Emodin Ameliorates LPS-Induced Acute Lung Injury,Involving the Inactivation of NF-κB in Mice

Acute lung injury (ALI) and its severe manifestation of acute respiratory distress syndrome (ARDS) are well-known illnesses. Uncontrolled and self-amplified pulmonary inflammation lies at the center of the pathology of this disease. Emodin, the bio-active coxund of herb Radix rhizoma Rhei, shows potent anti-inflammatory properties through inactivation of nuclear factor-κB (NF-κB). The aim of this study was to evaluate the effect of emodin on lipopolysaccharide (LPS)-induced ALI in mice, and its potential bio-mechanism. In our study, BALB/c mice were stimulated with LPS to induce ALI. After 72 h of LPS stimulation, pulmonary pathological changes, lung injury scores, pulmonary edema, myeloperoxidase (MPO) activity, total cells, neutrophils, macrophages, TNF-α, IL-6 and IL-1β in bronchoalveolar lavage fluid (BALF), and MCP-1 and E-selectin expression were notably attenuated by emodin in mice. Meanwhile, our data also revealed that emodin significantly inhibited the LPS-enhanced the phosphorylation of NF-κB p65 and NF-κB p65 DNA binding activity in lung. Our data indicates that emodin potently inhibits LPS-induced pulmonary inflammation, pulmonary edema and MCP-1 and E-selectin expression, and that these effects were very likely mediated by inactivation of NF-κB in mice. These results suggest a therapeutic potential of emodin as an anti-inflammatory agent for ALI/ARDS treatment.     

Glucagon Like Peptide-1 (GLP-1) Modulates OVA-Induced Airway Inflammation and Mucus Secretion Involving a Protein Kinase A (PKA)-Dependent Nuclear Factor-κB (NF-κB) Signaling Pathway in Mice

Asthma is a common chronic pulmonary inflammatory disease, featured with mucus hyper-secretion in the airway. Recent studies found that glucagon like peptide-1 (GLP-1) analogs, including liraglutide and exenatide, possessed a potent anti-inflammatory property through a protein kinase A (PKA)-dependent signaling pathway. Therefore, the aim of current study was to investigate the value of GLP-1 analog therapy liraglutide in airway inflammation and mucus secretion in a murine model of ovalbumin (OVA)-induced asthma, and its underlying molecular mechanism. In our study, BALB/c mice were sensitized and challenged by OVA to induce chronic asthma. Pathological alterations, the number of cells and the content of inflammatory mediators in bronchoalveolar lavage fluid (BALF), and mucus secretion were observed and measured. In addition, the mRNA and protein expression of E-selectin and MUC5AC were analyzed by qPCR and Western blotting. Then, the phosphorylation of PKA and nuclear factor-κB (NF-κB) p65 were also measured by Western blotting. Further, NF-κB p65 DNA binding activity was detected by ELISA. OVA-induced airway inflammation, airway mucus hyper-secretion, the up-regulation of E-selectin and MUC5AC were remarkably inhibited by GLP-1 in mice (all p < 0.01). Then, we also found that OVA-reduced phosphorylation of PKA, and OVA-enhanced NF-κB p65 activation and NF-κB p65 DNA binding activity were markedly improved by GLP-1 (all p < 0.01). Furthermore, our data also figured out that these effects of GLP-1 were largely abrogated by the PKA inhibitor H-89 (all p < 0.01). Taken together, our results suggest that OVA-induced asthma were potently ameliorated by GLP-1 possibly through a PKA-dependent inactivation of NF-κB in mice, indicating that GLP-1 analogs may be considered an effective and safe drug for the potential treatment of asthma in the future.     

Ultrasensitive Imaging of Ca2+ Dynamics in Pancreatic Acinar Cells of Yellow Cameleon-Nano Transgenic Mice

