scholarly journals Tissue Inhibitor of Metalloproteinase-3 Ameliorates Diabetes-Induced Retinal Inflammation

2022 ◽  
Vol 12 ◽  
Author(s):  
Ahmed M. Abu El-Asrar ◽  
Ajmal Ahmad ◽  
Mohd Imtiaz Nawaz ◽  
Mohammad Mairaj Siddiquei ◽  
Alexandra De Zutter ◽  
...  
Keyword(s):  
Diabetic Retinopathy ◽  
Matrix Metalloproteinase ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Tissue Inhibitor ◽  
Matrix Metalloproteinase 3 ◽  
Tnf Α ◽  
Brb Breakdown

Purpose: Endogenous tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) has powerful regulatory effects on inflammation and angiogenesis. In this study, we investigated the role of TIMP-3 in regulating inflammation in the diabetic retina.Methods: Vitreous samples from patients with proliferative diabetic retinopathy (PDR) and non-diabetic patients were subjected to Western blot analysis. Streptozotocin-treated rats were used as a preclinical diabetic retinopathy (DR) model. Blood-retinal barrier (BRB) breakdown was assessed with fluorescein isothiocyanate (FITC)-conjugated dextran. Rat retinas, human retinal microvascular endothelial cells (HRMECs) and human retinal Müller glial cells were studied by Western blot analysis and ELISA. Adherence of human monocytes to HRMECs was assessed and in vitro angiogenesis assays were performed.Results: Tissue inhibitor of matrix metalloproteinase-3 in vitreous samples was largely glycosylated. Intravitreal injection of TIMP-3 attenuated diabetes-induced BRB breakdown. This effect was associated with downregulation of diabetes-induced upregulation of the p65 subunit of NF-κB, intercellular adhesion molecule-1 (ICAM-1), and vascular endothelial growth factor (VEGF), whereas phospho-ERK1/2 levels were not altered. In Müller cell cultures, TIMP-3 significantly attenuated VEGF upregulation induced by high-glucose (HG), the hypoxia mimetic agent cobalt chloride (CoCl2) and TNF-α and attenuated MCP-1 upregulation induced by CoCl2 and TNF-α, but not by HG. TIMP-3 attenuated HG-induced upregulation of phospho-ERK1/2, caspase-3 and the mature form of ADAM17, but not the levels of the p65 subunit of NF-κB and the proform of ADAM17 in Müller cells. TIMP-3 significantly downregulated TNF-α-induced upregulation of ICAM-1 and VCAM-1 in HRMECs. Accordingly, TIMP-3 significantly decreased spontaneous and TNF-α- and VEGF-induced adherence of monocytes to HRMECs. Finally, TIMP-3 significantly attenuated VEGF-induced migration, chemotaxis and proliferation of HRMECs.Conclusion:In vitro and in vivo data point to anti-inflammatory and anti-angiogenic effects of TIMP-3 and support further studies for its applications in the treatment of DR.

scholarly journals Effects of Neuromedin S on the Proliferation of Splenic Lymphocytes and the Cytokine Secretion by Pulmonary Alveolar Macrophages in Pigs in vitro

2016 ◽  
Vol 19 (3) ◽  
pp. 485-494 ◽  
Author(s):  
R. Lin ◽  
Q. Wang ◽  
B. Qi ◽  
Y. Huang ◽  
G. Yang
Keyword(s):  
Immune Response ◽  
Western Blot ◽  
Alveolar Macrophages ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Splenic Lymphocytes ◽  
Rt Pcr ◽  
Tnf Α ◽  
Pulmonary Alveolar Macrophages

