Publications by authors named "Bhesh Raj Sharma"

26 Publications

  • Page 1 of 1

Synergism of TNF-α and IFN-γ Triggers Inflammatory Cell Death, Tissue Damage, and Mortality in SARS-CoV-2 Infection and Cytokine Shock Syndromes.

Cell 2021 01 19;184(1):149-168.e17. Epub 2020 Nov 19.

Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA. Electronic address:

COVID-19 is characterized by excessive production of pro-inflammatory cytokines and acute lung damage associated with patient mortality. While multiple inflammatory cytokines are produced by innate immune cells during SARS-CoV-2 infection, we found that only the combination of TNF-α and IFN-γ induced inflammatory cell death characterized by inflammatory cell death, PANoptosis. Mechanistically, TNF-α and IFN-γ co-treatment activated the JAK/STAT1/IRF1 axis, inducing nitric oxide production and driving caspase-8/FADD-mediated PANoptosis. TNF-α and IFN-γ caused a lethal cytokine shock in mice that mirrors the tissue damage and inflammation of COVID-19, and inhibiting PANoptosis protected mice from this pathology and death. Furthermore, treating with neutralizing antibodies against TNF-α and IFN-γ protected mice from mortality during SARS-CoV-2 infection, sepsis, hemophagocytic lymphohistiocytosis, and cytokine shock. Collectively, our findings suggest that blocking the cytokine-mediated inflammatory cell death signaling pathway identified here may benefit patients with COVID-19 or other infectious and autoinflammatory diseases by limiting tissue damage/inflammation.
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http://dx.doi.org/10.1016/j.cell.2020.11.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7674074PMC
January 2021

COVID-19 cytokines and the hyperactive immune response: Synergism of TNF-α and IFN-γ in triggering inflammation, tissue damage, and death.

bioRxiv 2020 Oct 29. Epub 2020 Oct 29.

Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA.

The COVID-19 pandemic has caused significant morbidity and mortality. Currently, there is a critical shortage of proven treatment options and an urgent need to understand the pathogenesis of multi-organ failure and lung damage. Cytokine storm is associated with severe inflammation and organ damage during COVID-19. However, a detailed molecular pathway defining this cytokine storm is lacking, and gaining mechanistic understanding of how SARS-CoV-2 elicits a hyperactive inflammatory response is critical to develop effective therapeutics. Of the multiple inflammatory cytokines produced by innate immune cells during SARS-CoV-2 infection, we found that the combined production of TNF-α and IFN-γ specifically induced inflammatory cell death, PANoptosis, characterized by gasdermin-mediated pyroptosis, caspase-8-mediated apoptosis, and MLKL-mediated necroptosis. Deletion of pyroptosis, apoptosis, or necroptosis mediators individually was not sufficient to protect against cell death. However, cells deficient in both RIPK3 and caspase-8 or RIPK3 and FADD were resistant to this cell death. Mechanistically, the STAT1/IRF1 axis activated by TNF-α and IFN-γ co-treatment induced iNOS for the production of nitric oxide. Pharmacological and genetic deletion of this pathway inhibited pyroptosis, apoptosis, and necroptosis in macrophages. Moreover, inhibition of PANoptosis protected mice from TNF-α and IFN-γ-induced lethal cytokine shock that mirrors the pathological symptoms of COVID-19. In vivo neutralization of both TNF-α and IFN-γ in multiple disease models associated with cytokine storm showed that this treatment provided substantial protection against not only SARS-CoV-2 infection, but also sepsis, hemophagocytic lymphohistiocytosis, and cytokine shock models, demonstrating the broad physiological relevance of this mechanism. Collectively, our findings reveal that blocking the COVID-19 cytokine-mediated inflammatory cell death signaling pathway identified in this study may benefit patients with COVID-19 or other cytokine storm-driven syndromes by limiting inflammation and tissue damage. The findings also provide a molecular and mechanistic description for the term cytokine storm. Additionally, these results open new avenues for the treatment of other infectious and autoinflammatory diseases and cancers where TNF-α and IFN-γ synergism play key pathological roles.
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http://dx.doi.org/10.1101/2020.10.29.361048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7605562PMC
October 2020

ZBP1 promotes fungi-induced inflammasome activation and pyroptosis, apoptosis, and necroptosis (PANoptosis).

J Biol Chem 2020 Dec 27;295(52):18276-18283. Epub 2020 Oct 27.

Department of Immunology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA

are dangerous fungal pathogens with high morbidity and mortality, particularly in immunocompromised patients. Innate immune-mediated programmed cell death (pyroptosis, apoptosis, necroptosis) is an integral part of host defense against pathogens. Inflammasomes, which are canonically formed upstream of pyroptosis, have been characterized as key mediators of fungal sensing and drivers of proinflammatory responses. However, the specific cell death pathways and key upstream sensors activated in the context of and infections are unknown. Here, we report that and infection induced inflammatory programmed cell death in the form of pyroptosis, apoptosis, and necroptosis (PANoptosis). Further, we identified the innate immune sensor Z-DNA binding protein 1 (ZBP1) as the apical sensor of fungal infection responsible for activating the inflammasome/pyroptosis, apoptosis, and necroptosis. The Zα2 domain of ZBP1 was required to promote this inflammasome activation and PANoptosis. Overall, our results demonstrate that and induce PANoptosis and that ZBP1 plays a vital role in inflammasome activation and PANoptosis in response to fungal pathogens.
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http://dx.doi.org/10.1074/jbc.RA120.015924DOI Listing
December 2020

Interferon regulatory factor 1 regulates PANoptosis to prevent colorectal cancer.

JCI Insight 2020 06 18;5(12). Epub 2020 Jun 18.

Department of Immunology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.