Yellow Cameleons are genetically encoded Ca²⁺ indicators in which cyan and yellow fluorescent proteins and calmodulin work together as a fluorescence (Förster) resonance energy transfer Ca²⁺-sensor probe. To achieve ultrasensitive Ca²⁺ imaging for low resting Ca²⁺ or small Ca²⁺ transients in various organs, we generated a transgenic mouse line expressing the highest-sensitive genetically encoded Ca²⁺ indicator (Yellow Cameleon-Nano 15) in the whole body. We then focused on the mechanism of exocytotic events mediated by intracellular Ca²⁺ signaling in acinar cells of the mice with an agonist and observed them by two-photon excitation microscopy. In the results, two-photon excitation imaging of Yellow Cameleon-Nano 15 successfully visualized intracellular Ca²⁺ concentration under stimulation with the agonist at nanomolar levels. This is the first demonstration for application of genetically encoded Ca²⁺ indicators to pancreatic acinar cells. We also simultaneously observed exocytotic events and an intracellular Ca²⁺ concentration under in vivo condition. Yellow Cameleon-Nano 15 mice are healthy and no significant deteriorative effect was observed on physiological response regarding the pancreatic acinar cells. The dynamic range of 165% was calculated from R_max and R_min values under in vivo condition. The mice will be useful for ultrasensitive Ca²⁺ imaging in vivo.     

Protective Role of Andrographolide in Bleomycin-Induced Pulmonary Fibrosis in Mice

Idiopathic pulmonary fibrosis (IPF) is a chronic devastating disease with poor prognosis. Multiple pathological processes, including inflammation, epithelial mesenchymal transition (EMT), apoptosis, and oxidative stress, are involved in the pathogenesis of IPF. Recent findings suggested that nuclear factor-κB (NF-κB) is constitutively activated in IPF and acts as a central regulator in the pathogenesis of IPF. The aim of our study was to reveal the value of andrographolide on bleomycin-induced inflammation and fibrosis in mice. The indicated dosages of andrographolide were administered in mice with bleomycin-induced pulmonary fibrosis. On day 21, cell counts of total cells, macrophages, neutrophils and lymphocytes, alone with TNF-α in bronchoalveolar lavage fluid (BALF) were measured. HE staining and Masson’s trichrome (MT) staining were used to observe the histological alterations of lungs. The Ashcroft score and hydroxyproline content of lungs were also measured. TGF-β1 and α-SMA mRNA and protein were analyzed. Activation of NF-κB was determined by western blotting and electrophoretic mobility shift assay (EMSA). On day 21 after bleomycin stimulation, andrographolide dose-dependently inhibited the inflammatory cells and TNF-α in BALF. Meanwhile, our data demonstrated that the Ashcroft score and hydroxyproline content of the bleomycin-stimulated lung were reduced by andrographolide administration. Furthermore, andrographloide suppressed TGF-β1 and α-SMA mRNA and protein expression in bleomycin-induced pulmonary fibrosis. Meanwhile, andrographolide significantly dose-dependently inhibited the ratio of phospho-NF-κB p65/total NF-κB p65 and NF-κB p65 DNA binding activities. Our findings indicate that andrographolide compromised bleomycin-induced pulmonary inflammation and fibrosis possibly through inactivation of NF-κB. Andrographolide holds promise as a novel drug to treat the devastating disease of pulmonary fibrosis.     

Modulation of Ca Signals by Epigallocatechin-3-gallate(EGCG) in Cultured Rat Hippocampal Neurons

Green tea has been receiving considerable attention as a possible neuroprotective agent against neurodegenerative disease. Epigallocatechin-3-gallate (EGCG) is the major compound of green tea. Calcium signaling has profound effects on almost all aspects of neuronal function. Using digital calcium imaging and patch-clamp technique, we determined the effects of EGCG on Ca(2+) signals in hippocampal neurons. The results indicated that EGCG caused a dose-dependent increase in intracellular Ca(2+) ([Ca(2+)](i)). This [Ca(2+)](i) increase was blocked by depleting intracellular Ca(2+) stores with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin and cyclopiazonic acid. Furthermore, EGCG-stimulated increase in [Ca(2+)](i) was abolished following treatment with a PLC inhibitor. However, EGCG inhibited high-voltage activated Ca(2+) currents (I(HVA)) and NMDA-induced inward currents (I(NMDA)). These data suggest that EGCG triggers a cascade of events: it activates phospholipase C (PLC), mobilizes intracellular Ca(2+) stores, raises the cytosolic Ca(2+) levels, and inhibits the VGCC and NMDA receptors-mediated Ca(2+) influx through a process that remains to be determined.     

