COX-2 mRNA Research Articles

Endarachne binghamiae Extract Ameliorates Inflammatory Responses in Macrophages Through Regulation of MAPK, NF-kB and PI3K/AKT Pathways, and Prevents Acute Lung Injury in Mice.

In this study, the anti-inflammatory effect of the hot water extract of Endarachne binghamiae (EB-WE), a type of marine brown algae, was investigated in LPS-stimulated RAW 264.7 cells and an acute lung injury (ALI) mouse model induced by intranasal LPS administration. Treatment with EB-WE significantly inhibited NO and pro-inflammatory cytokine (TNF-a and IL-6) production in LPS-stimulated RAW 264.7 cells. In mRNA analysis, the expression of pro-inflammatory cytokines, COX-2, and iNOS mRNAs, was down-regulated by EB-WE treatment. The phosphorylation of MAPK, IkB, and PI3K/AKT molecules responsible for signal pathways during inflammation in LPS-stimulated macrophages was also significantly inhibited by EB-WE. In an in vivo model for ALI, oral administration of EB-WE significantly reduced the level of pro-inflammatory cytokines (TNF-a, IL-1b, and IL-6) and chemokines (MCP-1, CXC-16, CXCL1, and TARC) in serum or bronchoalveolar lavage fluid (BALF) of mice. Similarly to the results in LPS-stimulated RAW 264.7 cells, treatment with EB-WE significantly inhibited intracellular signal pathways mediated by MAPK, IkB, and PI3K/AKT in lung tissues of mice with ALI, and also decreased the expression of mRNAs of inflammatory mediators such as TNF-a, IL-6, iNOS, and COX-2. Furthermore, the inhibitory effect of EB-WE on ALI was apparently confirmed in histological examination through lung tissue staining. Taken together, it is clear that EB-WE has potential activity to effectively ameliorate the inflammatory responses in macrophages through down-regulation of MAPK, NF-kB, and PI3K/AKT activation, and suppress acute lung injury induced by LPS. These findings strongly suggest that EB-WE is a promising natural product beneficial for developing preventive treatments and cures of inflammation-related diseases.

Cannabis sativa L. Extract Increases COX-1, COX-2 and TNF-α in the Hippocampus of Rats with Neuropathic Pain.

Inflammation is the critical component of neuropathic pain; therefore, this study aimed to assess the potential anti-inflammatory effects of Cannabis sativa L. extracts in a vincristine-induced model of neuropathic pain. The effects of different doses (5.0-40.0 mg/kg) of two Cannabis sativa L. extracts (B and D) on COX-1, COX-2, TNF-α, and NF-κB mRNA and protein levels were examined in the rat hippocampus, cerebral cortex, and blood lymphocytes. There were statistically significant differences in COX-1, COX-2, and TNF-α mRNA and protein expression in the hippocampus and cerebral cortex, with significant differences in COX-2 and TNF-α in the lymphocytes. Extract D dose-dependently increased COX-1 mRNA and protein in the hippocampus and cortex. In contrast, Extract B dose-dependently increased COX-1 mRNA and decreased COX-2 mRNA (in a dose of 7.5 mg/kg) and TNF-α protein levels in the cortex. Cannabis sativa L. extracts significantly influenced the expression of inflammatory genes and proteins, with effects varying based on dose and tissue type. The increased expression of COX-1, COX-2, and TNF-α (in comparison to groups receiving NaCl, vincristine, and gabapentin) in the rat hippocampus and COX-1 in the cerebral cortex suggests that Cannabis may have a pro-inflammatory effect. Due to species specificity, the results of our research based on rats require confirmation in humans. However, Cannabis sativa should be recommended with caution for treating pain with an inflammatory component.

Down-regulation of interlinked inflammatory signalling cascades by ethanolic extract of Suaeda fruticosa Forssk. ex J.F. Gmel. attenuated in vivo inflammatory and nociceptive responses.