Abstract Neuromedin S (NMS), a 36-amino acid neuropeptide, has been found to be involved in the regulation of the endocrine activity. It has been also detected in immune tissues in mammals, what suggests that NMS may play an important role in the regulation of immune response. The aim of this study was to demonstrate the presence of NMS receptor 1 (NMU1R) and effect of NMS in pig splenic lymphocytes (SPLs) and pulmonary alveolar macrophages (PAMs). The presence of NMU1R in pig SPLs and PAMs was respectively confirmed by reverse transcription-polymerase chain reaction (RT-PCR), western blot analysis and immunocytochemical methods. Furthermore, SPL proliferation was analyzed using the 3-(4,5)-dimethyl-thiahiazo-(-2-yl)-3,5-di-phenytetrazoliumromide (MTT) method. Additionally, the secretion of interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α) in PAMs was all measured by enzyme-linked immunosorbent assay (ELISA) kits. In the present study, the results of RT-PCR and western blot analysis revealed that NMU1R mRNA and protein were both expressed in pig SPLs and PAMs, and the immunocytochemical investigations further revealed that the positive signal of NMU1R immunoreactivity was observed in plasma membranes of both SPLs and PAMs. In the in vitro study, we found that at concentrations of 0.001-1000 nM NMS alone or combined with lipopolysaccharide or phytohemagglutinin significantly increased SPL proliferation. Application of ELISA method showed that NMS could induce the secretion of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α in PAMs. These results suggest that NMS can act as a potently positive pro-inflammatory factor and immunomodulatory agent that affects the immune response of immune cells by combining with its receptor NMU1R.

scholarly journals Mitochondrial-Targeting Antioxidant SS-31 Suppresses Airway Inflammation and Oxidative Stress Induced by Cigarette Smoke

2021 ◽  
Vol 2021 ◽  
pp. 1-16
Author(s):  
De-qing Yang ◽  
Qiu-nan Zuo ◽  
Tao Wang ◽  
Dan Xu ◽  
Liu Lian ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Western Blot ◽  
Airway Inflammation ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Mapk Signaling ◽  
Tnf Α ◽  
And Oxidative Stress

This study investigated whether the mitochondrial-targeted peptide SS-31 can protect against cigarette smoke- (CS-) induced airway inflammation and oxidative stress in vitro and in vivo. Mice were exposed to CS for 4 weeks to establish a CS-induced airway inflammation model, and those in the experimental group were pretreated with SS-31 1 h before CS exposure. Pathologic changes and oxidative stress in lung tissue, inflammatory cell counts, and proinflammatory cytokine levels in bronchoalveolar lavage fluid (BALF) were examined. The mechanistic basis for the effects of SS-31 on CS extract- (CSE-) induced airway inflammation and oxidative stress was investigated using BEAS-2B bronchial epithelial cells and by RNA sequencing and western blot analysis of lung tissues. SS-31 attenuated CS-induced inflammatory injury of the airway and reduced total cell, neutrophil, and macrophage counts and tumor necrosis factor- (TNF-) α, interleukin- (IL-) 6, and matrix metalloproteinase (MMP) 9 levels in BALF. SS-31 also attenuated CS-induced oxidative stress by decreasing malondialdehyde (MDA) and myeloperoxidase (MPO) activities and increasing that of superoxide dismutase (SOD). It also reversed CS-induced changes in the expression of mitochondrial fission protein (MFF) and optic atrophy (OPA) 1 and reduced the amount of cytochrome c released into the cytosol. Pretreatment with SS-31 normalized TNF-α, IL-6, and MMP9 expression, MDA and SOD activities, and ROS generation in CSE-treated BEAS-2B cells and reversed the changes in MFF and OPA1 expression. RNA sequencing and western blot analysis showed that SS-31 inhibited CS-induced activation of the mitogen-activated protein kinase (MAPK) signaling pathway in vitro and in vivo. Thus, SS-31 alleviates CS-induced airway inflammation and oxidative stress via modulation of mitochondrial function and regulation of MAPK signaling and thus has therapeutic potential for the treatment of airway disorders caused by smoking.

The anti-inflammatory effects of two ent-kaurane diterpenes from the stems and leaves of Gochnatia decora

2021 ◽  
Vol 25 ◽  
Author(s):  
Yanfang Liu ◽  
Weifeng Dai ◽  
Qihui Cao ◽  
Mi Zhang
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Chemical Constituents ◽  
Raw264.7 Cells ◽  
Inflammatory Factor ◽  
Tnf Α ◽  
Stems And Leaves ◽  
Anti Inflammatory

