scholarly journals Sensory Neuropeptides and their Receptors Participate in Mechano-Regulation of Murine Macrophages

2019 ◽  
Vol 20 (3) ◽  
pp. 503 ◽  
Author(s):  
Dominique Muschter ◽  
Anna-Sophie Beiderbeck ◽  
Tanja Späth ◽  
Christian Kirschneck ◽  
Agnes Schröder ◽  
...  
Keyword(s):  
Protein Expression ◽  
Mrna Level ◽  
Cyclic Stretch ◽  
Murine Bone Marrow ◽  
Raw264.7 Macrophages ◽  
Neurokinin Receptor ◽  
Calcitonin Gene ◽  
Gene And Protein Expression ◽  
Sensory Neuropeptides ◽  
Comparable Activity

This study aimed to analyze if the sensory neuropeptide SP (SP) and the neurokinin receptor 1 (NK1R) are involved in macrophage mechano-transduction, similar to chondrocytes, and if alpha-calcitonin gene-related peptide (αCGRP) and the CGRP receptor (CRLR/Ramp1) show comparable activity. Murine RAW264.7 macrophages were subjected to a cyclic stretch for 1–3 days and 4 h/day. Loading and neuropeptide effects were analyzed for gene and protein expression of neuropeptides and their receptors, adhesion, apoptosis, proliferation and ROS activity. Murine bone marrow-derived macrophages (BMM) were isolated after surgical osteoarthritis (OA) induction and proliferation, apoptosis and osteoclastogenesis were analyzed in response to loading. Loading induced NK1R and CRLR/Ramp1 gene expression and altered protein expression in RAW264.7 macrophages. SP protein and mRNA level decreased after loading whereas αCGRP mRNA expression was stabilized. SP reduced adhesion in loaded RAW264.7 macrophages and both neuropeptides initially increased the ROS activity followed by a time-dependent suppression. OA induction sensitized BMM to caspase 3/7 mediated apoptosis after loading. Both sensory neuropeptides, SP and αCGRP, and their receptors are involved in murine macrophage mechano-transduction affecting neuropeptide impact on adhesion and ROS activity. OA induction altered BMM apoptosis in response to loading indicate that OA-associated biomechanical alterations might affect the macrophage population.

Methamphetamine causes neurotoxicity by promoting polarization of macrophages and inflammatory response

2017 ◽  
Vol 37 (5) ◽  
pp. 486-495 ◽  
Author(s):  
X Li ◽  
F Wu ◽  
L Xue ◽  
B Wang ◽  
J Li ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Protein Expression ◽  
Macrophage Polarization ◽  
Enzyme Linked Immunosorbent Assay ◽  
Neuronal System ◽  
Activated Macrophages ◽  
Alternatively Activated Macrophages ◽  
Raw264.7 Macrophages ◽  
Gene And Protein Expression ◽  
Anti Inflammatory

Macrophages, especially their activation state, are closely related to the progression of neurotoxicity. Classically activated macrophages (M1) are proinflammatory effectors, while alternatively activated macrophages (M2) exhibit anti-inflammatory properties. As a powerful addictive psychostimulant drug, coupled with its neurotoxicity, methamphetamine (Meth) abuse may lead to long-lasting abnormalities in the neuronal system. The present study investigated the effect of Meth at subtoxic concentration on macrophage activation state and its underlying toxicity to neuronal cells. PC12 and Murine RAW264.7 cells were coincubated with Meth to test its toxicity. 3-(4,5-Dimethylthiazol)-2,5-diphenyltetrazolium-bromide, enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and Western blot assays were performed to evaluate the toxicity, cytokine secretion, gene, and protein expression. Results showed that cytotoxicity was enhanced on PC12 cells after coculturing with RAW264.7 stimulated with Meth. RAW264.7 macrophages tended to switch to the M1 phenotype, releasing more nitric oxide and proinflammatory cytokines, including tumor necrosis factor α (TNFα), interleukin (IL)-12, and IL-1β, while decreasing the release of anti-inflammatory cytokine IL-10 after treatment with Meth. Meth upregulated the gene expression of IL-6, IL-1β, and TNFα and downregulated the expression of Arg-1, IL-10, and KLF4. Meth could also upregulate the protein expression of IL-1β and TNF α and downregulate the expression of Arg-1 and KLF4. However, the abovementioned effects induced by Meth were abolished by the addition of dopamine receptor D3 antagonist. In conclusion, our study demonstrated that Meth promoted macrophage polarization from M0 to M1 and enhanced inflammatory response, which provided the scientific rationale for the neurotoxicity caused by the chronic use of Meth.