Juice and decoction of leaves of Suaeda fruticosa, a halophytic medicinal plant of Cholistan desert, is traditionally used to treat rheumatism. The current study was carried out to probe into in vivo anti-nociceptive, anti-inflammatory, and anti-arthritic potential of ethanolic extract of the whole plant of S. fruticosa (Et-SF)and its bioactive molecules. GC-MS screening of Et-SF revealed presence ofvarious bioactive compounds including phytol, thymol, n-hexadecanoic acid,farnesol, and 1-heptacosanol. DPPH in vitro radical scavengingassay demonstrated moderate antioxidant potential of Et-SF. Safety evaluation of Et-SF confirmed no lethal effects in female albino rats up to the singleoral dose of 5000mg/kg. In all invivo models, Et-SF was administered in three doses (125, 250, and 500mg/kg) and a single dose of flurbiprofen (FP) (10mg/kg). Et-SF significantly (p < 0.05) attenuated acute inflammation in carrageenan, histamine, and serotonin-induced rat paw oedema models in a time-dependent manner. Et-SF alleviated oedema, restored haematological parameters, and reduced severe pannus formation, inflammatory cell infiltration, and fibrous tissue proliferation inthe paws of CFA-induced arthritic rats.Moreover, treatment with thymol, farnesol and n-hexadecanoic acid alone and in combination also attenuated the arthritic progression in the arthritic rats indicating involvementof these compounds towards anti-arthritic potential of Et-SF. Et-SF and FP significantly (p < 0.05) down-regulated IL-1β, TNF-α, IL-6, NF-κB, and COX-2 mRNA expression, and up-regulated IL-4 and IL-10 mRNA expression in arthritic rats. Hot plate and acetic acid-induced writhing models results indicated the analgesic attributes of Et-SF in mice models. This study suggests that S. fruticosa ethanol extract may regulate the expression ofinflammatory markers involved in nociceptive, inflammatory, and arthritic disorders. Its phytochemicals could target multiple phases of these conditions at cellular and subcellular levels. Further research is needed to confirm this hypothesis.

Low Prostaglandin E2 but High Prostaglandin D2, a Paradoxical Dissociation in Arachidonic Acid Metabolism in Aspirin-Exacerbated Airway Disease: Role of Airway Epithelium.

In patients with aspirin-exacerbated respiratory disease (AERD), there is disparate regulation of prostaglandin E2 (PGE2) and prostaglandin D2 (PGD2). Both prostanoids are synthesised by cyclooxygenase 1 (COX-1) and cyclooxygenase 2 (COX-2). However, while the basal synthesis of PGE2 tends to decrease, that of PGD2 increases in patients with AERD. Furthermore, both behave differently in response to the inhibitory action of NSAIDs on COX-1: PGE2 levels decrease while PGD2 increases. Increased PGD2 release correlates with nasal, bronchial, and extra-pulmonary symptoms caused by aspirin in AERD. The proposed hypothesis establishes that the answer to this paradoxical dissociation can be found in the airway epithelium. This is based on the observation that reduced COX-2 mRNA and/or protein expression is associated with reduced PGE2 synthesis in cultured fibroblast and epithelial cells from AERD compared to patients with asthma who are aspirin-tolerant and healthy subjects. The low production of PGE2 by the airway epithelium in AERD results in an excessive release of alarmins (TSLP, IL-33), which in turn contributes to activating group 2 innate lymphoid cells (ILC2s) and PGD2 synthesis by mast cells and eosinophils. Aspirin, by further increasing the diminished PGE2 regulation capacity in AERD, leads to respiratory reactions associated with the surge in PGD2 from mast cells and eosinophils. In summary, the downregulation of COX-2 and the subsequent low production of PGE2 by airway cells account for the apparently paradoxical increased production of PGD2 by mast cells and eosinophils at the baseline and after aspirin provocation in patients with AERD. A better understanding of the role of the airway epithelium would contribute to elucidating the mechanism of AERD.

Dual anti-inflammatory activities of COX-2/5-LOX driven by kratom alkaloid extracts in lipopolysaccharide-induced RAW 264.7 cells

Cyclooxygenase (COX) and lipoxygenase (LOX) enzymes play a pivotal role in producing pro-inflammatory eicosanoids, including prostaglandins (PGs) and leukotrienes (LTs), in the inflammation process. Mitragynine is a primary alkaloid contained in the kratom’s leaves and has been reported to show anti-inflammatory activity by suppressing COX-2 mRNA translation to lowering PGs synthesis. In this study, the Kratom’s alkaloid extract containing ~ 46% mitragynine was found to exhibit dual inhibition activity towards COX-2/5-LOX enzymes at concentrations below 25 ppm in the LPS-induced RAW 264.7 macrophage cells. At these levels, no cell toxicity was observed while the cells became death (e.g., 10–46% viability at 50–100 ppm) and only COX-2 inhibition activity was observed after exposed with more than 25 ppm of alkaloid extract. In contrast, the methanolic-crude extract of Kratom’s leaf containing ~ 5% mitragynine showed no inhibition toward COX-2/5-LOX enzymes and did not toxic onto the cells, even after treated at 100 ppm. The alkaloid extract suppressed several antiinflammation parameters, including ROS (64% reduction at 25 ppm), NO (30% reduction at 25 ppm), TNF-α (~ 50% reduction at 25 ppm), and IL-6 production (60% reduction at 6.25 ppm). In silico molecular studies indicated strong binding affinity of Kratom alkaloids to COX-2 and 5-LOX active sites, supporting the Kratom’s alkaloids to have great potential dual inhibition activity towards COX-2/5-LOX enzymes and to be developed as a safer NSAIDs with fewer side effects.

