IL-4 mRNA Levels Research Articles

Effect of NLRP3 inflammasome induced astrocyte phenotype alteration in morphine tolerance.

Morphine is a widely used analgesic, but its prolonged use often leads to tolerance, limiting its therapeutic efficacy. Research implicates the NLRP3 inflammasome and reactive astrocytes in the development of morphine tolerance, with reactive astrocytes classified into A1 neurotoxic and A2 neuroprotective phenotypes. This study explores the role of the NLRP3 inflammasome and the transformation of astrocyte phenotypes in the progression of morphine tolerance. A model of morphine tolerance was established by administering morphine intrathecally for seven consecutive days. To inhibit NLRP3 inflammasome activation, we coadministered MCC950, a selective NLRP3 inhibitor. Thermal withdrawal latency was used to assess tolerance development. Protein and mRNA levels of GFAP, IL-18, NLRP3, C3 (A1 marker), and S100A10 (A2 marker) in the spinal cord were measured using Western blotting (WB) and real-time quantitative polymerase chain reaction (RT-qPCR). Immunofluorescence was employed to assess the colocalization of C3 and GFAP. Seven days of morphine administration induced tolerance, which was associated with increased levels of GFAP, IL-18, NLRP3, and C3, and a decreased level of S100A10. Coadministration of morphine and MCC950 significantly slowed the development of morphine tolerance and reversed changes in NLRP3, IL-18, GFAP, C3, and S100A10 protein levels. Our findings indicate a significant link between NLRP3 inflammasome activation and morphine tolerance, suggesting that NLRP3 contributes to the transformation of astrocytes to the A1 phenotype. Inhibiting NLRP3 inflammasome activation holds promise in reversing astrocyte phenotype changes, potentially mitigating morphine tolerance.

Biomimetic porphyrin-lipid nanoparticles - novel nanoscale theranostics for multimodal imaging and therapy in atherosclerotic cardiovascular disease

Abstract Background High-density lipoprotein (HDL) mimetic nanoagents have unrealized potential for atherosclerosis theranostics. Porphyrin-lipid HDL mimetic nanoparticles (Por-HDL-NPs) incorporate a porphyrin-lipid conjugate which permits near infrared fluorescence imaging and positron emission tomography (PET) through chelation of Copper-64 (64Cu). The outer shell contains apolipoprotein A-I mimetic peptide R4F designed to interact with scavenger receptor class B type I (SR-BI), enabling macrophage-targeting and therapeutic effects. Purpose To investigate the multimodal imaging, macrophage-targeting and therapeutic properties of Por-HDL-NPs and applications in atherosclerotic cardiovascular disease. Methods In vitro, the cholesterol efflux capacity of PBS (control), reconstituted HDL (rHDL) and Por-HDL-NPs was determined in immortalized bone marrow-derived macrophages (iBMDMs) loaded with [³H]-cholesterol. Real-time quantitative PCR (RT-qPCR) assessed mRNA levels of inflammatory mediators. ELISAs and Western blotting measured changes in protein levels. Apolipoprotein (Apo)e-/- mice were fed high-cholesterol diet (HCD) for 6 weeks, for early-stage plaque, and 13 weeks for the unstable plaque model in which two partial stenoses were made in the right carotid artery. PET and fluorescence (IVIS, flow cytometry, microscopy) imaging identified Por-HDL-NPs in plaques, circulating and aortic cells. Histological techniques assessed plaque area and composition in aortic sinus and carotid artery sections. Results Por-HDL-NPs were internalized by iBMDMs, visualized via fluorescence microscopy and flow cytometry. Por-HDL-NPs increased cholesterol efflux (49%, P<0.05), compared to rHDL. Por-HDL-NPs reduced macrophage mRNA levels of Il-1b (88%), Il-18 (54%) and Ccl5 (75%), and protein secretion of IL-1β (69%) and CCL5 (82%), P<0.05. Por-HDL-NPs suppressed inflammasome components Nlrp3 (69%) and Asc (36%), and activation of inflammatory transcription factor p65-NF-κB (53%), P<0.05. SR-BI siRNA knockdown and methyl-β-cyclodextrin, revealed that the anti-inflammatory properties of Por-HDL-NPs were independent of SR-BI and cholesterol efflux. In Apoe-/- mice, PET imaging showed 64Cu-Por-HDL-NPs localized in hearts and could detect increases in plaque size over time with HCD feeding. Por-HDL-NP fluorescence was detected within aortic sinus and unstable carotid plaques, co-localized with CD68+ macrophages. Por-HDL-NP-treated mice had smaller early-stage (22%) and unstable plaques (52%) than control PBS-treated mice, P<0.05. Por-HDL-NP mice had fewer circulating (32%) and aortic monocytes (81%), and a reduction in aortic arch Rela (26%), P<0.05. Conclusions Por-HDL-NPs present as potential nanoscale theranostics for atherosclerotic cardiovascular disease. Por-HDL-NPs can detect plaques using multiple imaging modalities and exhibit atheroprotective effects.Atherosclerosis imaging with Por-HDL-NPs

