NOS mRNA Research Articles

NOS3 regulates angiogenic potential of human induced pluripotent stem cell-derived endothelial cells

Incorporation of blood capillaries in engineered tissues and their functional connection to host blood vessels is essential for success in engineering vascularized tissues, a process which involves spatial patterning of endothelial cells (ECs) to form functional and integrated vascular networks. Different types of ECs have been employed for vascular network formation and each source offers advantages and disadvantages. While ECs derived from induced pluripotent stem cells (iPSC-ECs) offer advantages over primary ECs in that they can be generated in large quantities for autologous applications, their suitability for disease modelling and cell replacement therapies is not well-explored. The present study compares the angiogenic capacity of iPSC-ECs and primary ECs (cardiac microvascular ECs and lymphatic microvascular ECs) using an in vitro tubulogenesis assay, revealing comparable performance in forming a pseudo-capillary network on Matrigel. Analysis of genes encoding angiogenic factors (VEGFA, VEGFC, VEGFD and ANG), endothelial cell markers (PECAM1, PCDH12 and NOS3) and proliferation markers (AURKB and MKI67) indicates a significant positive correlation between NOS3 mRNA expression levels and various tubulogenic parameters. Further experimentation using a CRISPR activation system demonstrates a positive impact of NOS3 on tubulogenic activity of ECs, suggesting that iPSC-ECs can be enhanced with endogenous NOS3 activation. Collectively, these findings highlight the potential of iPSC-ECs in generating vascularized tissues and advancing therapeutic vascularization.

Acute Exercise-induced Endothelial Shear Stress Activates Nos3 Mrna Translation In Vitro

Acute Exercise-induced Endothelial Shear Stress Activates Nos3 Mrna Translation In Vitro

Prolonged extracellular low sodium concentrations and subsequent their rapid correction modulate nitric oxide production dependent on NFAT5 in microglia

Hyponatremia is the most common clinical electrolyte disorder. Chronic hyponatremia has been recently reported to be associated with falls, fracture, osteoporosis, neurocognitive impairment, and mental manifestations. In the treatment of chronic hyponatremia, overly rapid correction of hyponatremia can cause osmotic demyelination syndrome (ODS), a central demyelinating disease that is also associated with neurological morbidity and mortality. Using a rat model, we have previously shown that microglia play a critical role in the pathogenesis of ODS. However, the direct effect of rapid correction of hyponatremia on microglia is unknown. Furthermore, the effect of chronic hyponatremia on microglia remains elusive. Using microglial cell lines BV-2 and 6-3, we show here that low extracellular sodium concentrations (36 mmol/L decrease; LS) suppress Nos2 mRNA expression and nitric oxide (NO) production of microglia. On rapid correction of low sodium concentrations, NO production was significantly increased in both cells, suggesting that acute correction of hyponatremia partly directly contributes to increased Nos2 mRNA expression and NO release in ODS pathophysiology. LS also suppressed expression and nuclear translocation of nuclear factor of activated T cells-5 (NFAT5), a transcription factor that regulates the expression of genes involved in osmotic stress. Furthermore, overexpression of NFAT5 significantly increased Nos2 mRNA expression and NO production in BV-2 cells. Expressions of Nos2 and Nfat5 mRNA were also modulated in microglia isolated from cerebral cortex in chronic hyponatremia model mice. These data indicate that LS modulates microglial NO production dependent on NFAT5 and suggest that microglia contribute to hyponatremia-induced neuronal dysfunctions.

Evaluation of In Vitro Antihypertensive and Anti-Inflammatory Properties of Dairy By-Products