Interferon regulatory factor 1 (IRF1) regulates diverse biological functions, including modulation of cellular responses involved in tumorigenesis. Genetic mutations and altered IRF1 function are associated with several cancers. Although the function of IRF1 in the immunobiology of cancer is emerging, IRF1-specific mechanisms regulating tumorigenesis and tissue homeostasis in vivo are not clear. Here, we found that mice lacking IRF1 were hypersusceptible to colorectal tumorigenesis. IRF1 functions in both the myeloid and epithelial compartments to confer protection against AOM/DSS-induced colorectal tumorigenesis. We further found that IRF1 also prevents tumorigenesis in a spontaneous mouse model of colorectal cancer. The attenuated cell death in the colons of Irf1-/- mice was due to defective pyroptosis, apoptosis, and necroptosis (PANoptosis). IRF1 does not regulate inflammation and the inflammasome in the colon. Overall, our study identified IRF1 as an upstream regulator of PANoptosis to induce cell death during colitis-associated tumorigenesis.
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http://dx.doi.org/10.1172/jci.insight.136720DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7406299PMC
June 2020

Identification of the PANoptosome: A Molecular Platform Triggering Pyroptosis, Apoptosis, and Necroptosis (PANoptosis).

Front Cell Infect Microbiol 2020 29;10:237. Epub 2020 May 29.

Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, United States.

Programmed cell death plays crucial roles in organismal development and host defense. Recent studies have highlighted mechanistic overlaps and extensive, multifaceted crosstalk between pyroptosis, apoptosis, and necroptosis, three programmed cell death pathways traditionally considered autonomous. The growing body of evidence, in conjunction with the identification of molecules controlling the concomitant activation of all three pathways by pathological triggers, has led to the development of the concept of PANoptosis. During PANoptosis, inflammatory cell death occurs through the collective activation of pyroptosis, apoptosis, and necroptosis, which can circumvent pathogen-mediated inhibition of individual death pathways. Many of the molecular details of this emerging pathway are unclear. Here, we describe the activation of PANoptosis by bacterial and viral triggers and report protein interactions that reveal the formation of a PANoptosome complex. Infection of macrophages with influenza A virus, vesicular stomatitis virus, , or serovar Typhimurium resulted in robust cell death and the hallmarks of PANoptosis activation. Combined deletion of the PANoptotic components caspase-1 (CASP1), CASP11, receptor-interacting serine/threonine-protein kinase 3 (RIPK3), and CASP8 largely protected macrophages from cell death induced by these pathogens, while deletion of individual components provided reduced or no protection. Further, molecules from the pyroptotic, apoptotic, and necroptotic cell death pathways interacted to form a single molecular complex that we have termed the PANoptosome. Overall, our study identifies pathogens capable of activating PANoptosis and the formation of a PANoptosome complex.
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http://dx.doi.org/10.3389/fcimb.2020.00237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7274033PMC
May 2020

The nonreceptor tyrosine kinase SYK drives caspase-8/NLRP3 inflammasome-mediated autoinflammatory osteomyelitis.

J Biol Chem 2020 03 12;295(11):3394-3400. Epub 2019 Nov 12.

Department of Immunology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105

Chronic recurrent multifocal osteomyelitis (CRMO) in humans can be modeled in mice, which carry a missense mutation in the proline-serine-threonine phosphatase-interacting protein 2 () gene. As disease in mice, the experimental model analogous to human CRMO, is mediated specifically by IL-1β and not by IL-1α, delineating the molecular pathways contributing to pathogenic IL-1β production is crucial to developing targeted therapies. In particular, our earlier findings support redundant roles of NLR family pyrin domain-containing 3 (NLRP3) and caspase-1 with caspase-8 in instigating However, the signaling components upstream of caspase-8 and pro-IL-1β cleavage in mice are not well-understood. Therefore, here we investigated the signaling pathways in these mice and discovered a central role of a nonreceptor tyrosine kinase, spleen tyrosine kinase (SYK), in mediating osteomyelitis. Using several mutant mouse strains, immunoblotting, and microcomputed tomography, we demonstrate that absent in melanoma 2 (AIM2), receptor-interacting serine/ threonine protein kinase 3 (RIPK3), and caspase recruitment domain-containing protein 9 (CARD9) are each dispensable for osteomyelitis induction in mice, whereas genetic deletion of completely abrogates the disease phenotype. We further show that SYK centrally mediates signaling upstream of caspase-1 and caspase-8 activation and principally up-regulates NF-κB and IL-1β signaling in mice, thereby inducing These results provide a rationale for directly targeting SYK and its downstream signaling components in CRMO.
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http://dx.doi.org/10.1074/jbc.RA119.010623DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7076204PMC
March 2020

DDX3X acts as a live-or-die checkpoint in stressed cells by regulating NLRP3 inflammasome.

Nature 2019 09 11;573(7775):590-594. Epub 2019 Sep 11.

Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, USA.

The cellular stress response has a vital role in regulating homeostasis by modulating cell survival and death. Stress granules are cytoplasmic compartments that enable cells to survive various stressors. Defects in the assembly and disassembly of stress granules are linked to neurodegenerative diseases, aberrant antiviral responses and cancer. Inflammasomes are multi-protein heteromeric complexes that sense molecular patterns that are associated with damage or intracellular pathogens, and assemble into cytosolic compartments known as ASC specks to facilitate the activation of caspase-1. Activation of inflammasomes induces the secretion of interleukin (IL)-1β and IL-18 and drives cell fate towards pyroptosis-a form of programmed inflammatory cell death that has major roles in health and disease. Although both stress granules and inflammasomes can be triggered by the sensing of cellular stress, they drive contrasting cell-fate decisions. The crosstalk between stress granules and inflammasomes and how this informs cell fate has not been well-studied. Here we show that the induction of stress granules specifically inhibits NLRP3 inflammasome activation, ASC speck formation and pyroptosis. The stress granule protein DDX3X interacts with NLRP3 to drive inflammasome activation. Assembly of stress granules leads to the sequestration of DDX3X, and thereby the inhibition of NLRP3 inflammasome activation. Stress granules and the NLRP3 inflammasome compete for DDX3X molecules to coordinate the activation of innate responses and subsequent cell-fate decisions under stress conditions. Induction of stress granules or loss of DDX3X in the myeloid compartment leads to a decrease in the production of inflammasome-dependent cytokines in vivo. Our findings suggest that macrophages use the availability of DDX3X to interpret stress signals and choose between pro-survival stress granules and pyroptotic ASC specks. Together, our data demonstrate the role of DDX3X in driving NLRP3 inflammasome and stress granule assembly, and suggest a rheostat-like mechanistic paradigm for regulating live-or-die cell-fate decisions under stress conditions.
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http://dx.doi.org/10.1038/s41586-019-1551-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6980284PMC
September 2019