Aim and objectives: The Gochnatia decora (Kurz) A. L. Cabrera is a rare woody plant belonging to the family of Asteraceae. The bark of this plant is used in Chinese folk medicine to treat cough and pneumonia. However, the effective substance related to its efficacy remains unknown. This study aims to evaluate the potential anti-inflammatory activities of the chemicals isolated from this plant using a model of LPS-induced RAW264.7 cells. Material and Methods: Chemical constituents were isolated from the stems and leaves of G. decora by a series of chromatographic separation methods and identified by spectral analysis techniques. The model of inflammation in vitro was established by treatment of 1 µM LPS on RAW264.7 cells. The influence of tested compounds on inflammatory factor production, including NO, TNF-α, IL-1β, IL-6, IL-17, were determined by ELISA. The mechanisms involved were studied by western blot analysis. Results: Two known ent-kaurane diterpenes (1 and 2), identified as ent-17-hydroxy-15-oxokauran-19-oic acid (1) and ent-15α-hydroxy-16-kauran-19-oic acid (2), were isolated from the stems and leaves of G. decora. The bioassay showed both of them produced significant inhibition of LPS-induced release of NO, TNF-α, IL-1β, IL-6, IL-17, iNOS, and COX-2 expression. Western blot analysis showed that these same 2 chemicals blocked LPS-induced phosphorylation of NF-κB. Conclusions: Compounds 1 and 2 were obtained from the genus Gochnatia. These compounds demonstrated useful anti-inflammatory activities in the model of LPS-induced RAW264.7 cells. A potential action mechanism may be correlation of the NF-κB pathway.

Radiosensitivity of glioma to Gamma Knife treatment enhanced in vitro and in vivo by RNA interfering Ku70 that is mediated by a recombinant adenovirus

2010 ◽  
Vol 113 (Special_Supplement) ◽  
pp. 228-235 ◽  
Author(s):  
Qiang Jia ◽  
Yanhe Li ◽  
Desheng Xu ◽  
Zhenjiang Li ◽  
Zhiyuan Zhang ◽  
...  
Keyword(s):  
Western Blot ◽  
Gamma Knife ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Glioma Cells ◽  
C6 Glioma ◽  
C6 Glioma Cells ◽  
C6 Cells

Object The authors sought to evaluate modification of the radiation response of C6 glioma cells in vitro and in vivo by inhibiting the expression of Ku70. To do so they investigated the effect of gene transfer involving a recombinant replication-defective adenovirus containing Ku70 short hairpin RNA (Ad-Ku70shRNA) combined with Gamma Knife treatment (GKT). Methods First, Ad-Ku70shRNA was transfected into C6 glioma cells and the expression of Ku70 was measured using Western blot analysis. In vitro, phenotypical changes in C6 cells, including proliferation, cell cycle modification, invasion ability, and apoptosis were evaluated using the MTT (3′(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay, Western blot analysis, and cell flow cytometry. In vivo, parental C6 cells transfected with Ad-Ku70shRNA were implanted stereotactically into the right caudate nucleus in Sprague-Dawley rats. After GKS, apoptosis was analyzed using the TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling) method. The inhibitory effects on growth and invasion that were induced by expression of proliferating cell nuclear antigen and matrix metalloproteinase–9 were determined using immunohistochemical analyses. Results The expression of Ku70 was clearly inhibited in C6 cells after transfection with Ad-Ku70shRNA. In vitro following transfection, the C6 cells showed improved responses to GKT, including suppression of proliferation and invasion as well as an increased apoptosis index. In vivo following transfection of Ad-Ku70shRNA, the therapeutic efficacy of GKT in rats with C6 gliomas was greatly enhanced and survival times in these animals were prolonged. Conclusions Our data support the potential for downregulation of Ku70 expression in enhancing the radiosensitivity of gliomas. The findings of our study indicate that targeted gene therapy–mediated inactivation of Ku70 may represent a promising strategy in improving the radioresponsiveness of gliomas to GKT.

scholarly journals Punicalagin Prevents Inflammation in LPS-Induced RAW264.7 Macrophages by Inhibiting FoxO3a/Autophagy Signaling Pathway

Nutrients ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 2794 ◽  
Author(s):  
Cao ◽  
Chen ◽  
Ren ◽  
Zhang ◽  
Tan ◽  
...  
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Inflammatory Cytokines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Inflammatory Responses ◽  
Raw264.7 Macrophages ◽  
Tnf Α ◽  
Autophagy Inhibition ◽  
Pro Inflammatory Cytokines

