1), nuclear respiratory factor-1 (NRF-1), transcription factor A, mitochondrial (TFAM) and the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC), were determined in cellular or mitochondrial levels by quantitative polymerase chain reaction (qPCR) or Western blot, respectively. The glucose levels and total ATP contents were measured by the glucose consumption in a culture medium, cellular glucose uptake and ATP assays. LF extract at 4 mg/ml and betaine at 2 and 5 mM significantly increased the expression of MyHC in C2C12 myotubes, compared with non-treated cells. LF extract and betaine significantly increased the expression of PGC-1α, Sirt-1, NRF-1 and TFAM mRNA and protein in the myotubes, as well as phosphorylation of AMPK and ACC. Furthermore, LF extract and betaine significantly increased the mitochondrial protein contents, as the TFAM and NRF-1 expressions were increased. LF extract and betaine also significantly increased the glucose uptake and ATP contents in the myotubes. The LF extract contained 3.18% betaine was quantitated by HPLC. LF extract and betaine enhanced muscle differentiation and energy metabolism through the up-regulation of mitochondrial biogenesis-regulating factors, suggesting that LF extract and betaine can help to prevent the dysfunction of skeletal muscle.

Anti-inflammatory and antioxidant effects of MOK, a polyherbal extract, on lipopolysaccharide­stimulated RAW 264.7 macrophages.

MOK, a pharmacopuncture medicine consisting of 10 herbs, has a long history as treatment for various inflammatory conditions. To investigate the mechanisms of action of MOK, its anti­inflammatory and antioxidative effects were assessed in RAW 264.7 macrophages stimulated by lipopolysaccharide (LPS). RAW 264.7 cells were treated with different concentrations of MOK extract for 30 min prior to stimulation with or without LPS for the indicated times. Nitric oxide (NO) production was measured using Griess reagent, while the mRNA levels of inflammatory cytokines, tumor necrosis factor (TNF)­α, interleukin (IL)­1ß, IL­6 and the antioxidant enzymes Mn superoxide dismutase and heme oxygenase­1, were determined using reverse transcription­polymerase chain reaction analysis. Western blotting was used to determine the protein expression of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)­2, superoxide dismutase (SOD)2, catalase (CAT) and heme oxygenase­1 (HO­1), and the phosphorylation of mitogen­activated protein kinases (MAPKs), including ERK1/2, JNK and p38. Western blotting and immunocytochemistry were used to observe the nuclear expression of nuclear factor (NF)­κB p65. Additionally, reactive oxygen species (ROS) and prostaglandin (PG)E2 production were determined using the ROS assay and an enzyme immunoassay. With MOK treatment, there was a notable decrease in NO and PGE2 production induced by LPS in RAW 264.7 cells by downregulation of iNOS and COX­2 mRNA and protein expression. Furthermore, with MOK treatment, there was a decrease in the mRNA expression levels of TNF­α, IL­1ß and IL­6, as well as in the phosphorylation of ERK, JNK and p38 MAPK, by blocking the nuclear translocation of NF­κB p65 in LPS­stimulated cells. In addition, MOK treatment led to an increase in the antioxidant enzymes SOD, CAT and HO­1 in LPS­stimulated cells, with a concomitant decrease in ROS generation. These results indicate that the inflammatory responses in activated macrophages are inhibited by MOK through downregulation of the transcription levels of inflammatory mediators and inhibition of the MAPK/NF­κB pathway. Moreover, MOK protects against oxidative damage by upregulating the expression of antioxidant enzymes and generating ROS scavengers.

Effects of Rhizome Extract of Dioscorea batatas and Its Active Compound, Allantoin, on the Regulation of Myoblast Differentiation and Mitochondrial Biogenesis in C2C12 Myotubes.

With the aging process, a loss of skeletal muscle mass and dysfunction related to metabolic syndrome is observed in older people. Yams are commonly use in functional foods and medications with various effects. The present study was conducted to investigate the effects of rhizome extract of Dioscorea batatas (Dioscoreae Rhizoma, Chinese yam) and its bioactive compound, allantoin, on myoblast differentiation and mitochondrial biogenesis in skeletal muscle cells. Yams were extracted in water and allantoin was analyzed by high performance liquid chromatography (HPLC). The expression of myosin heavy chain (MyHC) and mitochondrial biogenesis-regulating factors, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), sirtuin-1 (Sirt-1), nuclear respiratory factor-1 (NRF-1) and transcription factor A, mitochondrial (TFAM), and the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) were determined in C2C12 myotubes by reverse transcriptase (RT)-polymerase chain reaction (RT-PCR) or western blot. The glucose levels and total ATP contents were measured by glucose consumption, glucose uptake and ATP assays, respectively. Treatment with yam extract (1 mg/mL) and allantoin (0.2 and 0.5 mM) significantly increased MyHC expression compared with non-treated myotubes. Yam extract and allantoin significantly increased the expression of PGC-1α, Sirt-1, NRF-1 and TFAM, as well as the phosphorylation of AMPK and ACC in C2C12 myotubes. Furthermore, yam extract and allantoin significantly increased glucose uptake levels and ATP contents. Finally, HPLC analysis revealed that the yam water extract contained 1.53% of allantoin. Yam extract and allantoin stimulated myoblast differentiation into myotubes and increased energy production through the upregulation of mitochondrial biogenesis regulators. These findings indicate that yam extract and allantoin can help to prevent skeletal muscle dysfunction through the stimulation of the energy metabolism.