Tinospora cordifolia preserves pancreatic beta cells and enhances glucose uptake in adipocytes to regulate glucose metabolism in diabetic rats.

Phytother Res 2019 Oct 5;33(10):2765-2774. Epub 2019 Aug 5.

Department of Oriental Medicine Resources and Institute of Korean Medicine Industry, Mokpo National University, Mokpo, Republic of Korea.

The purpose of this study was to evaluate the pancreatic beta cell protective and glucose uptake enhancing effect of the water extract of Tinospora cordifolia stem (TCSE) by using rat insulinoma (RIN)-m5F cells and 3 T3-L1 adipocytes. RIN-m5F cells were stimulated with interleukin-1β and interferon-γ, and the effect of TCSE on insulin secretion and cytokine-induced toxicity was measured by ELISA and MTT assay, respectively. The glucose uptake and protein expression were measured by fluorometry and western blotting. Antidiabetic effect of TCSE was measured using streptozotocin-induced diabetic rats. TCSE dose dependently increased cell viability and insulin secretion in RIN-m5F cells. In addition, TCSE increased both the glucose uptake and glucose transporter 4 translocation in 3 T3-L1 adipocytes via PI3K pathway. Finally, TCSE significantly lowered blood glucose and diet intake and increased body weight in streptozotocin-induced diabetic rats. The level of serum insulin and hepatic glycogen was increased, whereas the level of serum triglyceride, total cholesterol, dipeptidyl peptidase-4, and thiobarbituric acid reactive substances was decreased in TCSE-administered rats. TCSE also increased glucose transporter 4 protein expression in the adipose tissue and liver of TCSE-fed diabetic rats. Our results suggested that TCSE preserved RIN-m5F cells from cytokine-induced toxicity and enhanced glucose uptake in 3 T3-L1 adipocytes, which may regulate glucose metabolism in diabetic rats.
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http://dx.doi.org/10.1002/ptr.6462DOI Listing
October 2019

Role of AIM2 inflammasome in inflammatory diseases, cancer and infection.

Eur J Immunol 2019 11 14;49(11):1998-2011. Epub 2019 Aug 14.

Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, USA.

AIM2 is a cytosolic innate immune receptor which recognizes double-stranded DNA (dsDNA) released during cellular perturbation and pathogenic assault. AIM2 recognition of dsDNA leads to the assembly of a large multiprotein oligomeric complex termed the inflammasome. This inflammasome assembly leads to the secretion of bioactive interleukin-1β (IL-1β) and IL-18 and induction of an inflammatory form of cell death called pyroptosis. Sensing of dsDNA by AIM2 in the cytosol is crucial to mediate protection against the invading pathogens including bacteria, virus, fungi and parasites. AIM2 also responds to dsDNA released from damaged host cells, resulting in the secretion of the effector cytokines thereby driving the progression of sterile inflammatory diseases such as skin disease, neuronal disease, chronic kidney disease, cardiovascular disease and diabetes. Additionally, the protection mediated by AIM2 in the development of colorectal cancer depends on its ability to regulate epithelial cell proliferation and gut microbiota in maintaining intestinal homeostasis independently of the effector cytokines. In this review, we will highlight the recent progress on the role of the AIM2 inflammasome as a guardian of cellular integrity in modulating chronic inflammatory diseases, cancer and infection.
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http://dx.doi.org/10.1002/eji.201848070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7015662PMC
November 2019

Innate immune adaptor MyD88 deficiency prevents skin inflammation in SHARPIN-deficient mice.

Cell Death Differ 2019 03 23;26(4):741-750. Epub 2018 Jul 23.

Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA.

Mice deficient in SHANK-associated RH domain-interacting protein (SHARPIN), a component of the linear ubiquitin chain assembly complex (LUBAC), develop a spontaneous inflammatory disorder with pathologic hallmarks similar to atopic dermatitis and psoriasis in humans. Previous studies identified the crucial role of components of the TNF and IL-1 signaling pathways in the progression of disease in SHARPIN-deficient mice. However, an innate immune adaptor or sensor that relates to the disease progression has remained unknown. In this study, we found that the genetic ablation of myeloid differentiation primary response 88 (MyD88) completely rescued skin inflammation in SHARPIN-deficient (Sharpin) mice. Systemic inflammation and immune cell dysregulation were partially rescued. Fibroblasts derived from SharpinMyd88 mice failed to provide protection against TNF-induced cell death. SharpinMyd88 mice had reduced TNF production in their skin. Furthermore, depletion of the microbiota through the oral administration of antibiotics (ABX) partially rescued both the skin inflammation and systemic inflammation, demonstrating a role for the gut microbiota in SHARPIN-deficient mice. Our findings suggest a detrimental role for the innate immune adaptor MyD88 in instigating skin inflammation in Sharpin mice.
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http://dx.doi.org/10.1038/s41418-018-0159-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6460383PMC
March 2019

IRF8 Regulates Transcription of Naips for NLRC4 Inflammasome Activation.