scholarly journals Alterations in Gastric Mucosal Expression of Calcitonin Gene-Related Peptides, Vanilloid Receptors, and Heme Oxygenase-1 Mediate Gastroprotective Action of Carbon Monoxide against Ethanol-Induced Gastric Mucosal Lesions

2018 ◽  
Vol 19 (10) ◽  
pp. 2960 ◽  
Author(s):  
Katarzyna Magierowska ◽  
Dagmara Wojcik ◽  
Anna Chmura ◽  
Dominik Bakalarz ◽  
Mateusz Wierdak ◽  
...  
Keyword(s):  
Carbon Monoxide ◽  
Protein Expression ◽  
Alcian Blue ◽  
Heme Oxygenase ◽  
Mrna Level ◽  
Fold Change ◽  
Gastric Damage ◽  
Gastric Lesions ◽  
Calcitonin Gene ◽  
Enos Mrna

Carbon monoxide (CO) has been reported to contribute to the maintenance of gastric mucosal integrity, gastroprotection, and ulcer healing. However, involvement of transient receptor potential vanilloid receptor type 1 (TRPV1) located on afferent sensory fibers endings and sensory neuropeptide calcitonin gene-related peptide (CGRP) in CO-mediated gastroprotection against ethanol-induced gastric damage has not been explored. Male Wistar rats with and without denervation of afferent sensory neurons induced by capsaicin (total dose 125 mg/kg within 3 days) were pretreated with vehicle, CO donor tricarbonyldichlororuthenium (II) dimer (CORM-2, 5 mg/kg i.g.), administered alone or with CGRP-α (10 μg/kg i.p.) or TRPV1 antagonist capsazepine (5 mg/kg i.g.), followed 30 min later by intragastric (i.g.) administration of 75% ethanol. The area of gastric damage and gastric blood flow (GBF) were assessed planimetrically and by laser flowmetry, respectively. Microscopic evaluation of ethanol-induced gastric lesions was performed after haematoxylin/eosin (H&E) or alcian blue/periodic acid-Schiff/alcian blue (AB/PAS) staining. Gastric mucosal mRNA fold change for heme oxygenase (HMOX)-1, HMOX-2, CGRP-α, CGRP-β, inducible nitric oxide synthase (iNOS), endothelial (e)NOS, neuronal (n)NOS, cyclooxygenase (COX)-1, COX-2, and protein expression for HMOX-1 and TRPV1 was determined by real-time PCR or Western blot, respectively. Pretreatment with CORM-2 combined or not with CGRP reduced ethanol-induced gastric lesions and elevated GBF. Capsaicin-denervation or co-treatment with capsazepine or CGRP and CORM-2 in capsaicin-denervated animals failed to affect these beneficial effects of CO donor. In rats with intact sensory nerves, CORM-2 increased gastric mRNA level for HMOX-1 and CGRP-α. In capsaicin-denervated rats, CORM-2 increased eNOS mRNA fold change and TRPV1 protein expression while capsaicin denervation itself decreased HMOX-1 protein expression and eNOS mRNA level. We conclude that CO prevents gastric mucosa from ethanol-induced lesions due to activation of TRPV1/CGRP-α system and accompanying increase in gastric microcirculation but independently on afferent sensory nerve activity despite the stimulation of TRPV1 protein and CGRP-α mRNA expression.

scholarly journals The inhibitory effects of rosmarinic acid on catabolism induced by IL-1β in rat chondrocyte

2018 ◽  
Author(s):  
Zhong-Nan Hu ◽  
Li-Juan Huang ◽  
Wei-Ping Chen
Keyword(s):  
Gene Expression ◽  
Extracellular Matrix ◽  
Protein Expression ◽  
Rosmarinic Acid ◽  
Mrna Level ◽  
Inhibitory Effects ◽  
Gene And Protein Expression ◽  
Ecm Degradation ◽  
Interleukin 1Beta ◽  
A Disintegrin And Metalloproteinase

The effects of rosmarinic acid (RosA) on osteoarthritis (OA) was investigated in rat chondrocytes. Chondrocytes were isolated from rat cartilage, incubated with RosA in the presence of interleukin-1beta (IL-1β) (10 ng/ml). The production of IL-6, as well as the mRNA level of aggrecan (ACAN) and collagen 2 (COL2), were assessed. The gene and protein expression of A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, ADAMTS-5 were also measured. RosA inhibited the production of IL-6 as well as the gene and protein expression of ADAMTS-4 and ADAMTS-5, in addition, RosA abolished IL-1β-induced inhibition of ACAN and COL2 gene expression. Our results suggest that RosA can inhibit extracellular matrix (ECM) degradation in OA, thus, RosA may be a possible agent in the treatment of OA.