Punicalagin, a hydrolysable tannin of pomegranate juice, exhibits multiple biological effects, including inhibiting production of pro-inflammatory cytokines in macrophages. Autophagy, an intracellular self-digestion process, has been recently shown to regulate inflammatory responses. In this study, we investigated the anti-inflammatory potential of punicalagin in lipopolysaccharide (LPS) induced RAW264.7 macrophages and uncovered the underlying mechanisms. Punicalagin significantly attenuated, in a concentration-dependent manner, LPS-induced release of NO and decreased pro-inflammatory cytokines TNF-α and IL-6 release at the highest concentration. We found that punicalagin inhibited NF-κB and MAPK activation in LPS-induced RAW264.7 macrophages. Western blot analysis revealed that punicalagin pre-treatment enhanced LC3II, p62 expression, and decreased Beclin1 expression in LPS-induced macrophages. MDC assays were used to determine the autophagic process and the results worked in concert with Western blot analysis. In addition, our observations indicated that LPS-induced releases of NO, TNF-α, and IL-6 were attenuated by treatment with autophagy inhibitor chloroquine, suggesting that autophagy inhibition participated in anti-inflammatory effect. We also found that punicalagin downregulated FoxO3a expression, resulting in autophagy inhibition. Overall these results suggested that punicalagin played an important role in the attenuation of LPS-induced inflammatory responses in RAW264.7 macrophages and that the mechanisms involved downregulation of the FoxO3a/autophagy signaling pathway.

Regulation of Rhcg, an ammonia transporter, by aldosterone in the kidney

2021 ◽  
Author(s):  
Koji Eguchi ◽  
Yuichiro Izumi ◽  
Yukiko Yasuoka ◽  
Terumasa Nakagawa ◽  
Makoto Ono ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Membrane Fraction ◽  
Potassium Level ◽  
Collecting Duct ◽  
Subcutaneous Administration ◽  
Extracellular Potassium ◽  
Membrane Expression

Rh C glycoprotein (Rhcg), an ammonia transporter, is a key molecule in urinary acid excretion and is expressed mainly in the intercalated cells (ICs) of the renal collecting duct. In the present study we investigated the role of aldosterone in the regulation of Rhcg expression. In in vivo experiments using C57BL/6J mice, Western blot analysis showed that continuous subcutaneous administration of aldosterone increased the expression of Rhcg in membrane fraction of the kidney. Supplementation of potassium inhibited the effect of aldosterone on the Rhcg. Next, mice were subjected to adrenalectomy with or without administration of aldosterone, and then ad libitum 0.14M NH4Cl containing water was given. NH4Cl load increased the expression of Rhcg in membrane fraction. Adrenalectomy decreased NH4Cl-induced Rhcg expression, which was restored by administration of aldosterone. Immunohistochemical studies revealed that NH4Cl load induced the localization of Rhcg at the apical membrane of ICs in the outer medullary collecting duct. Adrenalectomy decreased NH4Cl-induced membrane localization of Rhcg, which was restored by administration of aldosterone. For in vitro experiments, IN-IC cells, an immortalized cell line stably expressing Flag-tagged Rhcg (Rhcg-Flag), were used. Western blot analysis showed that aldosterone increased the expression of Rhcg-Flag in membrane fraction, while the increase in extracellular potassium level inhibited the effect of aldosterone. Both spironolactone and Gӧ6983, a PKC inhibitor, inhibited the expression of Rhcg-Flag in the membrane fraction. These results suggest that aldosterone regulates the membrane expression of Rhcg through the mineralocorticoid receptor and PKC pathways, which is modulated by extracellular potassium level.