Amelioration of Toll-like Receptor-4 Signaling and Promotion of Mitochondrial Function by Mature Silkworm Extracts in Ex Vivo and in Vitro Macrophages.

The unknown immune-enhancing effects of steamed mature silkworms (Bombyx mori L.), known as HongJam (HJ), were investigated. Supercritical fluid extracts from the White Jade variety of HJ (WJ-SCEs) were applied to in vitro RAW264.7 macrophages (RAWMs) and ex vivo bone marrow-derived macrophages (BMDMs). WJ-SCE enhanced the proliferation and viability of both RAWMs and BMDMs. Supplementation with WJ-SCE significantly reduced the lipopolysaccharide (LPS)-induced expression of iNOS mRNA and protein, resulting in decreased nitric oxide (NO) production. Additionally, WJ-SCE lowered the mRNA and protein expression of COX-2 and reduced the levels of pro-inflammatory cytokines. The mitochondrial function, ATP levels, and reactive oxygen species levels in LPS-treated macrophages were restored following WJ-SCE treatment. WJ-SCE modulated LPS-Toll-like receptor-4 (TLR-4) signaling by reducing the levels of phosphorylated (p)-p38, p-ERK1/2, and p-p65. WJ-SCE also restored gene expression related to cytokines, chemokines, glucose transport, mitochondrial metabolism, and TLR-4 signaling, suggesting the inhibition of pro-inflammatory M1 macrophage polarization. Furthermore, WJ-SCE enhanced macrophage phagocytic and pinocytotic activity. WJ-SCE demonstrated anti-inflammatory effects by inhibiting LPS-induced M1 polarization in both macrophage types, potentially suppressing chronic inflammation while enhancing phagocytosis and pinocytosis.

Anti-inflammatory diterpenoids from the brown alga Dictyota coriacea.

A new xenicane diterpene named 4α-acetoxyisodictyohemiacetal (1) was isolated from the brown alga Dictyota coriacea, along with 11 known diterpenoids. The structure of 1 was established by spectroscopic analyses, and its absolute configuration was determined by comparing the experimental and theoretical electronic circular dichroism (ECD) spectra. Two dictyodiacetal diastereomers (5 and 6) were isolated and the full NMR assignments were performed. The anti-inflammatory activities of the isolated compounds were evaluated using the lipopolysaccharide-stimulated mouse macrophage cell line, RAW264. The hemiacetal-containing xenicanes and dictyol-type diterpene inhibited not only the production of nitric oxide, but also the expression of inducible nitric oxide synthase, interleukin-6, and cyclooxygenase-2 mRNA in RAW264 cells. This study demonstrated that diterpenes from Dictyota coriacea may be useful as natural anti-inflammatory agents.

Administration of a combination of COX-2/TGF-β1 siRNAs induces hypertrophic scar fibroblast apoptosis through a TP53 mediated caspase pathway

Hypertrophic scar (HTS) formation is a pathological fibrotic skin disease, with no satisfactory treatments available currently. Inducing apoptosis of HTS-derived fibroblasts (HSFs) are becoming promising approaches. In this research, we aim to improve the technology with co-delivery COX-2 and TGF-β1 siRNAs and further investigate the underlying mechanism. Firstly, the HSFs were transfected with 1 µg/ml COX-2 and/or TGF-β1 siRNAs, and proved that the apoptosis of HSFs was greater induced by COX-2/TGF-β1 siRNAs than either COX-2 or TGF-β1 siRNA alone by flow cytometry. To investigate the impact of co-silencing TGF-β1 and COX-2 mRNA expression in vivo, we established HTSs model in rat tails. Our results confirmed that co-silencing of TGF-β1 and COX-2 mRNA expression could significantly alleviate the HTS formation in vivo. Furthermore, we explored the potential molecular mechanism and revealed that the protein levels of TP53, Bcl-2 and Caspase-3 were downregulated while Bax and Cleaved Caspase-3 were upregulated in the COX-2/TGF-β1 siRNA groups compared with HKP group. Taken together, our results demonstrated that simultaneous silencing of COX-2 and TGF-β1 expression by siRNAs induced HSF apoptosis through a TP53 mediated caspase pathway. Therefore, COX-2/TGF-β1 siRNAs might serve as a novel and effective therapeutic alternative for HTSs treatments.