Sweet whey (SW) and yogurt acid whey (YAW) are dairy by-products of the cheese-making process and Greek-style yogurt production, respectively. Both of them are considered pollutants with huge volumes of SW and YAW produced due to the growing demand for dairy products worldwide. Moreover, whey-derived peptides, resulting from fermentation as well as from further hydrolysis during digestion, have been associated with various biological activities. In the present study, the angiotensin-converting enzyme (ACE)-inhibitory activity of 48 SW samples and 33 YAW samples from bovine, ovine, caprine, and ovine/caprine milk obtained were evaluated. Additionally, the SW and YAW digestates and two of their fractions (smaller than 10 kDa, SW-D-P10 and YAW-D-P10, and smaller than 3 kDa, SW-D-P3 and YAW-D-P3), which were obtained after in vitro digestion and subsequent ultrafiltration, were also subjected to evaluation. Our data indicated that the D-P10 and D-P3 fractions exhibited higher ACE-inhibitory activity compared to the corresponding values before digestion. The ACE-inhibitory capacity after in vitro digestion was higher for the ovine SW samples compared to their bovine and caprine counterparts. The effect of the D-P3 fraction on the inhibition of nitric oxide (NO) production and the expression of a selected panel of immune-response-related genes in LPS-stimulated RAW 264.7 macrophages was also evaluated. Fractions from both dairy by-products inhibited NO production in LPS-stimulated RAW 264.7 cells. Especially, ovine SW-D-P3 showed a strong NO inhibitory activity and suppressed inducible nitric oxide synthase (Nos2) mRNA levels. However, YAW-D-P3 could not trigger neither the gene expression of inflammatory macrophage mediators Nos2 and cyclooxygenase-2 (Ptgs2) nor tumor necrosis factor-α (Tnf) and interleukin 6 (Il6) in LPS-stimulated murine macrophages regardless of animal origin. These findings suggest that in vitro digestion could enhance the production of ACE-inhibitory peptides in both dairy by-products, while SW from ovine origin displays higher potential as an anti-inflammatory agent, effectively preventing excessive NO production.

Nobiletin restores HFD-induced enteric nerve injury by regulating enteric glial activation and the GDNF/AKT/FOXO3a/P21 pathway

BackgroundTo explore whether nobiletin has a protective effect on high-fat diet (HFD)-induced enteric nerve injury and its underlying mechanism.MethodsAn obesity model was induced by a HFD. Nobiletin (100 mg/kg and 200 mg/kg) and vehicle were administered by gastric gavage for 4 weeks. Lee’s index, body weight, OGTT and intestinal propulsion assays were performed before sacrifice. After sampling, lipids were detected using Bodipy 493/503; lipid peroxidation was detected using MDA and SOD kits and the expression of PGP 9.5, Trem2, GFAP, β-tubulin 3, Bax, Bcl2, Nestin, P75 NTR, SOX10 and EDU was detected using immunofluorescence. The GDNF, p-AKT, AKT, p-FOXO3a, FOXO3a and P21 proteins were detected using western blotting. The relative mRNA expression levels of NOS2 were detected via qPCR. Primary enteric neural stem cells (ENSCs) were cultured. After ENSCs were treated with palmitic acid (PA) and nobiletin, CCK-8 and caspase-3/7 activity assays were performed to evaluate proliferation and apoptosis.ResultsHFD consumption caused colon lipid accumulation and peroxidation, induced enteric nerve damage and caused intestinal motor dysfunction. However, nobiletin reduced lipid accumulation and peroxidation in the colon; promoted Trem2, β-tubulin 3, Nestin, P75NTR, SOX10 and Bcl2 expression; inhibited Bax and GFAP expression; reduced NOS2 mRNA transcription; and regulated the GDNF/AKT/FOXO3a/P21 pathway. Nobiletin also promoted PA-induced impairment of ENSCs.ConclusionsNobiletin restored HFD-induced enteric nerve injury, which may be associated with inhibiting enteric nerve apoptosis, promoting enteric nerve survival and regulating the GDNF/AKT/FOXO3a/P21 pathway.

The Extremely-Low-Frequency Electromagnetic Field Affects Apoptosis and Oxidative-Stress-Related Genes and Proteins in the Porcine Endometrium-An In Vitro Study.