Cell 2018 05 22;173(4):920-933.e13. Epub 2018 Mar 22.

Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA. Electronic address:

Inflammasome activation is critical for host defenses against various microbial infections. Activation of the NLRC4 inflammasome requires detection of flagellin or type III secretion system (T3SS) components by NLR family apoptosis inhibitory proteins (NAIPs); yet how this pathway is regulated is unknown. Here, we found that interferon regulatory factor 8 (IRF8) is required for optimal activation of the NLRC4 inflammasome in bone-marrow-derived macrophages infected with Salmonella Typhimurium, Burkholderia thailandensis, or Pseudomonas aeruginosa but is dispensable for activation of the canonical and non-canonical NLRP3, AIM2, and Pyrin inflammasomes. IRF8 governs the transcription of Naips to allow detection of flagellin or T3SS proteins to mediate NLRC4 inflammasome activation. Furthermore, we found that IRF8 confers protection against bacterial infection in vivo, owing to its role in inflammasome-dependent cytokine production and pyroptosis. Altogether, our findings suggest that IRF8 is a critical regulator of NAIPs and NLRC4 inflammasome activation for defense against bacterial infection.
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http://dx.doi.org/10.1016/j.cell.2018.02.055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5935577PMC
May 2018

Caulerpa okamurae extract inhibits adipogenesis in 3T3-L1 adipocytes and prevents high-fat diet-induced obesity in C57BL/6 mice.

Nutr Res 2017 Nov 14;47:44-52. Epub 2017 Sep 14.

Department of Oriental Medicine Resources and Institute of Korean Medicine Industry, Mokpo National University, Jeonnam 58554, Republic of Korea. Electronic address:

Seaweeds are considered a potential source of antiobesity agents. Because Caulerpa, a seaweed, has been consumed for food in Japan, China, South Korea, and Australia, we hypothesized that Caulerpa okamurae may have antiobesity effects in an animal model of high-fat diet (HFD)-induced obesity in C57BL/6 mice. Herein, we found that the ethanolic extract of C okamurae (COE) significantly inhibited lipid accumulation and reduced the expression of the master regulator of adipogenesis, peroxisome proliferator-activated receptor-γ, sterol regulatory element binding protein-1c, and CCAAT/enhancer-binding protein-α in 3T3-L1 adipocytes. Moreover, COE significantly decreased body weight, fat weight, and liver weight in HFD-fed mice. This effect is comparable to that of positive control Garcinia cambogia extract, which has been approved by the Korean Food and Drug Administration as a weight loss food supplement in South Korea. Similarly, markers of weight gain such as free fatty acids, triglyceride, total cholesterol, glucose, and insulin in the plasma and free fatty acid, triglyceride, total cholesterol, and total lipid in the liver are significantly reduced in COE-treated HFD-fed mice. We found significantly reduced peroxisome proliferator-activated receptor-γ, CCAAT/enhancer-binding protein-α, fatty acid synthase, sterol regulatory element binding protein-1c, cluster of differentiation 36, and acetyl-CoA synthetase in the adipose tissue of COE-treated HFD-fed mice. In conclusion, our results demonstrated that COE is effective in preventing body weight gain and fat accumulation and reduces plasma and hepatic lipid profiles. Together, these findings suggest that C okamurae may be used as a possible treatment option for the management of obesity and associated metabolic disorders.
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http://dx.doi.org/10.1016/j.nutres.2017.09.002DOI Listing
November 2017

Anti-nociceptive and anti-inflammatory effects of the methanolic extract of stem.

Avicenna J Phytomed 2017 Jul-Aug;7(4):366-375

Department of Oriental Medicine Resources Mokpo National University, Muan-gun, Jeonnam 58554, Republic of Korea.

Objective: () Raf. has been used for the prevention and treatment of rheumatoid arthritis, inflammation, and cancer. Our study was designed to unveil the anti-nociceptive and anti-inflammatory effects of the methanolic extract of Raf stem (OHS).

Materials And Methods: The anti-nociceptive effect was measured by hot plate, acetic acid-induced writhing, and tail flick assays in mice and rats. Moreover, the anti-inflammatory effect was measured by vascular permeability and carrageenan and serotonin-induced paw edema tests in rats. Furthermore, anti-inflammatory effect was also measured using macrophage-like LPS-induced RAW 264.7 cells.

Results: OHS extract inhibited acetic acid-induced writhing (p<0.0001), and delayed the reaction time of mice to the hot plate-induced thermal stimulation (p<0.0001) and tail flick tests (p<0.05). OHS extract attenuated the carrageenan and serotonin-induced paw edema in rats (p<0.001). Similarly, OHS extract significantly decreased Evans blue concentration in acetic acid induced vascular permeability test (p<0.0001), revealing its strong anti-inflammatory effect. Finally, among four different fractions of OHS extract, -butanol fraction strongly decreased NO production (p<0.0001) and iNOS expression in LPS-induced RAW 264.7 cells.

Conclusion: Our results suggest that the methanolic extract of stem can be used to develop a therapeutic or supportive drug and/or functional food against pain and inflammation related diseases.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5580874PMC
September 2017

Anti-inflammatory effects and mechanisms of Hizikia fusiformis via multicellular signaling pathways in lipopolysaccharide-induced RAW 264.7 cells.

Pak J Pharm Sci 2017 Jan;30(1):43-48

Department of Oriental Medicine Resources and Institute of Korean Medicine Industry, Mokpo National University, Jeonnam, Republic of Korea / Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, USA.