Effects of Flower Buds Extract of Tussilago farfara on Focal Cerebral Ischemia in Rats and Inflammatory Response in BV2 Microglia.

OBJECTIVE: To investigate the effects of the flower buds extract of Tussilago farfara Linné (Farfarae Flos; FF) on focal cerebral ischemia through regulation of inflammatory responses in activated microglia. METHODS: Brain ischemia was induced in Sprague-Dawley rats by a transient middle cerebral artery occlusion (tMCAO) for 90 min and reperfusion for 24 h. Twenty rats were randomly divided into 4 groups (n=5 per group): normal, tMCAO-induced ischemic control, tMCAO plus FF extract 300 mg/kg-treated, and tMCAO plus MK-801 1 mg/kg-treated as reference drug. FF extract (300 mg/kg, p.o.) or MK-801 (1 mg/kg, i.p.) was administered after reperfusion. Brain infarction was measured by 2,3,5,-triphenyltetrazolium chloride staining. Neuronal damage was observed by haematoxylin eosin, Nissl staining and immunohistochemistry using anti-neuronal nuclei (NeuN), anti-glial fibrillary acidic protein (GFAP), and anti-CD11b/c (OX42) antibodies in ischemic brain. The expressions of inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF-α), and hypoxia-inducible factor-1a (HIF-1α) were determined by Western blot. BV2 microglial cells were treated with FF extract or its main bioactive compound, tussilagone with or without lipopolysaccharide (LPS). Nitric oxide (NO) production was measured in culture medium by Griess assay. The expressions of iNOS, COX-2 and pro-inflammatory cytokines mRNA were analyzed by reverse transcription-polymerase chain reaction. The expression of iNOS, and COX-2 proteins, the phosphorylation of ERK1/2, JNK, and p38 MAPK and the nuclear expression of NF-κB p65 in BV2 cells were determined by Western blot. RESULTS: FF extract significantly decreased brain infarctions in ischemic rats (P<0.01). The neuronal death and the microglia/astrocytes activation in ischemic brains were inhibited by FF extract. FF extract also suppressed iNOS, TNF-α, and HIF-1α expression in ischemic brains. FF extract (0.2 and 0.5 mg/mL, P<0.01) and tussilagone 20 and 50 µmol/L, P<0.01) significantly decreased LPS-induced NO production in BV2 microglia through downregulation of iNOS mRNA and protein expression. FF extract and tussilagone significantly inhibited LPS-induced expression of TNF-α, IL-1ß, and IL-6 mRNA, and also suppressed the phosphorylation of ERK1/2, JNK and p38 MAPK and the nuclear expression of NF-κB in a dose-dependent manner. CONCLUSIONS: FF extract has a neuroprotective effect in ischemic stroke by the decrease of brain infarction, and the inhibition of neuronal death and microglial activation-mediated inflammatory responses.

Therapeutic effects of acupuncture with MOK, a polyherbal medicine, on PTU-induced hypothyroidism in rats.