Nowadays, the extremely-low-frequency electromagnetic field (ELF-EMF) is recognized as environmental pollution. The data indicate that the ELF-EMF may affect factors related to epigenetic regulation and alter important biological processes in the uterus. The impact of the ELF-EMF on apoptosis and oxidative-stress-related genes has not been documented in porcine endometrium. This raises the question of whether the exposure to the ELF-EMF can induce apoptosis and/or oxidative stress in the endometrium of pigs during the peri-implantation period. Porcine endometrial slices (100 ± 5 mg) collected (n = 5) during the peri-implantation period were treated in vitro with ELF-EMF at a frequency of 50 Hz and flux density of 8 × 104 mG for 2 h. To determine the effect of ELF-EMF on apoptosis and oxidative stress in the endometrium, CASP3, CASP7, CIDEB, GADD45G, NOS1, NOS2, NOS3, and TP53I3 mRNA transcript were analyzed using real-time PCR, and protein abundance of CASP3, CASP7 using Western blot, and eNOS using ELISA were determined. Moreover, CASP3/7 and NOS activity was analyzed using flow cytometry and colorimetry, respectively. The decreased CASP7 and increased NOS3 mRNA transcript and protein abundance in ELF-EMF-treated endometrium were observed. Moreover, CIDEB, GADD45G, and TP53I3 mRNA transcript abundance was increased. Only p ≤ 0.05 was considered a statistically significant difference. The documented alterations indicate the potential of the ELF-EMF to affect apoptosis and generate oxidative stress in the endometrium. The insight into observed consequences documents for the first time the fact that the ELF-EMF may influence endometrial cell proliferation, angiogenesis, and/or tissue receptivity during peri-implantation.

Chemical Characterization, Free Radical Scavenging, and Cellular Antioxidant Properties of the Egadi Island Endemic Brassica macrocarpa Guss Leaf Extract.

The genus Brassica is an important source of food in the Mediterranean diet with documented nutritional and medicinal properties. However, few studies have investigated the phytochemical composition and the biological activity of wild Sicilian taxa. Thus, we aimed to study the chemical profile and the antioxidant potential, in vitro and in LPS-stimulated RAW 264.7 cells, of a methanolic extract of leaves of wild Brassica macrocarpa Guss (B. macrocarpa) (Egadi Islands; Sicily-Italy). B. macrocarpa methanolic extract showed a large amount of glucosinolates and different phenolic compounds. It exhibited antioxidant activity in the DPPH assay and in LPS-stimulated RAW 264.7 cells, being able to reduce NO and ROS levels and NOS2 mRNA expression. Our study demonstrated that Sicilian B. macrocarpa methanolic extract, in LPS-stimulated macrophages, efficiently counteracts oxidative stress and displays radical scavenging activity. Future studies are required to identify the contribution of the single phytocomponents, to characterize the action mechanism, and to reveal possible applications in human health.

Neuroprotective Effect of Lithospermum officinale Callus Extract on Inflamed Primary Microglial Cells.

Plants that have therapeutic features for humans or animals are commonly referred to as "medicinal plants". They produce secondary metabolites with antioxidant, antimicrobial and/or anti-cancer effects. Lithospermum officinale, known as European stone seed, is a famous medicinal herb. However, due to the pyrrolizidine alkaloids (PzAl) in the root extract of L.officinal, there are therapeutic limitations to this herb. This research was devoted to the evaluation of the anti-inflammatory capacity of methanolic extracts of L. officinale callus (LoE) (fresh cells) on rat microglial cells, the immune cells of the Central Nervous System, which play an essential role in the responses to neuroinflammation. Primary microglia were obtained from neonatal Wistar rats (1 to 3-days old), and then treated with various concentration of CfA and methanolic extracts of 17 and 31-day-old L. officinale callus before LPS-stimulation. In addition to HPLC analysis of the extracts, viability, nitric oxide production, and evaluation of pro-inflammatory genes and cytokines in the inflamed microglia were investigated by MTT, Griess methos, qrt-PCR, and ELISA. Methanolic extract of the 17-day-old callus of L. officinale exhibited anti-inflammatory effects on LPS-stimulated microglial cells much higher than observed for CfA. The data were further supported by the decreased expression of Nos2, Tnf-α, and Cox-2 mRNA and the suppression of TNF-α and IL-1β release in the activated microglial cells pretreated with the effective dose of LoE (0.8 mg mL-1). It was assumed that the better anti-neuroinflammatory performance of LoE than CfA in LPS-activated primary microglia could be a result of the synergism of the components of the extract and the lipophilic nature of RsA as the main phenolic acid of LoE. Considering that LoE shows a high antioxidant capacity and lacks PzAl, it is anticipated that LoE extract might be considered a reliable substitute to play a key role in the preparation of neuroprotective pharmaceutical formulas, which require in vivo research and further experiments.

CD44 expression on murine hepatic stellate cells promotes the induction of monocytic and polymorphonuclear myeloid-derived suppressor cells.

