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 American wild ginseng and Korean cultivated wild ginseng pharmacopuncture extracts on the regulation of C2C12 myoblasts differentiation through AMPK and PI3K/Akt/mTOR signaling pathway.

Targeting impaired myogenesis and mitochondrial biogenesis offers a potential alternative strategy for balancing energy to fight muscle disorders such as sarcopenia. In traditional Korean medicine, it is believed that the herb wild ginseng can help restore energy to the elderly. The present study investigated whether American wild ginseng pharmacopuncture (AWGP) and Korean cultivated wild ginseng pharmacopuncture (KCWGP) regulate energy metabolism in skeletal muscle cells. C2C12 mouse myoblasts were differentiated into myotubes using horse serum for 5 days. An MTT colorimetric assay verified cell viability. AWGP, KCWGP (0.5, 1, or 2 mg/ml), or metformin (2.5 mM) for reference were used to treat the C2C12 myotubes. The expressions of differentiation and mitochondrial biogenetic factors were measured by western blotting in C2C12 myotubes. Treatment of C2C12 cells stimulated with AWGP and KCWGP at a concentration of 10 mg/ml did not affect cell viability. AWGP and KCWGP treatments resulted in significant increases in the myogenesis proteins, myosin heavy chain, myostatin, myoblast determination protein 1 and myogenin, as well as increases to the biogenic regulatory factors, peroxisome proliferator­activated receptor­Î³ coactivator­1­α, nuclear respiratory factor 1, mitochondrial transcription factor A and Sirtuin 1, in the myotubes through AMPK and PI3K/AKT/mTOR signaling pathway activation. These results suggest that AWGP and KCWGP may be beneficial to muscle function by improving muscle differentiation and energy metabolism.

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.

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.

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.

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.

Effect of the semen extract of Cuscuta chinensis on inflammatory responses in LPS-stimulated BV-2 microglia.

AIM: To investigate the anti-inflammatory activities of the semen extract of Cuscuta chinensis Lam. (Cuscutae Semen; CS) on the production of inflammatory mediators, nitric oxide (NO), prostaglandin 2 (PGE2), and proinflammatory cytokines in lipopolysaccharide (LPS)-stimulated BV-2 microglia. METHOD: BV-2 cells were treated with CS extract for 30 min, and then stimulated with LPS or without for 24 h. The levels of NO, PGE2 and proinflammatory cytokines were measured by Griess assay and ELISA. The expression of inducible nitric oxide synthase (iNOS), and cyclooxygenase (COX)-2 mRNA and protein was determined by RT-PCR and Western blot, respectively. The phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK), and the nuclear expression of nuclear factor (NF)-κB p65 were investigated by Western blot analysis. RESULTS: CS extract significantly decreased the production of NO and PGE2 by suppressing the expression of iNOS and COX-2 in activated microglia. CS extract decreased the production of TNF-α, IL-1ß, and IL-6 by down-regulating their transcription levels. In addition, CS extract suppressed the phosphorylation of ERK1/2, JNK, and p38 MAPK, and the nuclear translocation of NF-κB p65 in activated microglia. CONCLUSION: These results indicate that CS extract is capable of suppressing the inflammatory response by microglia activation, suggesting that CS extract has potential in the treatment of brain inflammation.

Anti-inflammatory effect of Lycii radicis in LPS-stimulated RAW 264.7 macrophages.

The root bark of Lycium barbarum (Lycii radicis cortex, LRC) is used as a cooling agent for fever and night sweats in East Asian traditional medicine. The inhibitory effect of LRC water extract on inflammation is unknown. In this study, the anti-inflammatory effect of LRC was investigated in lipopolysaccharide (LPS)-stimulated mouse macrophage, RAW 264.7 cells. LRC extract significantly decreased the LPS-induced production of inflammatory mediators, nitric oxide (NO), prostaglandin (PG) E2 and pro-inflammatory cytokines, interleukin (IL)-1ß and IL-6 in the cells. In addition, LRC extract inhibited the LPS-induced expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 mRNA and protein, and inflammatory cytokines mRNA in the cells. The action mechanism of LRC underlies the blocking of LPS-mediated p38 and Jun N-terminal kinase (JNK), mitogen-activated protein kinases (MAPKs), and the nuclear factor (NF)-κB signaling pathway. These results indicate that LRC extract inhibits the inflammatory response in activated macrophages by down-regulating the transcription levels of inflammatory mediators and blocking the MAPKs and NF-κB pathway.

Effect of the semen extract of Thuja orientalis on inflammatory responses in transient focal cerebral ischemia rat model and LPS-stimulated BV-2 microglia.

In the central nervous system inflammation is dependent upon the synthesis of various inflammatory mediators by local neurons, astrocytes and especially microglia. In this study, we investigated the anti-inflammatory activities of the semen extract of Thuja orientalis (Thujae Orientalis Semen; TOS) on transient middle cerebral artery occlusion (tMCAO)-induced ischemia in rats and the production of inflammatory mediators such as nitric oxide (NO), prostaglandin E(2) (PGE(2)) and proinflammatory cytokine, interleukin (IL)-1ß in lipopolysaccharide (LPS)-stimulated BV-2 mouse microglia. TOS extract significantly decreased the infarction volumes of ischemic brains and also inhibited microglia activation and neuronal death. In addition, TOS extract significantly decreased the production of NO, PGE(2) and IL-1ß in LPS-stimulated BV-2 microglia. TOS extract also attenuated the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and IL-1ß mRNAs and proteins in activated microglia. Furthermore, TOS extract significantly suppressed the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 MAPK, and the nuclear translocation of NF-κB p65 in activated microglia. Our results indicate that TOS extract is capable of inhibiting microglia activation in an ischemic brain through the down-regulation of inflammatory responses, suggesting that the TOS extract may have therapeutic potential as an anti-inflammatory drug for ischemic stroke.