Effect of nitrate on gene and protein expression of nitric oxide synthase enzymes in insulin-sensitive tissues of type 2 diabetic male rats

2021 ◽  
Vol 21 ◽  
Author(s):  
Majid Shokri ◽  
Sajad Jeddi ◽  
Hassan Faridnouri ◽  
Vajiheh Khorasani ◽  
Khosrow Kashfi ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Protein Expression ◽  
Mrna Level ◽  
Diabetic Rats ◽  
Metabolic Effects ◽  
Male Rats ◽  
Epididymal Adipose Tissue ◽  
Protein Levels ◽  
Gene And Protein Expression

Background and Objective: Decreased nitric oxide (NO) bioavailability contributes to the pathophysiology of type 2 diabetes mellitus (T2DM). This study aims to determine the effects of nitrate (NO3–) on gene and protein expression of NO synthase (NOS) enzymes in the liver, soleus muscle (SM), and epididymal adipose tissue (eAT) of rats with T2DM. Methods: Twenty-eight male rats were divided into 4 groups: Control, diabetes, control+NO3–, and diabetes+NO3– (n = 7/each group). NO3– was administered for 6 months, and mRNA and protein levels of NOS enzymes were measured at the end of the study. Results: mRNA and protein levels of inducible NOS (iNOS) were higher in the liver (475% and 73%), SM (271% and 43%), and eAT (543% and 24%) of rats with T2DM. In the case of the endothelial NOS (eNOS), diabetic rats had lower mRNA and protein levels in the liver (26% and 24%) and SM (60% and 62%) and lower mRNA level (30%) in eAT. mRNA and protein levels of neural NOS (nNOS) were lower in SM (69% and 73%) and eAT (25% and 31%) of rats with T2DM. NO3– administration restored disrupted iNOS and eNOS expressions to their near normal values in all the studied tissues; NO3– also increased nNOS mRNA and protein levels in SM and eAT but decreased nNOS protein level in the liver. Conclusion: Long-term NO3– administration restored disrupted expression of NOS enzymes in the liver, SM, and eAT of rats with T2DM; these findings partly explain the beneficial metabolic effects of nitrate in T2DM.

Inhibition of Migration and Invasion of Hepatocarcinoma HepG2 Cells by Dietary Saponins via Targeting HIF-1α

2018 ◽  
Vol 18 (7) ◽  
pp. 1025-1031
Author(s):  
Cheng Luo ◽  
Di Wu ◽  
Meiling Chen ◽  
Wenhua Miao ◽  
Changfeng Xue ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Confocal Microscopy ◽  
Protein Expression ◽  
Mtt Assay ◽  
Hepg2 Cells ◽  
Molecular Mechanisms ◽  
Migration And Invasion ◽  
Rt Pcr ◽  
Gene And Protein Expression ◽  
Simulated Hypoxia

Background: Different saponins from herbs have been used as tonic or functional foods, and for treatment of various diseases including cancers. Although clinical data has supported the function of these saponins, their underlying molecular mechanisms have not been well defined. Methods: With the simulated hypoxia created by 8 hours of Cu++ exposure and following 24 hour incubation with different concentration of saponins in HepG2 cells for MTT assay, migration and invasion assays, and for RT-PCR, and with each group of cells for immunofluorescence observation by confocal microscopy. Results： ZC-4 had the highest rate of inhibition of cell proliferation by MTT assay, and the highest inhibition of migration rate by in vitro scratch assay, while ZC-3 had the highest inhibition of invasion ratio by transwell assay. Under the same simulated hypoxia, the molecular mechanism of saponin function was conducted by measuring the gene expression of Hypoxia Inducible Factor (HIF)-1α through RT-PCR, in which ZC-3 showed a potent inhibition of gene HIF-1α. For the protein expression by immunofluorescence staining with confocal microscopy, HIF-1α was also inhibited by saponins, with the most potent one being ZC-4 after eight hours’ relatively hypoxia incubation. Conclusion: Saponins ZC-4 and ZC-3 have the potential to reduce HepG2 cell proliferation, migration and invasion caused by hypoxia through effectively inhibiting the gene and protein expression of HIF-1α directly and as antioxidant indirectly

scholarly journals Treadmill Running Changes Endothelial Lipase Expression: Insights from Gene and Protein Analysis in Various Striated Muscle Tissues and Serum