Interfering With Hyaluronic Acid Metabolism Suppresses Glioma Cell Proliferation by Regulating Autophagy

2020 ◽  
Author(s):  
Tao Yan ◽  
Xin Chen ◽  
Hua Zhan ◽  
Penglei Yao ◽  
Ning Wang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Hyaluronic Acid ◽  
Tumor Microenvironment ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Glioma Cell ◽  
Glioma Cells

Abstract BackgroundThe tumor microenvironment plays an important role in tumor progression. Hyaluronic acid (HA), an important component of the extracellular matrix in the tumor microenvironment, abnormally accumulates in a variety of tumors. Whereas the role of abnormal HA metabolism in glioma remains unclear. MethodsThe expression level of hyaluronic acid (HA) was analyzed by ELISA assay and proteins such as HAS3, CD44, P62, LC3, CCND1 and CCNB1 were measured with Western blot analysis. The cell viability and proliferation were measured by MTT and KI67 immunofluorescence staining respectively. Autophagic vesicles and autophagosomes were quantified by transmission electron microscopy (TEM) and GFP-RFP-LC3 fluorescence analysis respectively. Cell cycle was analyzed by flowcytometry and Western blot analysis. Immunohistochemical (IHC) staining was used to detect expression levels of HA, Ki67, HAS3 and CD44 in human and mouse tumor tissues. Lentivirus constructed HAS3 and CD44 knockout stable glioma cells were transplanted to BALB/C nude mice for in vivo experiments. 4-Methylumbelliferone (4MU) was also used to treat glioma bearing mice for verifing its anti-tumor ability. The expression curve of HAS3, CD44 and the disease-free survival (DFS) curves for HAS3, CD44 in patients with LGG and GBM was performed based on TCGA database. ResultsAs shown in the present study, HA, hyaluronic acid synthase 3 (HAS3) and a receptor of HA named CD44 are expressed at high levels in human glioma tissues and negatively correlated with the prognosis of patients with glioma. Silencing HAS3 or blocking CD44 inhibited the proliferation of glioma cells in vitro and in vivo. The underlying mechanism was attributed to the inhibition of autophagy flux and further maintaining glioma cell cycle arrest in G1 phase. More importantly, 4-Methylumbelliferone (4-MU), a small competitive inhibitor of UDP with the ability to penetrate the blood-brain barrier (BBB), also inhibited the proliferation of glioma cells in vitro and in vivo. ConclusionApproaches that interfere with HA metabolism by altering the expression of HAS3 and CD44 and the administration of 4-MU potentially represent effective strategies for glioma treatment.

scholarly journals A39 NOVEL GAIN OF FUNCTION NON-RECEPTOR TYROSINE KINASE MUTATIONS ARE LINKED TO THE PATHOGENESIS OF VEOIBD

2020 ◽  
Vol 3 (Supplement_1) ◽  
pp. 46-48
Author(s):  
M Mehta ◽  
L Wang ◽  
C Guo ◽  
N Warner ◽  
Q Li ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Western Blot ◽  
Receptor Tyrosine Kinase ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Intestinal Inflammation ◽  
De Novo ◽  
Single Gene ◽  
Gain Of Function

Abstract Background Very early-onset inflammatory bowel disease (VEOIBD) is an emerging global disease, that results in inflammation of the digestive tract. Severe forms of VEOIBD can be caused by mutations in a single gene (monogenic variants) and, can result in death. A candidate gene which codes for a non-receptor tyrosine kinase (nRTK) has recently been implicated as a monogenic cause of IBD (unpublished). Whole exome sequencing was performed in two unrelated children who presented with symptoms of IBD identifying two distinct de novo gain of function mutations (S550Y and P342T). Both mutations are located in the highly conserved region of the nRTK, and were predicted to have similar downstream effects. Furthermore, four other patients with a variety of adult-onset immune disorders have recently been identified with rare variants in the same gene (M450I, R42P, A353T, V433M, S550F) but, their potential gain of function status remains to be determined. Studies show that this nRTK is an essential mediator in inflammation. It is expressed in both intestinal epithelial and immune cells however, its role in infantile IBD is unclear. This protein is first activated by phosphorylation and is linked to activating downstream transcription factors such as ERK and JNK. All these target proteins play a meaningful role in intestinal inflammation in patients with IBD. Aims Since we identified P342T and S550Y to be gain of function, we wanted to determine if the new variants exhibit a similar downstream impact on target protein expression levels when compared with S550Y and P342T. We also wanted to identify if all variants can be rescued with a known nRTK inhibitor. It is hypothesized that the new variants are gain of function and that all variants can be rescued with the inhibitor. Methods Using western blot analysis, the activation of ERK, JNK and nRTK was compared between wildtype (WT) and mutants. This in vitro method helped identify the degree of activation. For the second part of the study, HEK293T cells were treated with inhibitor to test for a rescue of phenotypes via western blot analysis. Results Results show an increased activation of nRTK, ERK and JNK in all variants with S550Y and S550F having the highest activation. Furthermore, pharmacological inhibition using small molecular kinase inhibitors resulted in decreased activation of nRTK, ERK and JNK suggesting a rescue of phenotypes. Conclusions Characterizing the downstream functional impact of these nRTK variants is an important first step to determine if gain of function nRTK mutations drive IBD. With a rising prevalence of IBD worldwide, these findings may lead to the development of pharmacological nRTK inhibitors as a novel personalized therapeutic approach for these patients and possibly for the broader IBD population. Funding Agencies CIHR