Acupuncture with MOK, a polyherbal medicine (MOK pharmacopuncture), has been used for the treatment of thyroid syndromes including hypothyroidism and hyperthyroidism in traditional Korean medicine. The present study investigated the effect of MOK pharmacopuncture on hypothyroidism and the mechanism underlying its antioxidation and immune regulation effects. Hypothyroidism was induced in Sprague-Dawley rats by subcutaneous injection of Propylthiouracil (PTU; 10 mg/kg) once daily for 4 weeks. MOK was administered by acupuncture on the acupoints around the thyroid gland of PTU-induced hypothyroidism rats once daily for 2 weeks following hypothyroidism induction. Administration of MOK pharmacopuncture significantly increased the PTU-induced decrease in body temperature of hypothyroidism rats. The weights of the spleen were also significantly decreased in hyperthyroidism rats following MOK pharmacopuncture. MOK pharmacopuncture significantly decreased the thyroid stimulating hormone level and increased the T3 and T4 levels in hypothyroidism rats. Administration of MOK pharmacopuncture significantly increased the glucose levels and decreased the levels of triglycerides, total cholesterol, low-density lipoprotein-cholesterol, and alanine transaminase in the sera of hypothyroidism rats. The expression of transient receptor potential cation channel subfamily V member 1 was increased in dorsal root ganglion and brain tissues by administration of MOK pharmacopuncture, and glutathione levels and the expression of superoxide dismutase 1 and catalase were increased in the liver and brain tissues. Administration of MOK pharmacopuncture significantly inhibited interferon-γ expression and increased the expression of interleukin (IL)-4, IL-10, and Forkhead Box P3 in the spleen tissues of hypothyroidism rats. In histological analysis, the administration of MOK pharmacopuncture improved the pathological features in the thyroid glands of hypothyroidism rats. The results suggested that the administration of pharmacopuncture may ameliorate the pathological progression of hypothyroidism by multiple actions, including normalization of the hypothyroidism-induced thyroid hormone imbalance, stimulation of the antioxidant defense system, and regulation of the T helper (Th)1/Th2 imbalance. Therefore, MOK extract may be used for the treatment of hypothyroidism in Korean clinics as a useful pharmacopuncture medicine.

MOK, a pharmacopuncture medicine, regulates thyroid dysfunction in L-thyroxin-induced hyperthyroidism in rats through the regulation of oxidation and the TRPV1 ion channel.

BACKGROUND: In this study, we evaluated the therapeutic effect of MOK, a pharmacopuncture medicine, on thyroid dysfunction in L-thyroxin (LT4)-induced hyperthyroidism rats. METHODS: The experimental hyperthyroidism model was prepared by the intraperitoneal injection of LT4 (0.5 mg/kg) once daily for 2 weeks in SD rats. MOK extract was injected at doses of 0.3 or 3 mg/kg on acupuncture points in the thyroid glands of LT4-induced hypothyroidism rats once a day for 2 weeks. The body temperature, body weight, and food/water intake were measured once a week for 2 weeks. The levels of thyroid hormones, total cholesterol, LDL-cholesterol, GOT, and GPT were measured in the sera of rats using ELISA and an automatic blood analyzer. The histological changes of thyroid tissues were observed by H&E staining. The expression of thermo-regulating protein, TRPV1 was determined by western blot in dorsal root ganglion (DRG) and brain tissues. We also measured the contents of GSH in the liver and antioxidant enzymes, SOD, and catalase in the liver, heart, and brain tissues by enzyme-based assay and Western blot, respectively. RESULTS: The acupuncture of MOK extract on the thyroid gland of LT4-induced hyperthyroidism rats significantly decreased the body temperature, and did not change body weight and food and water intakes. MOK acupuncture significantly increased the level of TSH, and decreased the levels of T3 and T4 in hyperthyroidism rats. The expression of TRPV1 was inhibited in both DRG and brain tissues after MOK acupuncture, and the levels of GOT, GPT, total cholesterol, and LDL-cholesterol were also decreased. MOK acupuncture also inhibited the pathological feature with follicular lining epithelial thicknesses and increased follicular colloid depositions in the thyroid glands of hypothyroidism. MOK acupuncture significantly increased hepatic GSH levels and decreased the expression of SOD and catalase in the liver, heart, and brain tissues of hyperthyroidism rats. CONCLUSIONS: These results suggest that the pharmacopuncture with MOK extract in hyperthyroidism can improve the pathophysiological changes through regulating the body temperature, thyroid hormones imbalance, lipid accumulation, and oxidation. This anti-hyperthyroidism effect of MOK pharmacopuncture is thought to be related to the control of thermo-regulating protein TRPV1 in DRG and brain.

Cinnamomum cassia Prevents High-Fat Diet-Induced Obesity in Mice through the Increase of Muscle Energy.

The cortex of Cinnamomum cassia Presl (Cinnamomi Cortex: CC) has commonly been used for weight control in traditional medicines, but without a scientific basis. Therefore, this study was undertaken to investigate the anti-obesity effect of CC extract in a high-fat diet (HFD)-induced obese mouse model and in C2C12 mouse skeletal muscle cells. Male C57BL/6 mice were fed a normal diet or a HFD for 16 consecutive weeks, and orally administered CC extract (100 or 300[Formula: see text]mg/kg) or metformin (250[Formula: see text]mg/kg; positive control) daily for 16 weeks. CC extract administration significantly decreased body weights, food intakes, and serum levels of glucose, insulin, total cholesterol and ALT levels, prevented oral glucose tolerance and insulin resistance, inhibited the protein expressions of MyHC and PGC1[Formula: see text] and the phosphorylation of AMPK, suppressed lipid accumulation in liver, decreased adipocyte size and increased muscle mass in obese mice. For this in vitro study, C2C12 myoblasts were differentiated into the myotubes for five days, and then treated with CC extract (0.1 or 0.2[Formula: see text]mg/ml) for 24[Formula: see text]h. CC extract significantly increased ATP levels by increasing the mRNA expressions of mitochondrial biogenesis-related factors, such as, PGC1[Formula: see text], NRF-1, and Tfam, and the phosphorylations of AMPK and ACC. Our results suggest CC extract controls weight gain in obese mice by inhibiting lipid accumulation and increasing energy expenditure, and that its action mechanism involves the up-regulation of mitochondrial biogenesis in skeletal muscle cells.