High Humidity Alters Myeloid-Derived Suppressor Cells in Spleen Tissue: Insights into Rheumatoid Arthritis Progression.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by joint inflammation and bone destruction, leading to severe complications. Previous research has suggested that high humidity conditions may exacerbate RA, however, the underlying mechanisms remain unclear. Furthermore, there is a lack of evidence linking humidity to the worsening of RA symptoms in animal models. The Collagen-induced arthritis (CIA) mouse model was established using C57BL/6 mice. The arthritis status of the mice was evaluated under two distinct humidity conditions (50% and 80%). The aim of the present study was to investigate the impact of elevated humidity levels on the types of splenic cells present using mass spectrometry flow. Additionally, the study utilized MDSCs, which are significantly upregulated by high humidity, to assess the levels of oxidative stress and conducted mRNA sequencing of sorted MDSCs to investigate their impact on arthritis in CIA mice. Compared to normal humidity, high humidity exacerbated arthritis incidence in mice, resulting in increased arthritis scores, swelling, serum autoantibodies (anti-COII and anti-CCP), and upregulation of pro-inflammatory cytokines. Significant variations were observed in the spleen index under high humidity condition, accompanied by noticeable inflammatory alterations. Moreover, elevated humidity levels induced a substantial modulation in MDSCs population in the spleen of CIA mice, along with alterations in oxidative stress markers such as heightened serum ROS levels, and increased expression of COX, SOD, and Nrf2 mRNA. Following successful sorting of MDSCs, mRNA sequencing revealed a decrease in the expression of Rap1 signaling pathway under high humidity environment, which may contribute to the increase of MDSCs cells and aggravate the progression of RA disease. A comprehensive analysis of the available data reveals a detrimental impact of high humidity on MDSCs numbers within spleen tissue, with potential implications for the development of RA.

SR-16234, a Unique Selective Estrogen Receptor Modulator, Suppressed Proliferation and Pain-Related Factor Expression by Inhibition of the Nuclear Factor-kappa B Pathway in Endometriotic Stromal Cells.

What is the effect of SR-16234 (SR), a selective estrogen receptor (ER) modulator, on human endometriotic stromal cells (ESCs)? Endometriotic tissues were obtained from 21 patients undergoing laparoscopic surgery for ovarian endometriomas (OEs). Normal eutopic endometrium during the luteal phase was obtained from 18 patients without endometriosis. ESCs isolated from OEs and normal eutopic endometrial stromal cells (NESCs) were cultured with SR and subsequently exposed to tumor necrosis factor (TNF)-α. After 48 h of incubation, the effect of SR on cell proliferation was evaluated by the WST-8 assay. The gene expressions of inflammatory and pain-related factors, including interleukin (IL)-6, IL-8, cyclooxygenase (COX)-2, transient receptor potential vanilloid (TRPV)1, ESR1, and ESR2, were evaluated by real-time RT-PCR. The phosphorylation of Inhibitor κBα (IκBα), extracellular signal-regulated kinase (ERK)1/2, and Protein Kinase B (AKT) were evaluated by western blot analysis. ILs, prostaglandin (PG) E2, and intranuclear p65 syntheses were assessed by ELISA. SR treatment repressed TNF-α-induced proliferation by 20% in ESCs but not NESCs. SR also reduced IL-6, IL-8, COX-2, TRPV1, ESR1, and ESR2 mRNA expressions and ILs protein, and PGE2 synthesis in ESCs, whereas in NESCs, only TRPV1 mRNA expression was decreased. SR suppressed TNF-α-induced phosphorylated IκBα levels by approximately 50%, and intranuclear p65 protein was reduced by 30% compared to addition of only TNF-α in ESCs. However, SR did not affect the phosphorylation of AKT and ERK1/2. SR appears to be a potential therapeutic agent for endometriosis by suppressing inflammatory and pain-related factor expressions by inhibiting the nuclear factor-kappa B pathway.

Physicochemical properties, antioxidant and anti-inflammatory activities of polysaccharides from lotus leaves by pectinase-assisted extraction

In order to improve the extraction yield of lotus leaf polysaccharide (LLP) and offer scientific foundation for utilization of LLPs in the food and pharmaceutical industries, pectinase-assisted extraction method was employed for the preparation of LLP. The chemical composition, physicochemical properties (e.g. rheological and thermal properties etc.), antioxidant and anti-inflammatory effects of LLP and its two purified fractions (LLP1 and LLP3) obtained using DEAE-52 and subsequent Sephadex G-100 column chromatography were also investigated. Antioxidant assays indicated that the polysaccharides had remarkable DPPH• and ABTS+• scavenging activities. In H2O2-induced RAW264.7 cells, LLPs showed marked protective effect on oxidative stress, reducing the MDA and ROS contents and increasing the CAT and SOD activities. LLPs were also found to effectively modulate the expressions of CAT, SOD1, Nrf-2, HO-1, NQO1 and Keap1 mRNA. Thus, the potent antioxidant effects of LLPs were achieved possibly by regulating the Keap1/Nrf-2/HO-1 signaling pathways. In lipopolysaccharide-treated RAW264.7 macrophages, LLPs reduced phagocytic ability, decreased release of NO, TNF-α, IL-1β and IL-6, and increased release of IL-10 and TGF-β1, indicating their potent anti-inflammatory effect. LLPs downregulated expressions of iNOS, IL-1β, IL-6, TNF-α, COX-2, and TLR-4 mRNA. Therefore, modulation of NF-κB signaling pathway is possibly the important anti-inflammatory mechanism of LLPs.