In chronic inflammation, regulatory immune cells, such as regulatory T cells and myeloid-derived suppressor cells, can develop. Local signals in the inflamed tissue, such as cytokines and eicosanoids, but also contact-dependent signals, can promote myeloid-derived suppressor cell development. In the liver, hepatic stellate cells may provide such signals via the expression of CD44. Myeloid-derived suppressor cells generated in the presence of hepatic stellate cells and anti-CD44 antibodies were functionally and phenotypically analyzed. We found that both monocytic and polymorphonuclear myeloid-derived suppressor cells generated in the presence of αCD44 antibodies were less suppressive toward T cells as measured by T-cell proliferation and cytokine production. Moreover, both monocytic and polymorphonuclear myeloid-derived suppressor cells were phenotypically altered. Monocytic myeloid-derived suppressor cells mainly changed their expression of CD80 and CD39, and polymorphonuclear myeloid-derived suppressor cells showed altered expression of CD80/86, PD-L1, and CCR2. Moreover, both polymorphonuclear and monocytic myeloid-derived suppressor cells lost expression of Nos2 messenger RNA, whereas monocytic myeloid-derived suppressor cells showed reduced expression of TGFb messenger RNA and polymorphonuclear myeloid-derived suppressor cells reduced expression of Il10 messenger RNA. In summary, the presence of CD44 in hepatic stellate cells promotes the induction of both monocytic and polymorphonuclear myeloid-derived suppressor cells, although the mechanisms by which these myeloid-derived suppressor cells may increase suppressive function due to interaction with CD44 are only partially overlapping.

Novel Multitarget ACE Inhibitory Peptides from Bovine Colostrum Immunoglobulin G: Cellular Transport, Efficacy in Regulating Endothelial Dysfunction, and Network Pharmacology Studies.

In this study, we used traditional laboratory methods, bioinformatics, and cellular models to screen novel ACE inhibitory (ACEI) peptides with strong ACEI activity, moderate absorption rates, and multiple targets from bovine colostrum immunoglobulin G (IgG). The purified fraction of the compound proteinase hydrolysate of IgG showed good ACEI activity. After nano-UPLC-MS/MS identification and in silico analysis, eight peptides were synthesized and verified. Among them, SFYPDY, TSFYPDY, FSWF, WYQQVPGSGL, and GVHTFP were identified as ACEI peptides, as they exhibited strong ACEI activity (with IC50 values of 104.7, 80.0, 121.2, 39.8, and 86.3 μM, respectively). They displayed good stability in an in vitro simulated gastrointestinal digestion assay. In a Caco-2 monolayer model, SFYPDY, FSWF, and WYQQVPGSGL exhibited better absorption rates and lower IC50 values than the other peptides and were thereby identified as novel ACEI peptides. Subsequently, in a H2O2-induced endothelial dysfunction (ED) model based on HUVECs, SFYPDY, FSWF, and WYQQVPGSGL regulated ED by reducing apoptosis and ROS accumulation while upregulating NOS3 mRNA expression. Network pharmacology analysis and RT-qPCR confirmed that they regulated multiple targets. Overall, our results suggest that SFYPDY, FSWF, and WYQQVPGSGL can serve as novel multitarget ACEI peptides.

Analysis of the liver-gut axis including metabolomics and intestinal flora to determine the protective effects of kiwifruit seed oil on CCl4-induced acute liver injury.

The hepatoprotective effects of kiwifruit seed oil (KSO) were evaluated on acute liver injury (ALI) induced by carbon tetrachloride (CCl4) in vivo. Network pharmacology was used to predict active compounds and targets. Metabolomics and gut microbiota analyses were used to discover the activity mechanism of KSO. KSO improved the liver histological structure, significantly reduced serum proinflammatory cytokine levels, and increased liver antioxidant capacity. The metabolomics analysis showed that KSO may have hepatoprotective effects by controlling metabolites through its participation in signaling pathways like tryptophan metabolism, glycolysis/gluconeogenesis, galactose metabolism, and bile secretion. The gut microbiota analysis demonstrated that KSO improved the composition and quantity of the gut flora. Network pharmacological investigations demonstrated that KSO operated by altering Ptgs2, Nos2, Ppara, Pparg and Serpine1 mRNA levels. All evidence shows that KSO has a hepatoprotective effect, and the mechanism is connected to the regulation of metabolic disorders and intestinal flora.