Preventive effect of imperatorin against doxorubicin-induced cardiotoxicity through suppression of NLRP3 inflammasome activation.

Cardiotoxicity is one of the major obstacles to anthracycline chemotherapy. Anthracycline cardiotoxicity is closely associated with inflammation. Imperatorin (IMP), a furocoumarin ingredient extracted from Angelica dahurica, might have potential activity in preventing anthracycline cardiotoxicity due to its anti-cancer, anti-inflammatory, anti-oxidant, cardioprotective properties. This study aims to reveal the effect of IMP on doxorubicin (DOX)-induced cardiotoxicity and its underlying mechanism. We established a rat model of DOX-induced cardiotoxicity by intraperitoneal injection with DOX (1.25mg/kg twice weekly for 6weeks), and found that both IMP (25mg/kg and 12.5mg/kg) and dexrazoxane 12.5mg/kg relieved DOX-induced reductions in heart weight, change in cardiac histopathology, and elevated serum levels of LDH, AST and CK-MB. Moreover, DOX upregulated mRNA levels of NLRP3, CASP1, GSDMD, ASC, IL-1β and IL-18, elevated protein expressions of NLRP3, ASC, GSDMD-FL, GSDMD-N, pro‑caspase‑1, caspase‑1 p20, pro‑IL‑1β and IL‑1β in heart tissues, as well as increased serum levels of pro-inflammatory cytokines including IL-1β and IL-18, however both of IMP and dexrazoxane suppressed these alterations. In addition, we carried out neonatal rat cardiomyocytes experiments to confirm the results of the in vivo study. Consistently, pretreatment with IMP 25µg/mL relieved DOX (1μg/mL)-induced cardiomyocytes injury, including decreased cell viability and reduced supernatant LDH. IMP inhibited DOX-induced activation of NLRP3 inflammasome in cardiomyocytes. In conclusion, IMP had a protective effect against DOX-induced cardiotoxicity via repressing the activation of NLRP3 inflammasome. These findings suggest that IMP may be a promising alternative or adjunctive drug for the prevention of anthracycline cardiotoxicity.

Efficacy of Topical Cyclosporine Combined with Punctal Plugs in Treating Dry Eye Disease and Inflammation