Pharmacological investigations of newly synthesized oxazolones and imidazolones as COX-2 inhibitors

Oxazoles and Imidazoles are heterocyclic compounds with significant biological activities. The present study explores the pharmacological effects of some new oxazole and imidazole derivatives as potential COX-2 inhibitors. Docking studies of the compounds against targeted proteins COX-2 and TACE manifested good binding affinities for both the targets supporting their anti-inflammatory potential. Compounds (3F-A, 3F-B, N-A, N-B) were evaluated for in vivo anti-inflammatory effects by carrageenan-induced paw edema. Among all, compound N-A was found to be the most effective as it displayed most pronounced reduction in inflammation that was comparable to indomethacin. The in vivo tissue antioxidant activity was performed for estimation of the level of catalase, GSH, GST, and thiobarbituric acid in paw tissue. The results exhibited that targeted compounds improved the oxidative stress and restored the expression of enzymes. H &E staining revealed that aforesaid compounds displayed well-defined restoration of cellular damage. Compound NA exhibited maximum structural and functional preservation. Reduction in the expression of inflammatory markers was also analyzed by ELISA and maximum reduction in protein expression (COX-2 and TNF-a) was observed for compound N-B. Quantification of mRNA was done using PCR and a decrease in the expression of COX-2 mRNA level in treatment groups was depicted by all the new compounds but N-B showed maximum reduction in enzyme expression. All the results obtained from the present study have shown the significant anti-inflammatory potential of new compounds via the COX-2 inhibition pathway.

Multifunctional poloxamer-based thermo-responsive hydrogel loaded with human lactoferricin niosomes: In vitro study on anti-bacterial activity, accelerate wound healing, and anti-inflammation

Chronic wound infections are attributed to delayed tissue repair, which remains a major clinical challenge in long-term health care. Particularly, infections with antibiotic resistance have more serious effects on health, often resulting in unsuccessful treatments. Thus, antimicrobial peptide (AMP)-based therapy holds promise as a potential therapeutic approach to overcoming drug resistance. Conventional wound dressing is a passive strategy for wound care that is not capable of eradicating pathogens and promoting tissue repair. In this study, we aim to construct an advanced wound dressing; a thermo-responsive hydrogel incorporated with lactoferricin (Lfcin) niosome (Lfcin-Nio/hydrogel) for bacterial pathogen treatment. The Lfcin-loaded niosome (Lfcin-Nio) has a particle size of 396.91 ± 20.96 nm, 0.38 ± 0.01 of PdI, −10.5 ± 0.3 mV of ζ potential, and 72.30 ± 7.05 % Lfcin entrapment efficiency. Lfcin-Nio exhibited broad antibacterial activity on both drug-susceptible and drug-resistant strains, and also on bacteria residing in the biofilm matrix. The Lfcin-Nio/hydrogel was fabricated from 0.5 % w/v poloxamer 188–20 % w/v poloxamer 407, and supplemented with Lfcin-Nio and epidermal growth factor (EGF). The physical properties of Lfcin-Nio/hydrogels showed elasticity, swelling ability, and strong injectability with responsiveness to 33–37 °C temperatures. The biological properties of Lfcin-Nio/hydrogels exhibited a bactericidal effect against drug-resistant strains of S. aureus and P. aeruginosa, and showed less toxicity to the human skin fibroblast. It also promoted the healing of scratches by 55 % within 6 h, compared to the wound closure rate of 20 % in the cell control. The inflammatory response of the Lfcin-Nio/hydrogel-treated cells was reduced via suppression of IL-1β and COX-2 mRNA expressions. From this study, Lfcin-Nio/hydrogels can be suggested as a modern wound dressing that possesses multifunctional and beneficial properties for the management of chronic wound infections.

Demethylzeylasteral alleviates inflammation and colitis via dual suppression of NF-κB and STAT3/5 by targeting IKKα/β and JAK2