Study on the hypotensive effect and mechanism of hawthorn (Crataegus pinnatifida) fruits and hyperoside in spontaneously hypertensive rats.

Hawthorn fruits have a sweet and sour taste, besides having beneficial therapeutic effects on hyperlipidemia, hypertension, and coronary heart disease, making them widely used in food and clinical medicine. However, their hypotensive effects and potential mechanisms of anti-hypertension still need to be elucidated. This study aims to explore the antihypertensive effect of hawthorn and its monomer hyperoside on spontaneously hypertensive rats through pharmacodynamics, serum metabolomics, and in vivo mechanism studies. After 7 weeks of intervention with hawthorn extract and hyperoside, the blood pressure was significantly reduced. Aortic vascular staining results showed that the injury was significantly improved after intervention with hawthorn extract and hyperoside. According to the serum metabolomics study, the main metabolic pathway regulating blood pressure in hawthorn extract and hyperoside groups was the primary bile acid biosynthesis pathway. Quantitative experiments confirmed that the level of bile acid in the model group was significantly different from that in the normal group, while that in the hawthorn group and the hyperoside group was close to that in the normal group. Based on the prediction of bile acid-hypertension related targets and the literature, nine genes involved in bile acid metabolism and inflammatory pathways were selected for further study. The FXR, TGR5, ET-1, NOS3, Akt1, TNF-α, Ptgs2, ACE2 and Kdr mRNA expression levels in the hawthorn extract and hyperoside groups were significantly different from those in the model groups. In summary, hawthorn extract and hyperoside have a hypotensive effect on spontaneously hypertensive rats through bile acid and inflammation related targets. Hence, hawthorn extract has the potential to become a functional food or an alternative therapy for hypertension.

Recovery from indomethacin-induced gastrointestinal bleeding by treatment with teprenone

BackgroundGastrointestinal injuries caused by nonsteroidal anti-inflammatory drugs (NSAIDs) is a serious side effect in patients with rheumatoid arthritis (RA). However, effective therapeutic strategies have yet to be established. In this study, we investigated the therapeutic effects of teprenone (TEP), a gastric mucosal protective drug, on NSAID-induced gastrointestinal injuries in rats with RA (AA rats).MethodsGastrointestinal injury was induced by oral administration of indomethacin (IMC), a typical NSAID. TEP was orally administered after IMC-induced gastrointestinal bleeding, and the stomach, jejunum, and ileum were excised.ResultsOn day 14 of IMC administration, lesion areas in the stomach, jejunum, and ileum were significantly larger in AA rats than in normal rats. When TEP was orally administered to AA rats, the lesion areas in the stomach, jejunum, and ileum significantly decreased compared with those in control rats (IMC-induced AA rats). Therefore, we measured NOS2 mRNA and NO levels, which were significantly decreased in rats with IMC-induced AA after treatment with TEP.ConclusionsThese results suggest that the oral administration of TEP may be useful for the treatment of NSAID-induced gastrointestinal injuries in patients with RA.

Internal ribosomal entry site-mediated translational activity of nitric oxide synthase 2

ABSTRACT The internal ribosome entry site (IRES) is a unique structure found in the 5’ untranslated region (5'-UTR) of specific messenger RNAs (mRNAs) that allows ribosomes to bind and initiate translation without the need for a cap structure. In this study, we investigated the presence and functional properties of the IRES activity of nitric oxide synthase 2 (NOS2) mRNA, which encodes an enzyme that produces nitric oxide in response to various stimuli such as inflammation. Nitric oxide is a signaling molecule that plays a crucial role in various physiological processes, including immune responses and neuronal signaling. Our results showed the existence of IRES activity in the 5'-UTR of Nos2 mRNA in various cell types. IRES-mediated translation of NOS2 mRNA was higher in neuronal cells and its activity increased in response to lipopolysaccharide (LPS). Despite inhibition of cap-dependent translation, nitrite production was partially maintained. These results demonstrate the presence of IRES activity in the 5'-UTR of NOS2 mRNA and suggest that IRES-mediated translation plays a key role in controlling nitric oxide production in response to LPS, an inflammatory stimulus.