Purpose To evaluate the effect of punctal plugs combined with cyclosporine eye drops on dry eye disease (DED) and ocular surface inflammation. Methods In a clinical trial, 73 patients were randomly allocated into three groups: punctal plug group, combination therapy group, and cyclosporine group. At the baseline and four weeks after treatment, the Schirmer I test score, fluorescein tear film break-up time (FBUT), ocular surface staining score and dry eye symptoms were assessed. Tear samples were collected to detect the level of inflammatory factors (interleukins, matrix metalloproteinase 9 (MMP-9) and tumor necrosis factor alpha (TNF-α)). In an animal experiment, a New Zealand rabbit dry eye model was induced. The rabbits were randomly divided into control group, punctal plug group, and combination therapy group (n = 6). Conjunctival goblet cell density, protein level of MMP-9 in conjunctiva and mRNA levels of inflammatory factors in conjunctiva and cornea were measured before and after treatment. Results In combination therapy group of the clinical trial, the following results were observed: significant improvement in Schirmer I test scores and FBUT compared to the cyclosporine group and punctal plug group, respectively; a decrease in the tear levels of IL-6, IL-1, and MMP-9 compared to the punctal plug group; and a decrease in the tear levels of IL-1α, IL-6, and IL-17 compared to the baseline (all p < 0.05). In the animal experiment, rabbits in combination therapy group had a higher goblet cell density (p < 0.01) and lower mRNA levels of IL-16 (p < 0.05), IL-17 (p < 0.05), and MMP-9 (p < 0.01) in conjunctiva and that of MMP-9 (p < 0.01) in cornea compared to punctal plug group. Conclusion Cyclosporine eye drops combined with degradable punctal plugs is a more optimized clinical treatment strategy for DED compared with degradable punctal plugs or cyclosporine eye drops alone, considering the influence of comprehensive clinical efficacy and ocular surface inflammation.

Immunostimulatory Activity of a Mixture of Platycodon grandiflorum, Pyrus serotine, Chaenomeles sinensis, and Raphanus sativus in RAW264.7 Macrophages.

In this study, a mixture of Platycodon grandiflorum, Pyrus serotina, Chaenomeles sinensis, and Raphanus sativus (PPCRE) was investigated for their immuno-enhancing effects, as well as the molecular mechanism of PPCRE in RAW264.7 cells. PPCRE dramatically increased nitric oxide (NO) and prostaglandin E2 (PGE2) generation depending on the concentration while exhibiting no cytotoxicity. PPCRE markedly upregulated the mRNA and protein expression of immune-related cytotoxic factors such as cyclooxygenase (COX)-1, COX-2, and inducible nitric oxide synthase (iNOS) and pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor alpha (TNF-α), as well as the mRNA level of IL-4. PPCRE increased the mitogen-activated protein kinase (MAPK) signaling pathway by upregulating the phosphorylation of extracellular signal-regulated kinase (ERK), stress-activated protein kinase/Jun N-terminal-kinase (SAPK/JNK), and p38. Furthermore, PPCRE considerably activated the nuclear factor kappa B (NF-κB) signaling pathway by increasing phosphorylation of NF-κB-p65. PPCRE-stimulated RAW264.7 cells increased macrophage phagocytic capacity. In conclusion, our study found that PPCRE improved immune function by modulating inflammatory mediators and regulating the MAPK and NF-κB pathway of signaling in macrophages.

Daidzein attenuates high glucose-induced inflammatory injury in macrophages by regulating NLRP3 inflammasome signaling pathway