In this study, we investigated the anti-inflammatory effects and mechanisms of Hizikia fusiformis (HF) extracts in lipopolysaccharide (LPS)-induced RAW 264.7 cells. We extracted HF using solvent and sub-critical water techniques. In results, HF extracts inhibited nitric oxide (NO) production in cell-free and LPS-stimulated RAW 264.7 cells. HF210 (extract prepared with sub critical water at 210oC) was most effective. The HF210 extract dose-dependently inhibited inducible nitric oxide synthase expression (iNOS) and nuclear factor kappa (NF-B) p65 translocation from cytosol to the nucleus. Furthermore, HF210 extract dose-dependently inhibited the phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), Jun N-terminal kinase (JNK), and signal transducers and activators of transcription (STAT)-1in LPS-induced RAW 264.7 cells. Thus, our results suggest that anti-inflammatory effects of HF210 extract showed a noticeable distinction by regulation of multiple signaling pathways in LPS-induced RAW 264.7 cells.
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January 2017

inhibits protein-tyrosine phosphatase 1B and protects pancreatic beta cell via its insulin mimetic effect.

Food Sci Biotechnol 2017 30;26(2):495-499. Epub 2017 Apr 30.

1Department of Oriental Medicine Resources and Institute of Korean Medicine Industry, Mokpo National University, Muan, Jeonnam, 58554 Korea.

The aim of this study was to determine whether extract (CLE) can protect pancreatic beta cells and enhance insulin signaling in adipocytes. We measured the protein tyrosine phosphatase (PTP)-1B inhibitory effect of CLE using an in-vitro enzyme assay. Proteins involved in the pancreatic beta-cell death and insulin signaling were measured by western blotting. Oil-red O staining was used to measure the insulin mimetic effect of CLE. CLE strongly inhibited the PTP1B enzyme. In rat insulinoma (RIN)-m5F cells, CLE decreased the activation of extracellular regulated kinase (ERK)-1/2, P38 mitogen activated protein kinase (P38), c-Jun NH2-terminal kinase (JNK), and nuclear factor kappa-light-chain-enhancer of the activated B cells (NF-κB). Furthermore, CLE showed insulin-mimetic effect and enhanced the activation of insulin-signaling molecules including IRS, AKT, PI3K, and GSK-3β in 3T3-L1 adipocytes. Our results suggested that CLE-inhibited PTP1B, protected the pancreatic beta cells, and enhanced insulin sensitization in the adipocytes.
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http://dx.doi.org/10.1007/s10068-017-0068-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6049453PMC
April 2017

Korean Chungtaejeon tea extract attenuates body weight gain in C57BL/6J-Lep ob/ob mice and regulates adipogenesis and lipolysis in 3T3-L1 adipocytes.

J Integr Med 2017 Jan;15(1):56-63

Department of Oriental Medicine Resources and Institute of Korean Medicine Industry, Mokpo National University, Jeonnam 534-729, Republic of Korea.

Objective: Traditional Korean Chungtaejeon (CTJ) tea is a type of fermented tea, which has received increasing attention in recent years because of its purported health benefits. The present study was designed to investigate the effect and mechanism of CTJ tea extract on body weight gain using C57BL/6J-Lep ob/ob mice and 3T3-L1 adipocytes, respectively.

Methods: The effects of CTJ on cell viability, lipid accumulation, and expression of protein and mRNA were measured in 3T3-L1 adipocytes by using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, oil red O staining, Western blotting, and reverse transcriptase-polymerase chain reaction analyses. C57BL6J-Lep ob/ob mice were administered with CTJ (200 or 400 mg/kg body weight) for ten weeks. Then, body weight, food intake, total cholesterol, and triglyceride were measured in ob/ob mice.

Results: CTJ tea extract treated at 250 μg/mL (CTJ250) significantly suppressed lipid accumulation in the differentiated 3T3-L1 adipocytes. Likewise, CTJ250 significantly decreased the protein expression of peroxisome proliferator-activated receptorγ (PPARγ), CCAAT/enhancer-binding protein α, and adipocyte lipid-binding protein, and regulated the mRNA expression of PPARγ, sterol regulatory element-binding protein-1c gene, fatty acid synthase, adipocyte lipid-binding protein, hormone-sensitive lipase, carnitine palmitoyl transferase 1, cluster of differentiation 36, and acetyl-CoA carboxylase in the differentiated 3T3-L1 adipocytes. Mice administered with CTJ showed dose-dependent decrease in body weight gain, starting from week 4 of the experiment. CTJ tea extract administered at 400 mg/kg body weight significantly decreased fat mass, food efficacy ratio, and levels of plasma triglyceride and total cholesterol.

Conclusion: CTJ attenuated weight gain in ob/ob mice and regulated the activity of the molecules involved in adipogenesis and lipolysis in 3T3-L1 adipocytes. CTJ is a potentially valuable herbal therapy for the prevention of obesity and/or obesity-related disorders.
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http://dx.doi.org/10.1016/S2095-4964(17)60321-2DOI Listing
January 2017

IL-1β and Caspase-1 Drive Autoinflammatory Disease Independently of IL-1α or Caspase-8 in a Mouse Model of Familial Mediterranean Fever.

Am J Pathol 2017 Feb 18;187(2):236-244. Epub 2016 Dec 18.