GLP1 receptor agonists exhibit antioxidant and anti-inflammatory properties in human heart tissue via the canonical GLP1 receptor expression through a AMPK-mediated pathway

Abstract Background Clinical research has demonstrated that glucagon-like peptide-1 receptor (GLP1R) agonists (GLP1RA) can decrease the occurrence of major cardiovascular events in diabetic patients, regardless of their glycemic control. However, a significant challenge in understanding the positive effects of GLP1RA has been the lack of knowledge regarding the specific cellular types that express GLP1R. Despite the established cardiovascular benefits of GLP1RA, previous studies have been unable to confirm the expression of GLP1R in the heart. Aim The study aimed to investigate the expression and function of GLP1R in heart tissue. Methods We collected human cardiac tissue samples from 30 patients who underwent left ventricle (LV) biopsies (n=21) or right atrial (RA) appendage closure (n=9) at a university hospital. Gene expression levels were assessed using RT-qPCR, protein levels by Western blot analysis, in situ tissue localization of proteins by immunofluorescence (IF) staining, and the level of oxidative stress using dihydroethidium. Results LV biopsies and RA appendages showed a substantial heterogeneity in GLP1R mRNA levels. GLP1R mRNA levels exhibited a positive correlation with IL1B, IL6, TNFα, CCL2, CD68, ACE1, AT1R, and VCAM1 mRNA levels, but a negative correlation with NOS3 mRNA levels. High GLP1R expression in both RA appendages and LV biopsies was associated with increased oxidative stress levels, which were mitigated by the antioxidant N-acetylcysteine (NAC), NADPH oxidase inhibitor (VAS-2870), ACE inhibitor (perindoprilat), AT1R antagonist (losartan), TNF-α receptor neutralizing antibody (infliximab) and by GLP1R agonists (liraglutide and semaglutide) with inhibitory effects amounting to about 50-60 %. Exendin, a GLP1R antagonist reversed the effect of liraglutide and semaglutide which accounted for a receptor-mediated antioxidant effect. H-89, a PKA inhibitor equally reversed liraglutide and semaglutide effect on ROS generation suggesting a canonical GLP1R mediated effect involving the AMPK pathway. Conclusion These findings indicate that human cardiac GLP1R expression levels are correlated to low-grade inflammation and endothelial dysfunction. Increased expression levels of GLP1R were associated with higher levels of oxidative stress sensitive to GLP1RA in a canonical GLP1R AMPK-mediated manner. Therefore, the cardiovascular beneficial effects of GLP1RA might be attributable to their antioxidant and anti-inflammatory properties, specifically within the cardiac system.

BRD4 Regulates Glycolysis-Dependent Nos2 Expression in Macrophages Upon H pylori Infection

Metabolic reprogramming is essential for the activation and functions of macrophages, including bacterial killing and cytokine production. Bromodomain-containing protein 4 (BRD4) has emerged as a critical regulator of innate immune response. However, the potential role of BRD4 in the metabolic reprogramming of macrophage activation upon Helicobacter pylori infection remains unclear. Bone marrow-derived macrophages (BMDMs) from wild-type (WT) and Brd4-myeloid deletion conditional knockout (Brd4-CKO) mice were infected with H pylori. RNA sequencing was performed to evaluate the differential gene expression between WT and Brd4-deficient BMDMs upon infection. An invivo model of H pylori infection using WT and Brd4-CKO mice was used to confirm the role of BRD4 in innate immune response to infection. Depletion of Brd4 in BMDMs showed impaired H pylori-induced glycolysis. In addition, H pylori-induced expression of glycolytic genes, including Slc2a1 and Hk2, was decreased in Brd4-deficient BMDMs. BRD4 was recruited to the promoters of Slc2a1 and Hk2 via hypoxia-inducible factor-1α, facilitating their expression. BRD4-mediated glycolysis stabilized H pylori-induced nitric oxide synthase (Nos2) messenger RNA to produce nitric oxide. The NO-mediated killing of H pylori decreased in Brd4-deficient BMDMs, which was rescued by pyruvate. Furthermore, Brd4-CKO mice infected with H pylori showed reduced gastric inflammation and increased H pylori colonization with reduced inducible NO synthase expression in gastric macrophages. Our study identified BRD4 as a key regulator of hypoxia-inducible factor-1α-dependent glycolysis and macrophage activation. Furthermore, we show a novel regulatory role of BRD4 in innate immunity through glycolysis to stabilize Nos2 messenger RNA for NO production to eliminate H pylori infection.