Biomolecules ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 906
Author(s):  
Agnieszka Mikłosz ◽  
Bartłomiej Łukaszuk ◽  
Adrian Chabowski ◽  
Jan Górski
Keyword(s):  
Protein Expression ◽  
Density Lipoprotein ◽  
Treadmill Running ◽  
Endothelial Lipase ◽  
Type I ◽  
Fiber Types ◽  
Gene And Protein Expression ◽  
Single Bout ◽  
The Impact ◽  
White Gastrocnemius

Endothelial lipase (EL) is an enzyme capable of HDL phospholipids hydrolysis. Its action leads to a reduction in the serum high-density lipoprotein concentration, and thus, it exerts a pro-atherogenic effect. This study examines the impact of a single bout exercise on the gene and protein expression of the EL in skeletal muscles composed of different fiber types (the soleus—mainly type I, the red gastrocnemius—mostly IIA, and the white gastrocnemius—predominantly IIX fibers), as well as the diaphragm, and the heart. Wistar rats were subjected to a treadmill run: 1) t = 30 [min], V = 18 [m/min]; 2) t = 30 [min], V = 28 [m/min]; 3) t = 120 [min], V = 18 [m/min] (designated: M30, F30, and M120, respectively). We established EL expression in the total muscle homogenates in sedentary animals. Resting values could be ordered with the decreasing EL protein expression as follows: endothelium of left ventricle > diaphragm > red gastrocnemius > right ventricle > soleus > white gastrocnemius. Furthermore, we observed that even a single bout of exercise was capable of inducing changes in the mRNA and protein level of EL, with a clearer pattern observed for the former. After 30 min of running at either exercise intensity, the expression of EL transcript in all the cardiovascular components of muscles tested, except the soleus, was reduced in comparison to the respective sedentary control. The protein content of EL varied with the intensity and/or duration of the run in the studied whole tissue homogenates. The observed differences between EL expression in vascular beds of muscles may indicate the muscle-specific role of the lipase.

scholarly journals Toll-like receptor 2 induced senescence in intervertebral disc cells of patients with back pain can be attenuated by o-vanillin

2021 ◽  
Vol 23 (1) ◽  
Author(s):  
Matthew Mannarino ◽  
Hosni Cherif ◽  
Li Li ◽  
Kai Sheng ◽  
Oded Rabau ◽  
...  
Keyword(s):  
Gene Expression ◽  
Back Pain ◽  
Protein Expression ◽  
Disc Degeneration ◽  
Cell Senescence ◽  
Enzyme Linked Immunosorbent Assay ◽  
Matrix Synthesis ◽  
Gene And Protein Expression ◽  
Pain Patients ◽  
In Cells

Abstract Background There is an increased level of senescent cells and toll-like teceptor-1, -2, -4, and -6 (TLR) expression in degenerating intervertebral discs (IVDs) from back pain patients. However, it is currently not known if the increase in expression of TLRs is related to the senescent cells or if it is a more general increase on all cells. It is also not known if TLR activation in IVD cells will induce cell senescence. Methods Cells from non-degenerate human IVD were obtained from spine donors and cells from degenerate IVDs came from patients undergoing surgery for low back pain. Gene expression of TLR-1,2,4,6, senescence and senescence-associated secretory phenotype (SASP) markers was evaluated by RT-qPCR in isolated cells. Matrix synthesis was verified with safranin-O staining and Dimethyl-Methylene Blue Assay (DMMB) confirmed proteoglycan content. Protein expression of p16INK4a, SASP factors, and TLR-2 was evaluated by immunocytochemistry (ICC) and/or by enzyme-linked immunosorbent assay (ELISA). Results An increase in senescent cells was found following 48-h induction with a TLR-2/6 agonist in cells from both non-degenerate and degenerating human IVDs. Higher levels of SASP factors, TLR-2 gene expression, and protein expression were found following 48-h induction with TLR-2/6 agonist. Treatment with o-vanillin reduced the number of senescent cells, and increased matrix synthesis in IVD cells from back pain patients. Treatment with o-vanillin after induction with TLR-2/6 agonist reduced gene and protein expression of SASP factors and TLR-2. Co-localized staining of p16INK4a and TLR-2 demonstrated that senescent cells have a high TLR-2 expression. Conclusions Taken together our data demonstrate that activation of TLR-2/6 induce senescence and increase TLR-2 and SASP expression in cells from non-degenerate IVDs of organ donors without degeneration and back pain and in cells from degenerating human IVD of patients with disc degeneration and back pain. The senescent cells showed high TLR-2 expression suggesting a link between TLR activation and cell senescence in human IVD cells. The reduction in senescence, SASP, and TLR-2 expression suggest o-vanillin as a potential disease-modifying drug for patients with disc degeneration and back pain.

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