BackgroundUlcerative colitis (UC) is a common inflammatory bowel disease and a risk factor of colorectal cancer. Demethylzeylasteral (DZT), a bioactive component mainly isolated from Tripterygium wilfordii, has been shown to inhibit inflammation and cancer. However, its anti-UC function and molecular mechanisms have not been well characterized. This study aims to explore the therapeutic effect and functional targets of demethylzeylasteral against UC. MethodsRT-qPCR, Western blot and ELISA were used to detect the generation of pro-inflammatory cytokines and chemokines in murine macrophage cells. Luciferase reporter gene, Western blot, pull-down, CETSA, DARTS, and virtual docking were employed to detect the anti-inflammatory targets and molecular mechanisms of demethylzeylasteral. The anti-inflammatory and anti-colitis effects of demethylzeylasteral were further determined in DSS-challenged mice. ResultsIn vitro, demethylzeylasteral inhibited NO and PGE2 production by suppressing the mRNA and protein expression of iNOS and COX-2, and suppressed the mRNA expression of TNF-α, IL-1β, IL-6, MCP-1, CXCL9, and CXCL10 in RAW264.7 macrophages stimulated by LPS/IFNγ. Furthermore, demethylzeylasteral was not only capable of inhibiting IKKα/β-NF-κB activation, but also able to block JAKs-STAT3/5 activation in LPS/INFγ-incubated RAW264.7 cells or DSS-exposed colon tissues of mice. Mechanistically, demethylzeylasteral was found to directly bind to IKKα/β and JAK2 kinases, leading to inactivation of pro-inflammatory signaling cascades and reduced generation of cytokines and chemokines. In vivo, oral administration of demethylzeylasteral significantly attenuated DSS-induced colitis, which was mainly manifested as mitigated symptoms of colitis, colonic mucosal barrier damage, and colonic inflammation. ConclusionWe demonstrated that demethylzeylasteral alleviated UC pathology by blocking NF-κB and STAT3/5 pathways via targeting IKKα/β and JAK2 kinases, raising the possibility that demethylzeylasteral could act as a candidate for the treatment of UC.

Abstract MP18: Evidence for An Important Physiological Role of Bradykinin B 2 Receptors in The Renomedullary Interstitial cells of The Kidney in Angiotensin II-induced Hypertension in Mice

Bradykinin is a potent vasoactive peptide of the kallikrein-kinin system and activates two different classes of G protein-coupled receptors, B 1 and B 2 , in the kidney. In the present study, we tested the hypothesis that deletion of B 2 receptors selectively in renomemdullary interstitial cells (RMICs) of the kidney medulla lowers basal blood pressure and attenuates angiotensin II (Ang II)-induced hypertension. To test the hypothesis, we generated a novel mouse model with deletion of B 2 receptors selectively in RMICs of the medulla (RMIC- Bdkrb2 -/- ) using inducible Tenascin-C-CreER2/Bdkrb2 fl/fl recombination approach. Mice with deletion of B 2 receptors selectively in RMICs developed and grew normally without structural abnormalities in the kidney medulla. Basal telemetry systolic (SBP), diastolic, and mean arterial blood pressure were significantly lower in RMIC- Bdkrb2 -/- than WT mice (WT: 123 ± 3 mmHg vs. RMIC- Bdkrb2 -/- : 106 ± 3 mmHg, P &lt;0.01) (n=12-15). The hypotensive phenotype in RMIC- Bdkrb2 -/- mice was associated with significant decreases in 24 h urine output (WT: 1.86 ± 0.15 mL/24 h vs. RMIC- Bdkrb2 -/- : 1.09 ± 0.15 mL/24 h, P &lt;0.05) and urine osmolality (WT: 1893 ± 441 mOsm/Kg H 2 O vs. RMIC- Bdkrb2 -/- : 1311 ± 78 mOsm/Kg H 2 O, P &lt;0.01) (n=12=15). Deletion of Bdkrb2 receptors in RMICs significantly decreases COX-2, eNOS, ENaC, and AQP2 mRNA expression ( P &lt;0.01 vs. WT), but not PGE 2 or prorenin receptor mRNA expression in the kidney medulla (n.s.) (n=6-10). In response to Ang II, SBP increased to 166 ± 6 mmHg in WT mice ( P &lt;0.01 vs. Basal), but it increased only to 139 ± 5 mmHg in RMIC- Bdkrb2 -/- mice ( P &lt;0.01 vs. WT+Ang II) (n=10-12). Ang II-induced hypertension was completely blocked by oral administration of AT 1 receptor blocker losartan (20 mg/kg/day, p.o.) in WT and RMIC- Bdkrb2 -/- mice (n=10). By comparison, infusion of DDAVP (1-deamino-8-D-arginine vasopressin, ~0.5 mg/kg/day, i.p., for 2 weeks) had no effect on blood pressure in WT and RMIC- Bdkrb2 -/- mice (n.s.) (n=8). Similarly, infusion of Ang II or DDAVP for 2 weeks significantly increased urine osmolality in WT mice ( P &lt;0.01), and restored urine osmolality in RMIC- Bdkrb2 -/- mice to the WT basal level (1995 ± 315 mOsm/Kg H 2 O, P &lt;0.01) (n=8-12). In conclusion, the present study demonstrates for the 1 st time that bradykinin B 2 receptors in RMICs of the kidney medulla plays an important role in maintaining basal blood pressure and urine osmolality homeostasis and the development of Ang II-induced hypertension.