Duhuo Jisheng decoction alleviates neuroinflammation and neuropathic pain by suppressing microglial M1 polarization: a network pharmacology research

BackgroundNeuropathic pain (NP) is the most prevalent form of chronic pain resulting from nerve damage or injury. Despite the widespread use of Duhuo Jisheng decoction (DHJSD) in traditional Chinese medicine (TCM) to treat chronic pain, the mechanism underlying its analgesic action remains unclear.MethodsUsing network pharmacology, we obtained DHJSD and NP-related target information from public databases to construct protein–protein interactions (PPI) and compound-target networks based on common target genes. These networks were further analyzed using gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG). The interaction between molecules was verified through molecular docking using AutoDock Tools software. Additionally, we treated a chronic constriction injury (CCI) rat model with DHJSD and determined the mechanical withdrawal threshold (MWT). We used an enzyme-linked immunosorbent assay kit to determine the levels of inflammatory cytokines. Furthermore, qRT-PCR was employed to analyze ACHE, NOS2, MAPK3, PTGS2, AKT1, and PPARG mRNA expression, and immunofluorescence was used to evaluate changes in microglia.ResultsOur screening of compounds and targets identified 252 potential targets of DHJSD associated with NP. PPI analysis, along with GO and KEGG analyses, revealed that the potential mechanism of DHJSD in NP treatment may be related to inflammatory reactions, the IL-17 signaling pathway, MAP kinase activity, and endocrine activity. Based on molecular docking, the core target showed significant affinity for DHJSD's active components. Moreover, DHJSD treatment repaired the CCI-induced inflammatory reaction in the spinal cord while regulating the expression of ACHE, NOS2, MAPK3, PTGS2, AKT1, and PPARG mRNA. Immunofluorescence results indicated that the active components of DHJSD may regulate microglial M1 polarization to improve neuroinflammation, PPARG may have been involved in the process.ConclusionThe multi-component, multi-target, and multi-pathway actions of DHJSD provide new insights into its therapeutic mechanism in NP.

P38γ and p38δ modulate innate immune response by regulating MEF2D activation.

Evidence implicating p38γ and p38δ (p38γ/p38δ) in inflammation are mainly based on experiments using Mapk12/Mapk13-deficient (p38γ/δKO) mice, which show low levels of TPL2, the kinase upstream of MKK1-ERK1/2 in myeloid cells. This could obscure p38γ/p38δ roles, since TPL2 is essential for regulating inflammation. Here, we generated a Mapk12D171A/D171A/Mapk13-/- (p38γ/δKIKO) mouse, expressing kinase-inactive p38γ and lacking p38δ. This mouse exhibited normal TPL2 levels, making it an excellent tool to elucidate specific p38γ/p38δ functions. p38γ/δKIKO mice showed a reduced inflammatory response and less susceptibility to lipopolysaccharide (LPS)-induced septic shock and Candida albicans infection than wild-type (WT) mice. Gene expression analyses in LPS-activated wild-type and p38γ/δKIKO macrophages revealed that p38γ/p38δ-regulated numerous genes implicated in innate immune response. Additionally, phospho-proteomic analyses and in vitro kinase assays showed that the transcription factor myocyte enhancer factor-2D (MEF2D) was phosphorylated at Ser444 via p38γ/p38δ. Mutation of MEF2D Ser444 to the non-phosphorylatable residue Ala increased its transcriptional activity and the expression of Nos2 and Il1b mRNA. These results suggest that p38γ/p38δ govern innate immune responses by regulating MEF2D phosphorylation and transcriptional activity.