This study aims to explore the inhibitory effect of daidzein on macrophage inflammation induced by high glucose via regulating the NOD-like receptor protein 3(NLRP3) inflammasome signaling pathway. The cell counting kit-8(CCK-8) assay was employed to detect the effects of daidzein at different concentrations on the viability of RAW264.7 cells. Western blot was employed to determine the protein level of tumor necrosis factor(TNF)-α in macrophages exposed to different concentrations of glucose for different time periods as well as the expression levels of proteins involved in the polarization and Toll-like receptor 4(TLR4)-myeloid differentiation factor(MyD88)-NLRP3 inflammasome pathway of the macrophages exposed to high glucose. Enzyme-linked immunosorbent assay was employed to measure the levels of TNF-α, interleukin(IL)-18, and IL-1β secreted by macrophages. The expression level of nuclear factor-kappa B(NF-κB) p65 in macrophages exposed to high glucose was detected by immunofluorescence, and the level of intracellular reactive oxygen species(ROS) was detected by the DCFH-DA fluorescent probe. The mRNA levels of NLRP3, TNF-α, and IL-18 in macrophages were determined by qRT-PCR. The results showed that treatment with 30 mmol·L~(-1) glucose for 48 h was the best condition for the modeling of macrophage injury. Compared with the blank group, the model group showed improved polarization of macrophages, increased secretion of TNF-α, IL-18, and IL-1β, elevated ROS level, and up-regulated expression of NF-κB p65. In addition, the modeling up-regulated the mRNA levels of NLRP3, TNF-α, and IL-18 and the protein levels of TLR4, MyD88, NLRP3, NF-κB p65, p-NF-κB p65, I-κB, p-I-κB, ASC, pro-caspase-1, pro-IL-1β, cleaved IL-1β, and pro-IL-18. Compared with the model group, daidzein(10, 20, and 40 μmol·L~(-1)) lowered the levels of inflammatory cytokines and down-regulated the mRNA levels of NLRP3, TNF-α, and IL-18 as well as the protein levels of TLR4, MyD88, NLRP3, NF-κB p65, p-NF-κB p65, I-κB, p-I-κB, ASC, pro-caspase-1, pro-IL-1β, cleaved IL-1β, and pro-IL-18. In addition, daidzein reduced intracellular ROS. According to the available reports and the experimental results, high glucose can induce the polarization of macrophages and promote the secretion of inflammatory cytokines. Daidzein can inhibit the expression of ROS in macrophages by regulating the NLRP3 inflammasome signaling pathway, thereby reducing the inflammation of macrophages exposed to high glucose.

Overexpression of the β-Subunit of Acid Ceramidase in the Epidermis of Mice Provokes Atopic Dermatitis-like Skin Symptoms.

We previously reported that a pathogenic abnormality in the barrier and water-holding functions of the stratum corneum (SC) in the skin of patients with atopic dermatitis (AD) is mainly attributable to significantly decreased levels of total ceramides in the SC. That decrease is mediated by the abnormal expression of a novel ceramide-reducing enzyme, sphingomyelin/glucosylceramide deacylase (SGDase), which is the β-subunit (ASAH1b) of acid ceramidase. In this study, we determined whether mice overexpressing ASAH1b in their epidermis develop AD-like skin symptoms. We generated transgenic (TG) mice overexpressing ASAH1b, regulated by the involucrin promoter, to localize its expression in the upper epidermis. After hair removal using a depilatory cream containing glycolic acid, the TG mice without any visible skin inflammation at 8 weeks of age had increased levels of ASAH1b and decreased levels of SC ceramide, with disrupted barrier functions measured by trans-epidermal water loss compared to the wild-type (WT) mice. Interestingly, enzymatic assays revealed that SGDase activity was not detectable in the skin of the TG mice compared to WT mice. Immunological staining revealed that there was an increased expression level of IL-33 in the epidermis and an accumulation of macrophages in the dermis of TG mice compared to WT mice, which are phenotypic characteristics of AD, that were exacerbated by tape-stripping of the skin. In the skin of the TG mice, the mRNA levels of IL-5, CCL11, IL-22, CXCL10, and IFNγ were significantly upregulated compared to the WT mice, and tape-stripping significantly increased the mRNA levels of IL-4, IL-33, CXCL1, CXCL12, TLR9, and CD163 compared to WT mice. These findings strongly indicate that the skin of the depilatory cream-treated TG mice exists in an atopic dry skin condition that is highly sensitive to various environmental stimuli. The sum of our results suggests that ASAH1b itself, even in the absence of its enzymatic activity, is a major etiologic factor for atopic dry skin symptoms via an unknown mechanism.