Renal Protective Effects of Low Molecular Weight of Inonotus obliquus Polysaccharide (LIOP) on HFD/STZ-Induced Nephropathy in Mice

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease in diabetes mellitus. Oxidative stress, insulin resistance and pro-inflammatory cytokines have been shown to play an important role in pathogeneses of renal damage on type 2 diabetes mellitus (DM). Inonotus obliquus (IO) is a white rot fungus that belongs to the family Hymenochaetaceae; it has been used as an edible mushroom and exhibits many biological activities including anti-tumor, anti-oxidant, anti-inflammatory and anti-hyperglycemic properties. Especially the water-soluble Inonotus obliquus polysaccharides (IOPs) have been previously reported to significantly inhibit LPS-induced inflammatory cytokines in mice and protect from streptozotocin (STZ)-induced diabetic rats. In order to identify the nephroprotective effects of low molecular weight of IOP fraction (LIOP), from the fruiting bodies of Inonotus obliquus, high-fat diet (HFD) plus STZ-induced type 2-like diabetic nephropathy C57BL/6 mice were investigated in this study. Our data showed that eight weeks of administration of 10–100 kDa, LIOP (300 mg/kg) had progressively increased their sensitivity to glucose (less insulin tolerance), reduced triglyceride levels, elevated the HDL/LDL ratio and decreased urinary albumin/creatinine ratio(ACR) compared to the control group. By pathological and immunohistochemical examinations, it was indicated that LIOP can restore the integrity of the glomerular capsules and increase the numbers of glomerular mesangial cells, associated with decreased expression of TGF-β on renal cortex in mice. Consistently, three days of LIOP (100 μg/mL) incubation also provided protection against STZ + AGEs-induced glucotoxicity in renal tubular cells (LLC-PK1), while the levels of NF-κB and TGF-β expression significantly decreased in a dose-dependent manner. Our findings demonstrate that LIOP treatment could ameliorate glucolipotoxicity-induced renal fibrosis, possibly partly via the inhibition of NF-κB/TGF-β1 signaling pathway in diabetic nephropathy mice.     

Ghrelin Attenuates Liver Fibrosis through Regulation of TGF-β1 Expression and Autophagy

Ghrelin is a stomach-derived growth hormone secretagogue that promotes various physiological effects, including energy metabolism and amelioration of inflammation. The purpose of this study was to investigate the protective mechanism of ghrelin against liver fibrosis. Liver fibrosis was induced in C57BL/6 mice by intraperitoneal injection of CCl₄ (2.0 mL/kg of 10% CCl₄ v/v solution in peanut oil) two times per week for eight weeks. Ghrelin (10 μg/kg) was intraperitoneally injected two times per week for eight weeks. A second murine liver fibrosis model was induced by bile duct ligation (BDL) and concurrent ghrelin administration for four weeks. Hematoxylin eosin (H&E), and Masson’s trichrome were used to detect pathological changes to liver tissue. Western blotting was used to detect protein levels of transforming growth factor (TGF)-β1, phosphorylated Smad3 (p-Smad3), I-collage, α-smooth muscle actin (α-SMA), matrix metalloproteinases (MMPs) 2, tissue inhibitor of matrix metalloproteinases (TIMPs) 1, phosphorylated NF-κB (p-NF-κB), and microtubule-associated protein light chain 3 (LC3). In addition, qRT-PCR was used to detect mRNA levels of TGF-β1, I-collage, α-SMA, MMP2, TIMP1 and LC3, while levels of TGF-β1, p-Smad3, I-collage, α-SMA, and LC3 were detected immunohistochemically. Levels of aspartate aminotransferase and alanine aminotransferase were significantly decreased by ghrelin treatment. Ghrelin administration also significantly reduced the extent of pathological changes in both murine liver fibrosis models. Expression levels of I-collage and α-SMA in both models were clearly reduced by ghrelin administration. Furthermore, ghrelin treatment decreased protein expression of TGF-β1 and p-Smad3. The protein levels of NF-κB and LC3 were increased in the CCl₄- and BDL-treatment groups but were significantly reduced following ghrelin treatment. In addition, ghrelin inhibited extracellular matrix formation by decreasing NF-κB expression and maintaining the balance between MMP2 and TIMP1. Our results demonstrated that ghrelin attenuates liver fibrosis via inhibition of the TGF-β1/Smad3 and NF-κB signaling pathways, as well as autophagy suppression.