Уровень мРНК гена Nos2 как маркер фиброгенеза печени крыс, индуцированного тиоацетамидом

The aim of this paper was to evaluate the potential of the mRNA level of the Nos2 gene as a marker of fibrogenesis in rats at different stages of thioacetamide-induced liver fibrosis and cirrhosis. Materials and methods. The experiment involved 117 mature male Wistar rats weighing 190–210 g. Liver fibrosis and cirrhosis were induced by a thioacetamide solution administered through a gastric catheter at a dose of 200 mg per 1 kg of body weight 2 times a week. The dynamics of the process was studied at 9 timepoints over the course of 17 weeks. Nos2 mRNA level in the liver was detected by means of real-time polymerase chain reaction. The stage of fibrosis was determined in histological sections stained using the Mallory method according to the Ishak semi-quantitative scale. Results. Throughout the experiment, Nos2 mRNA had practically no reaction to the thioacetamide-induced liver fibrogenesis. At the mild fibrosis stage (F1), an insignificant rise in the level of Nos2 mRNA was noted (within 5 %). Intensive development of fibrosis (F2–F4/F5) and an increase in the production of extracellular matrix components were accompanied by an increase in the level of Nos2 mRNA with a peak value exceeding the initial level (control group value) by the factor of 1.69 (р < 0.05). At the point of transition of fibrosis to cirrhosis (F5), a decrease in the level of mRNA of the target gene was observed, and at the stage of definite cirrhosis (F6), a subsequent drop below the initial level was detected. Thus, according to the data obtained, Nos2 mRNA has the greatest potential as a marker of liver fibrogenesis at the stage of advanced fibrosis, but cannot act as a marker at the early stages. Moreover, Nos2 mRNA level cannot be used as a marker when assessing the degree of cirrhosis and its development dynamics.

Effect of spermidine on endothelial function in Systemic Lupus Erythematosus (SLE)

Impaired endothelial function has been shown in SLE patients and lupus-prone mice. Emerging evidence supports impaired mitochondrial dynamics and mitophagy contribute to SLE and endothelial dysfunction. Spermidine is a natural polyamine that stimulates mitophagy by activating the PINK1-Parkin pathway and can improve endothelial function. However, the effect of spermidine on mitophagy and vascular function in SLE has not been explored. To address this gap, 9-week-old female lupus-prone (MRL/lpr) and healthy control (MRL/MpJ) mice were randomly assigned into one of two groups, spermidine treatment (Lpr T and MpJ T) or control (Lpr C and MpJ C). Treated mice received spermidine (3mM) via drinking water for 8 weeks. After 8 weeks of treatment, endothelium-dependent vasorelaxation to acetylcholine (ACh) and endothelium-independent vasorelaxation to sodium nitroprusside (SNP) were measured in thoracic aortas using a wire myograph. Maximal responses to ACh were significantly impaired in Lpr C (67.1 ± 8.7, n = 11) compared to Mp C (90.3 ± 3.1, n = 13) (p = 0.01). Spermidine improved endothelial function in thoracic aorta from Lpr T (90.3 ± 2.8, n = 13) compared to Lpr C (p = 0.03). Maximal responses to SNP were not significantly different across the groups. Thoracic and abdominal aortas were used for measurements of protein content (n = 3-4/group) and gene expression (n = 4/group), respectively. There were strain differences in the mRNA expression of endothelial nitric oxide synthase ( Nos3) between Lpr C (0.65 ± 0.14) and MpJ C (1.00 ± 0.16) (p = 0008). In MpJ mice, Nos3 mRNA expression was significantly higher in the aorta from the MpJ T (1.47 ± 0.39) compared to MpJ C (p = 0.04). No significant differences were found in the Nos3 expression between Lpr groups. Vascular cell adhesion molecule 1 (Vcam1) is an indicator of inflammation and is associated with atherosclerosis susceptibility. Protein content of Vcam1 was significantly lower in Lpr T (3.73 ± 0.61) compared to Lpr C (8.49 ± 2.58) (p = 0.04) and gene expression of Vcam1 was also lower in Lpr T compared to Lpr C, suggesting spermidine decreases the inflammatory responses in vessels from SLE mice. Protein content of Parkin was measured as a marker of mitophagy. Parkin was significantly lower in Lpr C (0.38 ± 0.61) compared to MpJ C (1.00 ± 0.09) (p = 0.004), suggesting lower mitophagy in aorta from SLE mice. Parkin content in Lpr T was similar to MpJ C. Collectively, these results demonstrate the beneficial effects of spermidine treatment on endothelial function in SLE mice and suggest inflammation and altered mitophagy contribute to endothelial dysfunction in SLE. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.