Atractylenolide III Enhances Energy Metabolism by Increasing the SIRT-1 and PGC1α Expression with AMPK Phosphorylation in C2C12 Mouse Skeletal Muscle Cells.

Targeting energy expenditure provides a potential alternative strategy for achieving energy balance to combat obesity and the development of type 2 diabetes mellitus (T2DM). In the present study, we investigated whether atractylenolide III (AIII) regulates energy metabolism in skeletal muscle cells. Differentiated C2C12 myotubes were treated with AIII (10, 20, or 50 µM) or metformin (2.5 mM) for indicated times. The levels of glucose uptake, the expressions of key mitochondrial biogenesis-related factors and their target genes were measured in C2C12 myotubes. AIII significantly increased the glucose uptake levels, and significantly increased the expressions of peroxisome proliferator-activated receptor coactivator-1α (PGC1α) and mitochondrial biogenesis-related markers, such as, nuclear respiratory factor-1 (NRF-1), and mitochondrial transcription factor A (TFAM) and mitochondrial mass and total ATP contents. In addition, AIII significantly increased the phosphorylation of AMP-activated protein kinase (AMPK) and the expression of sirtuin1 (SIRT1). These results suggest that AIII may have beneficial effects on obesity and T2DM by improving energy metabolism in skeletal muscle.

The Root Extract of Pueraria lobata and Its Main Compound, Puerarin, Prevent Obesity by Increasing the Energy Metabolism in Skeletal Muscle.

Radix Pueraria lobata (RP) has been reported to prevent obesity and improve glucose metabolism; however, the mechanism responsible for these effects has not been elucidated. The mechanism underlying anti-obesity effect of RP was investigated in high-fat diet (HFD) induced obese mice and skeletal muscle cells (C2C12). Five-week-old C5BL/6 mice were fed a HFD containing or not containing RP (100 or 300 mg/kg) or metformin (250 mg/kg) for 16 weeks. RP reduced body weight gain, lipid accumulation in liver, and adipocyte and blood lipid levels. In addition, RP dose-dependently improved hyperglycemia, insulinemia, and glucose tolerance, and prevented the skeletal muscle atrophy induced by HFD. Furthermore, RP increased the peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) expression and phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) in skeletal muscle tissues. RP and its main component, puerarin, increased mitochondrial biogenesis and myotube hypertrophy in C2C12 cells. The present study demonstrates that RP can prevent diet-induced obesity, glucose tolerance, and skeletal muscle atrophy in mouse models of obesity. The mechanism responsible for the effect of RP appears to be related to the upregulation of energy metabolism in skeletal muscle, which at the molecular level may be associated with PGC-1α and AMPK activation.

Effects of the Fruit Extract of Tribulus terrestris on Skin Inflammation in Mice with Oxazolone-Induced Atopic Dermatitis through Regulation of Calcium Channels, Orai-1 and TRPV3, and Mast Cell Activation.

ETHNOPHARMACOLOGICAL RELEVANCE: In this study, we investigated the effects of Tribulus terrestris fruit (Leguminosae, Tribuli Fructus, TF) extract on oxazolone-induced atopic dermatitis in mice. MATERIALS AND METHODS: TF extract was prepared with 30% ethanol as solvent. The 1% TF extract with or without 0.1% HC was applied to the back skin daily for 24 days. RESULTS: 1% TF extract with 0.1% HC improved AD symptoms and reduced TEWL and symptom scores in AD mice. 1% TF extract with 0.1% HC inhibited skin inflammation through decrease in inflammatory cells infiltration as well as inhibition of Orai-1 expression in skin tissues. TF extract inhibited Orai-1 activity in Orai-1-STIM1 cooverexpressing HEK293T cells but increased TRPV3 activity in TRPV3-overexpressing HEK293T cells. TF extract decreased ß-hexosaminidase release in RBL-2H3 cells. CONCLUSIONS: The present study demonstrates that the topical application of TF extract improves skin inflammation in AD mice, and the mechanism for this effect appears to be related to the modulation of calcium channels and mast cell activation. This outcome suggests that the combination of TF and steroids could be a more effective and safe approach for AD treatment.