scholarly journals In Vitro and In Vivo Effects of Fermented Oyster-Derived Lactate on Exercise Endurance Indicators in Mice

2020 ◽  
Vol 17 (23) ◽  
pp. 8811
Author(s):  
Storm N. S. Reid ◽  
Joung-Hyun Park ◽  
Yunsook Kim ◽  
Yi Sub Kwak ◽  
Byeong Hwan Jeon
Keyword(s):  
Lactate Dehydrogenase ◽  
Protein Expression ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Biochemical Analysis ◽  
Positive Control ◽  
Exercise Endurance

Exogenous lactate administration has more recently been investigated for its various prophylactic effects. Lactate derived from potential functional foods, such as fermented oyster extract (FO), may emerge as a practical and effective method of consuming exogenous lactate. The current study endeavored to ascertain whether the lactate derived from FO may act on muscle cell biology, and to what extent this may translate into physical fitness improvements. We examined the effects of FO in vitro and in vivo, on mouse C2C12 cells and exercise performance indicators in mice, respectively. In vitro, biochemical analysis was carried out to determine the effects of FO on lactate content and muscle cell energy metabolism, including adenosine triphosphate (ATP) activity. Western blot analysis was also utilized to measure the protein expression of total adenosine monophosphate-activated protein kinase (AMPK), p-AMPK (Thr172), lactate dehydrogenase (LDH), succinate dehydrogenase (SDHA) and peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) in response to FO administration. Three experimental groups were formed: a positive control (PC) treated with 1% horse serum, FO10 treated with 10 μg/mL and FO50 treated with 50 μg/mL. In vivo, the effects of FO supplementation on exercise endurance were measured using the Rota-rod test, and Western blot analysis measured myosin heavy-chain 2 (MYH2) to assess skeletal muscle growth, alongside p-AMPK, total-AMPK, PGC-1α, cytochrome C and UCP3 protein expression. Biochemical analysis was also performed on muscle tissue to measure the changes in concentration of liver lactate, lactate dehydrogenase (LDH), glycogen and citrate. Five groups (n = 10/per group) consisted of a control group (CON), exercise group (Ex), positive control treated with Ex and 500 mg/kg Taurine (Ex-Tau), Ex and 100 mg/kg FO supplementation (Ex-FO100) and Ex and 200 mg/kg FO supplementation (Ex-FO200) orally administered over the 4-week experimental period.FO50 significantly increased PGC-1α expression (p < 0.001), whereas both FO10 and FO50 increased the expression of p-AMPK (p < 0.001), in C2C12 muscle cells, showing increased signaling important for mitochondrial metabolism and biogenesis. Muscle lactate levels were also significantly increased following FO10 (p < 0.05) and FO50 (p < 0.001). In vivo, muscle protein expression of p-AMPK (p < 0.05) and PGC-1α were increased, corroborating our in vitro results. Cytochrome C also significantly increased following FO200 intake. These results suggest that the effects of FO supplementation may manifest in a dose-response manner. FO administration, in vitro, and supplementation, in vivo, both demonstrate a potential for improvements in mitochondrial metabolism and biogenesis, and even for potentiating the adaptive effects of endurance exercise. Mechanistically, lactate may be an important molecule in explaining the aforementioned positive effects of FO.

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