Histological and Molecular Biological Changes in Canine Skin Following Acute Radiation Therapy-Induced Skin Injury.

Radiation therapy is a crucial cancer treatment, but it can damage healthy tissues, leading to side effects like skin injuries and molecular alterations. This study aimed to elucidate histological and molecular changes in canine skin post-radiation therapy (post-RT) over nine weeks, focusing on inflammation, stem cell activity, angiogenesis, keratinocyte regeneration, and apoptosis. Four male beagles received a cumulative radiation dose of 48 Gy, followed by clinical observations, histological examinations, and an RT-qPCR analysis of skin biopsies. Histological changes correlated with clinical recovery from inflammation. A post-RT analysis revealed a notable decrease in the mRNA levels of Oct4, Sox2, and Nanog from weeks 1 to 9. VEGF 188 levels initially saw a slight increase at week 1, but they had significantly declined by week 9. Both mRNA and protein levels of COX-2 and Keratin 10 significantly decreased over the 9 weeks following RT, although COX-2 expression surged in the first 2 weeks, and Keratin 10 levels increased at weeks 4 to 5 compared to normal skin. Apoptosis peaked at 2 weeks and diminished, nearing normal by 9 weeks. These findings offer insights into the mechanisms of radiation-induced skin injury and provide guidance for managing side effects in canine radiation therapy.

Nutraceutical formulation based on a synergic combination of melatonin and palmitoylethanolamide for the management of allergic events.

The management of allergic events is a growing global health issue, especially in industrialized countries. This disease is an immune-mediated process, regulated by the interaction of IgE with an allergen, resulting in mast cell activation, which concerns the release of several immune-inflammatory modulators, i.e., histamine, β-hexosaminidase, COX-2, IL-6, and TNF-α, responsible for the main allergic-reaction associated symptoms. The aim of the present study was the efficacy evaluation of an alternative remedy, an innovative nutraceutical formulation (NF) based on the synergic combination of melatonin (MEL) and palmitoylethanolamide (PEA) for the prevention and treatment of immune disease. At first, the intestinal bioaccessibility of PEA and MEL in NF was assessed at 1.6 and 36%, respectively. Then the MEL and PEA ability to modulate the release of immune-inflammatory modulators in the human mast cell line (HMC-1.2) at their bioaccessible concentration was investigated. Our results underline that NF treatment was able to reduce COX-2 mRNA transcription levels (-30% vs. STIM, p < 0.0001) in stimulated HMC-1.2 and to contract COX-2 enzymatic activity directly (IC50: 152 μg/mL). Additionally, NF showed valuable ability in reducing histamine and β-hexosaminidase release in stimulated HMC-1.2, as well as in decreasing TNF-α and IL-6 mRNA transcription levels and protein production.

Berberine Attenuates Acetamiprid Exposure-Induced Mitochondrial Dysfunction and Apoptosis in Rats via Regulating the Antioxidant Defense System.

Acetamiprid (ACMP) is a neonicotinoid insecticide that poses a significant threat to the environment and mankind. Oxidative stress and mitochondrial dysfunction are considered prime contributors to ACMP-induced toxic effects. Meanwhile, berberine (BBR) a natural plant alkaloid, is a topic of interest because of its therapeutic and prophylactic actions. Therefore, this study evaluated the effects of BBR on ACMP-mediated alterations in mitochondrial functions and apoptosis in rat liver tissue. Male Wistar rats were divided into four groups: (I) control, (II) BBR-treated, (III) ACMP-exposed, and (IV) BBR+ACMP co-treated groups. The doses of BBR (150 mg/kg b.wt) and ACMP (1/10 of LD50, i.e., 21.7 mg/kg b.wt) were given intragastrically for 21 consecutive days. The results showed that the administration of ACMP diminished mitochondrial complex activity, downregulated complex I (ND1 and ND2) and complex IV (COX1 and COX4) subunit mRNA expression, depleted the antioxidant defense system, and induced apoptosis in rat liver. BBR pre-treatment significantly attenuated ACMP-induced mitochondrial dysfunction by maintaining mitochondrial complex activity and upregulating ND1, ND2, COX1, and COX4 mRNA expression. BBR reversed ACMP-mediated apoptosis by diminishing Bax and caspase-3 and increasing the Bcl-2 protein level. BBR also improved the mitochondrial antioxidant defense system by upregulating mRNA expression of PGC-1α, MnSOD, and UCP-2 in rat liver tissue. This study is the first to evaluate the protective potential of BBR against pesticide-induced mitochondrial dysfunction in liver tissue. In conclusion, BBR offers protection against ACMP-induced impairment in mitochondrial functions by maintaining the antioxidant level and modulating the apoptotic cascade.