Department of Immunology, St. Jude Children's Research Hospital, Memphis, Tennessee. Electronic address:

Mutations in the gene encoding pyrin are associated with autoinflammatory disorder Familial Mediterranean Fever (FMF). A FMF-knock-in mouse strain that expresses chimeric pyrin protein with a V726A mutation (Mefv) was generated to model human FMF. This mouse strain shows an autoinflammatory disorder that is prevented by genetic deletion of IL-1 (IL-1) receptor or apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC). ASC-mediated cell death leads to the release of IL-1α and IL-1β, both of which signal through IL-1 receptor. Furthermore, caspase-1 and caspase-8 can interact with ASC to mediate secretion of IL-1 cytokines. The specific IL-1 cytokine instigating development of FMF and the enzymatic caspase involved in its secretion currently are unknown. In this study, we show that the autoinflammation observed in Mefv mice is mediated specifically by IL-1β and not IL-1α. Furthermore, the disorder is dependent on the caspase-1-ASC axis, whereas caspase-8 is dispensable. Concurrently, aberrant IL-1β release by Mefv monocytes in response to stimulation with lipopolysaccharide also is dependent on the caspase-1-ASC axis. In conclusion, our studies have uncovered a specific role for caspase-1-mediated IL-1β release in the manifestation of FMF.
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http://dx.doi.org/10.1016/j.ajpath.2016.10.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5389372PMC
February 2017

NLRC3 is an inhibitory sensor of PI3K-mTOR pathways in cancer.

Nature 2016 Dec 12;540(7634):583-587. Epub 2016 Dec 12.

Department of Immunology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.

NLRs (nucleotide-binding domain and leucine-rich repeats) belong to a large family of cytoplasmic sensors that regulate an extraordinarily diverse range of biological functions. One of these functions is to contribute to immunity against infectious diseases, but dysregulation of their functional activity leads to the development of inflammatory and autoimmune diseases. Cytoplasmic innate immune sensors, including NLRs, are central regulators of intestinal homeostasis. NLRC3 (also known as CLR16.2 or NOD3) is a poorly characterized member of the NLR family and was identified in a genomic screen for genes encoding proteins bearing leucine-rich repeats (LRRs) and nucleotide-binding domains. Expression of NLRC3 is drastically reduced in the tumour tissue of patients with colorectal cancer compared to healthy tissues, highlighting an undefined potential function for this sensor in the development of cancer. Here we show that mice lacking NLRC3 are hyper-susceptible to colitis and colorectal tumorigenesis. The effect of NLRC3 is most dominant in enterocytes, in which it suppresses activation of the mTOR signalling pathways and inhibits cellular proliferation and stem-cell-derived organoid formation. NLRC3 associates with PI3Ks and blocks activation of the PI3K-dependent kinase AKT following binding of growth factor receptors or Toll-like receptor 4. These findings reveal a key role for NLRC3 as an inhibitor of the mTOR pathways, mediating protection against colorectal cancer.
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http://dx.doi.org/10.1038/nature20597DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5468516PMC
December 2016

Distinct role of IL-1β in instigating disease in Sharpin mice.

Sci Rep 2016 11 28;6:36634. Epub 2016 Nov 28.

Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA.

Mice deficient in SHARPIN (Sharpin mice), a member of linear ubiquitin chain assembly complex (LUBAC), develop severe dermatitis associated with systemic inflammation. Previous studies have demonstrated that components of the TNF-signaling pathway, NLRP3 inflammasome and IL-1R signaling are required to provoke skin inflammation in Sharpin mice. However, whether IL-1α or IL-1β, both of which signals through IL-1R, instigates skin inflammation and systemic disease is not known. Here, we have performed extensive cellular analysis of pre-diseased and diseased Sharpin mice and demonstrated that cellular dysregulation precedes skin inflammation. Furthermore, we demonstrate a specific role for IL-1β, but not IL-1α, in instigating dermatitis in Sharpin mice. Our results altogether demonstrate distinct roles of SHARPIN in initiating systemic inflammation and dermatitis. Furthermore, skin inflammation in Sharpin mice is specifically modulated by IL-1β, highlighting the importance of specific targeted therapies in the IL-1 signaling blockade.
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http://dx.doi.org/10.1038/srep36634DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5125001PMC
November 2016

A Comprehensive Review on Chemical Profiling of Nelumbo Nucifera: Potential for Drug Development.

Phytother Res 2017 Jan 26;31(1):3-26. Epub 2016 Sep 26.

Department of Oriental Medicine Resources, College of Natural Sciences, Mokpo National University, 61, Muan-gun, Jeonnam, 534-729, Korea.

Nelumbo nucifera, also known as sacred lotus, has primarily been used as food throughout the Asian continent, and its medicinal values have been described in Ayurvedic and Traditional Chinese Medicine. The purpose of this study is to systematically characterize the chemical profiling and pharmacological activities of N. nucifera. Herein, we critically reviewed and analysed the phytochemical and pharmacological reports of N. nucifera. Our search for the keyword 'Nelumbo nucifera pharmacology' in all databases reported in Web of Science yielded 373 results excluding reviews and abstracts in document types. Two hundred and forty-three spectrum natural compounds from different parts of N. nucifera belonging to diverse chemical groups, including alkaloids, flavonoids, glycosides, terpenoids, steroids, fatty acids, proteins, minerals, and vitamins have been reported. In addition, distinct pharmacological activities, mainly against cancer, microbial infection, diabetes, inflammation, atherosclerosis, and obesity, have been associated with crude extracts, fractions, and isolated compounds. This review highlights potential use of neferine, liensinine, isoliensinine, and nuciferine in clinical trials. In depth, mechanism of the potential chemical entities from N. nucifera via structure activity relationship needs to be explored to guarantee the stability and safety for the clinical use. Copyright © 2016 John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/ptr.5732DOI Listing
January 2017

Alkaloid rich fraction from Nelumbo nucifera targets VSMC proliferation and migration to suppress restenosis in balloon-injured rat carotid artery.

Atherosclerosis 2016 May 21;248:179-89. Epub 2016 Mar 21.

Department of Oriental Medicine Resources, Mokpo National University, Muan-gun, Jeonnam 534-729, Republic of Korea. Electronic address:

Aims: Restenosis- an adverse consequence following angioplasty, and atherosclerosis are characterized by abnormal vascular smooth muscle cell (VSMC) proliferation and migration leading to neo-intima formation. In the present study, we investigated the inhibitory effects of alkaloid rich fraction (ARF) from Nelumbo nucifera and isolated compound neferine on platelet-derived growth factor (PDGF-BB) induced VSMC proliferation and migration in vitro and neo-intima formation in a rat carotid artery injury model.

Methods: PDGF-BB induced VSMC proliferation and migration was assessed using colorimetric assay and modified Boyden chamber method respectively. Gene expression of cell cycle associated molecules was determined by reverse transcription-polymerase chain reaction (RT-PCR). The signaling molecules such as PDGF-Rβ, extracellular regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK), P38, metalloproteinase (MMP)-9 and nuclear factor-kappa B (NF-κB) were determined by western blot analysis. Stress fiber formation was evaluated using immunofluorescence microscopy. The rat carotid artery balloon injury model was performed to assess the effect of ARF on neo-intima formation.

Results: ARF possessed the strongest anti-oxidant activities. The anti-proliferative activity of both ARF and neferine was due to suppression of cyclin D1, cyclin E and cyclin-dependent kinase (Cdk) gene expression. Moreover, ARF and neferine inhibited PDGF-Rβ, ERK1/2, JNK and P38 activations and NF-κB translocation. Also, ARF and neferine inhibited VSMC migration by inhibiting MMP-9 activity without affecting cytoskeleton remodeling. In a rat carotid artery injury model, ARF inhibited neo-intima formation.

Conclusion: Our results indicate that ARF targets VSMC proliferation and migration to attenuate neo-intima formation by inhibition of PDGF-Rβ mediated signaling.
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http://dx.doi.org/10.1016/j.atherosclerosis.2016.03.020DOI Listing
May 2016

Nelumbo Nucifera leaf extract attenuated pancreatic β-cells toxicity induced by interleukin-1β and interferon-γ, and increased insulin secrection of pancreatic β-cells in streptozotocin-induced diabetic rats.

J Tradit Chin Med 2016 Feb;36(1):71-7

Objective: To evaluate the effect of Nelumbo Nucifera leaf water extract (NNLE) on insulinoma (RIN) cells induced by interleukin-1β (IL-1β) and interferon-g (IFN-γ), and injured pancreatic β-cells induced by Streptozotocin (STZ) in rats.

Methods: The anti-oxidative effects of NNLE were assessed using 1,1-diphenyl-2-picryl hydrazyl (DPPH) and nitric oxide (NO) scavenging assays. The inhibitory effect of NNLE on α-glucosidase and DPP (dipeptidyl peptidase)-IV was measured in vitro. Pancreatic β-cell protective and insulin secretory effects were assessed, using IL-1β and IFN-γ-induced rat RIN cells. STZ-induced diabetic rats were treated with 50, 100, and 400 mg/kg NNLE for 4 weeks. The effects of NNLE on blood glucose (BG), body weight (BW), and lipid profiles were measured.

Results: NNLE inhibited DPPH, NO, α-glucosidase, and DPP-IV which were directly linked to the function of β-cells. Furthermore, NNLE protected RIN cells from toxicity induced by IL-1β and IFN-γ, decreased NO production, and increased insulin secretion. NNLE caused a significant reduction in blood glucose, triglyceride (TG), total cholesterol (TC), blood urea nitrogen (BUN), and creatinine in STZ-induced diabetic rats. Furthermore, it significantly decreased BW loss in STZ-induced diabetic rats.

Conclusion: Our results suggest that NNLE reduced the toxicity in insulinoma cells and increased insulin secretion in pancreatic β-cells in STZ-induced diabetic rats.
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http://dx.doi.org/10.1016/s0254-6272(16)30011-5DOI Listing
February 2016

Anti-Obesity Effects of the Mixture of Eriobotrya japonica and Nelumbo nucifera in Adipocytes and High-Fat Diet-Induced Obese Mice.

Am J Chin Med 2015 1;43(4):681-94. Epub 2015 Jul 1.

Department of Oriental Medicine Resources and Institute of Korean Medicine Industry, Mokpo National University, Jeonnam 534-729, Republic of Korea.

The present study is to evaluate the anti-obesity effects of Eriobotrya japonica (EJ), Nelumbo nucifera (NN), and their mixture (MIX, 1:1 ratio) in 3T3-L1 adipocytes and high-fat diet-induced obese mice. The treatment of EJ, NN, and MIX in 3T3-L1 adipocytes effectively inhibited lipid accumulation, significantly decreased expression of peroxisome proliferator-activated receptor gamma (PPARγ), sterol regulatory element binding protein (SREBP1c), and adipocyte lipid-binding protein (aP2), and significantly increased phosphorylation of AMP-activated protein kinase (AMPK). Moreover, oral treatment of MIX showed stronger effects than individual treatment. C57BL/6J mice (6 week old) were divided into two groups; low fat diet (LFD) containing 10% calories from fat and high fat diet (HFD) containing 60% calories from fat. The HFD groups were further divided into five subgroups; treated with distilled water (HFD), treated with 400 mg/kg EJ (EJ400), treated with 400 mg/kg NN (NN400), treated with 200 mg/kg MIX (MIX200), and treated with 400 mg/kg MIX (MIX400) during 13 weeks. In our results, the administration of EJ, NN, and MIX significantly decreased body weight (BW), fat weight, liver weight, hepatic triglyceride (TG) and total cholesterol (TC), lipid droplets in the liver, food efficacy ratio, and the plasma TG, TC, glucose, insulin, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in a dose-dependent manner, and MIX treatment showed stronger effect than their individual treatments. Similarly, MIX treatment decreased the expression of PPARγ, SREBP-1c, FAS, and ACC more strongly in the adipose tissue than single treatments. In conclusion, the MIX of EJ and NN extract may strongly regulate BW gain than EJ or NN alone, and its anti-obesity effect is associated with the control of lipid metabolism, including adipogenesis and lipogenesis.
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http://dx.doi.org/10.1142/S0192415X15500421DOI Listing
April 2016

Caulerpa lentillifera extract ameliorates insulin resistance and regulates glucose metabolism in C57BL/KsJ-db/db mice via PI3K/AKT signaling pathway in myocytes.