Anti-inflammatory and DNA Repair Effects of Astragaloside IV on PC12 Cells Damaged by Lipopolysaccharide.

This study aimed to establish a neural cell injury model in vitro by stimulating PC12 cells with lipopolysaccharide (LPS) and to examine the effects of astragaloside IV on key targets using high-throughput sequence technology and bioinformatics analyses. PC12 cells in the logarithmic growth phase were treated with LPS at final concentrations of 0.25, 0.5, 0.75, 1, and 1.25 mg/mL for 24 h. Cell morphology was evaluated, and cell survival rates were calculated. A neurocyte inflammatory model was established with LPS treatment, which reached a 50% cell survival rate. PC12 cells were treated with 0.01, 0.1, 1, 10, or 100 µmol/L astragaloside IV for 24 h. The concentration of astragaloside IV that did not affect the cell survival rate was selected as the treatment group for subsequent experiments. NOS activity was detected by colorimetry; the expression levels of ERCC2, XRCC4, XRCC2, TNF-α, IL-1β, TLR4, NOS and COX-2 mRNA and protein were detected by RT-qPCR and Western blotting. The differentially expressed genes (DEGs) between the groups were screened using a second-generation sequence (fold change>2, P<0.05) with the following KEGG enrichment analysis, RT-qPCR and Western blotting were used to detect the mRNA and protein expression of DEGs related to the IL-17 pathway in different groups of PC12 cells. The viability of PC12 cells was not altered by treatment with 0.01, 0.1, or 1 µmol/L astragaloside IV for 24 h (P>0.05). However, after treatment with 0.5, 0.75, 1, or 1.25 mg/mL LPS for 24 h, the viability steadily decreased (P<0.01). The mRNA and protein expression levels of ERCC2, XRCC4, XRCC2, TNF-α, IL-1β, TLR4, NOS, and COX-2 were significantly increased after PC12 cells were treated with 1 mg/mL LPS for 24 h (P<0.01); however, these changes were reversed when PC12 cells were pretreated with 0.01, 0.1, or 1 µmol/L astragaloside IV in PC12 cells and then treated with 1 mg/mL LPS for 24 h (P<0.05). Second-generation sequencing revealed that 1026 genes were upregulated, while 1287 genes were downregulated. The DEGs were associated with autophagy, TNF-α, interleukin-17, MAPK, P53, Toll-like receptor, and NOD-like receptor signaling pathways. Furthermore, PC12 cells treated with a 1 mg/mL LPS for 24 h exhibited increased mRNA and protein expression of CCL2, CCL11, CCL7, MMP3, and MMP10, which are associated with the IL-17 pathway. RT-qPCR and Western blotting analyses confirmed that the DEGs listed above corresponded to the sequence assay results. LPS can damage PC12 cells and cause inflammatory reactions in nerve cells and DNA damage. astragaloside IV plays an anti-inflammatory and DNA damage protective role and inhibits the IL-17 signaling pathway to exert a neuroprotective effect in vitro.

MiR-21 attenuated inflammation targeting MyD88 in human chondrocytes stimulated with Hyaluronan oligosaccharides

Inflammation is the body's response to injuries, which depends on numerous regulatory factors. Among them, miRNAs have gained much attention for their role in regulating inflammatory gene expression at multiple levels. In particular, miR-21 is up-regulated during the inflammatory response and reported to be involved in the resolution of inflammation by down-regulating pro-inflammatory mediators, including MyD88. Herein, we evaluated the regulatory effects of miR-21 on the TLR-4/MyD88 pathway in an in vitro model of 6-mer HA oligosaccharides-induced inflammation in human chondrocytes.The exposition of chondrocytes to 6-mer HA induced the activation of the TLR4/MyD88 pathway, which culminates in NF-kB activation. Changes in miR-21, TLR-4, MyD88, NLRP3 inflammasome, IL-29, Caspase1, MMP-9, iNOS, and COX-2 mRNA expression of 6-mer HA-stimulated chondrocytes were examined by qRT-PCR. Protein amounts of TLR-4, MyD88, NLRP3 inflammasome, p-ERK1/2, p-AKT, IL-29, caspase1, MMP-9, p-NK-kB p65 subunit, and IKB-a have been evaluated by ELISA kits. NO and PGE2 levels have been assayed by colorimetric and ELISA kits, respectively.HA oligosaccharides induced a significant increase in the expression of the above parameters, including NF-kB activity. The use of a miR-21 mimic attenuated MyD88 expression levels and the downstream effectors. On the contrary, treatment with a miR-21 inhibitor induced opposite effects. Interestingly, the use of a MyD88 siRNA confirmed MyD88 as the target of miR-21 action.Our results suggest that miR-21 expression could increase in an attempt to reduce the inflammatory response, targeting MyD88.