J Transl Med 2015 Feb 15;13:62. Epub 2015 Feb 15.

Department of Oriental Medicine Resources and Institute of Korean Medicine Industry, Mokpo National University, 1666 Youngsan-ro, Muan-gun, 534-729, Jeonnam, Republic of Korea.

Background: Glucose homeostasis is distorted by defects of the PI3K/AKT and AMPK pathways in insulin-sensitive tissues, allowing the accumulation of glucose in the blood. The purpose of this study was to assess the effects and mechanisms by which ethanol extract of Caulerpa lentillifera (CLE) regulates glucose metabolism in C57BL/KsJ-db/db (db/db) mice.

Methods: Mice were administered CLE (250 or 500 mg/kg BW) or rosiglitazone (RSG, 10 mg/kg BW) for 6 weeks. Then, oral glucose tolerance test (OGTT) and intraperitoneal insulin tolerance test (IPITT) were performed, and blood glucose was measured in db/db mice. Levels of insulin and insulin resistance factors in plasma, glycogen content in the liver, and IRS, PI3K, AKT, and GLUT4 expressions in skeletal muscles were measured in db/db mice. Glucose uptake and insulin signaling molecules were measured in L6 myocytes, using fluorometry and Western blotting.

Results: CLE significantly decreased fasting blood glucose, glucose level in OGTT and IPITT, plasma insulin, homeostatic model assessment-insulin resistant (HOMA-IR), TNF-α, IL-6, FFA, TG and TC levels, and hepatic glycogen content in db/db mice. CLE significantly increased the activation of IRS, AKT, PI3K, and GLUT4, which are the key effector molecules of the PI3K/AKT pathway in L6 myocytes and the skeletal muscles of db/db mice. The enhanced glucose uptake by CLE was abolished by treatment with a PI3K inhibitor (LY294002), but not by an AMPK inhibitor (compound C) in L6 myocytes. CLE regulated glucose uptake and homeostasis via the PI3K/AKT pathway in myocytes and db/db mice, respectively.

Conclusion: Our results suggest that CLE could be a potential candidate for the prevention of diabetes.
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http://dx.doi.org/10.1186/s12967-015-0412-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4350654PMC
February 2015

Lespedeza davurica (Lax.) Schindl. extract protects against cytokine-induced β-cell damage and streptozotocin-induced diabetes.

Biomed Res Int 2015 22;2015:169256. Epub 2015 Feb 22.

Department of Oriental Medicine Resources and Institute of Korean Medicine Industry, College of Natural Science, Mokpo National University, 1666 Youngsan-ro, Muan-gun, Jeonnam 534-729, Republic of Korea ; Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR 97331, USA.

Lespedeza has been used for the management of diabetes in folklore medicine. The purpose of this study is to investigate the protective effects of the methanol extract of Lespedeza davurica (LD) on cytokine-induced β-cell damage and streptozotocin- (STZ-) induced diabetes. RINm5F cells were treated with interleukin- (IL-) 1β and interferon- (IFN-) γ to induce pancreatic β-cell damage. The exposure of LD extract significantly decreased cell death, nitric oxide (NO) production, nitric oxide synthase (iNOS) expression, and nucleus factor-kappa B (NF-κB) p65 activation. Antidiabetic effects of LD extract were observed by oral glucose tolerance test (OGTT) in normal rats and by checking the biochemical, physiological, and histopathological parameters in STZ-induced diabetic rats. In OGTT, glucose clearance levels improved by oral treatment of LD extract. The water intake, urine volume, blood glucose, and serum TG, TC, TBARS, and DPP-IV levels were significantly decreased, and liver glycogen content was significantly increased by treatment of LD extract (250 mg/kg BW) in STZ-induced diabetic rats. Also, insulin immunoreactivity of the pancreases was increased in LD extract administrated rats compared with diabetic control rats. These results indicate that LD extract may protect pancreatic β-cell damage and regulate the blood glucose in STZ-induced diabetic rats.
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http://dx.doi.org/10.1155/2015/169256DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4352516PMC
January 2016

Anti-diabetic effects of Caulerpa lentillifera: stimulation of insulin secretion in pancreatic β-cells and enhancement of glucose uptake in adipocytes.

Asian Pac J Trop Biomed 2014 Jul;4(7):575-80

Department of Oriental Medicine Resources and Institute of Korean Medicine Industry, Mokpo National University, Jeonnam 534-729, Republic of Korea.

Objective: To evaluate anti-diabetic effect of Caulerpa lentillifera (C. lentillifera).

Methods: The inhibitory effect of C. lentillifera extract on dipeptidyl peptidase-IV and α-glucosidase enzyme was measured in a cell free system. Then, interleukin-1β and interferon-γ induced cell death and insulin secretion were measured in rat insulinoma (RIN) cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and ELISA kit, respectively. Glucose uptake and glucose transporter expression were measured by fluorometry and western blotting, using 3T3-L1 adipocytes.

Results: C. lentillifera extract significantly decreased dipeptidyl peptidase-IV and α-glucosidase enzyme activities, and effectively inhibited cell death and iNOS expression in interleukin-1β and interferon-γ induced RIN cells. Furthermore, C. lentillifera extract significantly enhanced insulin secretion in RIN cells and glucose transporter expression and glucose uptake in 3T3-L1 adipocytes.

Conclusions: Thus, our results suggest that C. lentillifera could be used as a potential anti-diabetic agent.
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http://dx.doi.org/10.12980/APJTB.4.2014APJTB-2014-0091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4032834PMC
July 2014