Anisodamine hydrobromide ameliorates acute lung injury via inhibiting pyroptosis in murine sepsis model

Objective Sepsis can have severe implications on lung function, leading to acute lung injury (ALI), a major contributor to sepsis-related mortality. Anisodamine hydrobromide (Ani HBr), a bioactive constituent derived from the root of Scopolia tangutica Maxim, a plant endemic to China, has demonstrated efficacy in treating septic shock. We aim to explore whether Ani HBr can alleviate sepsis-triggered ALI and elucidate the fundamental mechanisms involved. Materials and method The protective effects of Ani HBr were assessed in two models: in vitro, lipopolysaccharide (LPS)-stimulated RAW264.7 cells, and in vivo, cecal ligation puncture (CLP)-induced sepsis. To measure the cell viability of RAW264.7 cells after Ani HBr treatment, we used the CCK-8 assay. We quantified the levels of pro-inflammatory cytokines expression using ELISA. We also measured the expression of pyrotosis indicators by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), Western blotting, and immunofluorescence. Results Our study demonstrates that Ani HBr can alleviate pulmonary edema, bleeding, and excessive inflammation induced by CLP. Additionally, it exhibits protective effects against cytotoxicity induced by LPS in RAW264.7 macrophage cells. Furthermore, Ani HBr downregulates the mRNA and protein levels of NLRP3, Caspase-1, GSDMD, IL-18, and IL-1β in both animal models and cell cultures, thereby inhibiting pyroptosis in a similar mechanism to AC-YVAD-CMK (AYC)’s blockade of Caspase-1. Moreover, Ani HBr suppresses the production and release of proinflammatory cytokines. Conclusion These findings suggest that Ani HBr could serve as a protective agent against sepsis-induced ALI by suppressing pyroptosis.

Baicalin and probenecid protect against Glaesserella parasuis challenge in a piglet model

Glaesserella parasuis (G. parasuis) induces vascular damage and systemic inflammation. However, the mechanism by which it causes vascular damage is currently unclear. Baicalin has important anti-inflammatory, antibacterial and immunomodulatory functions. In this study, we explored the ability of baicalin and probenecid to protect against G. parasuis challenge in a piglet model. Sixty piglets were randomly divided into a control group; an infection group; a probenecid group; and 25 mg/kg, 50 mg/kg and 100 mg/kg baicalin groups. The probenecid group and the 25 mg/kg, 50 mg/kg and 100 mg/kg baicalin groups were injected intramuscularly with 20 mg/kg body weight (BW) probenecid and 25 mg/kg BW, 50 mg/kg BW and 100 mg/kg BW baicalin, respectively. All piglets except those from the control group were injected intraperitoneally with 1 × 108 CFU of G. parasuis. The control group was injected intraperitoneally with TSB. The results showed baicalin and probenecid protected piglets against G. parasuis challenge, improved body weight and decreased temperature changes in piglets. Baicalin and probenecid attenuated IL-1β, IL-10, IL-18, TNF-α and IFN-γ mRNA levels in the blood for 48 h, inhibited the production of the nucleosides ATP, ADP, AMP and UMP from 24 to 72 h, reduced Panx-1/P2Y6/P2X7 expression, weakened NF-kB, AP-1, NLRP3/Caspase-1 and ROCK/MLCK/MLC signalling activation, and upregulated VE-cadherin expression in the blood vessels of piglets challenged with G. parasuis. Baicalin and probenecid alleviated pathological tissue damage in piglets induced by G. parasuis. Our results might provide a promising strategy to control and treat G. parasuis infection in the clinical setting.

Porphyromonas gingivalis promote microglia M1 polarization through the NF-кB signaling pathway

BackgroundPorphyromonasgingivalis (P.gingivalis) is associated with the onset of Alzheimer's disease (AD), but the underlying molecular mechanism is unclear. Neuroinflammation in the brain from the microglial immune response induces the pathological progression of AD. In this study, the roles and molecular mechanism of P.gingivalis in microglial inflammation in vitro were investigated. MethodsIn this study, a P.gingivalis oral administration mouse model was generated, and microglia were stimulated with P.gingivalis in vitro. The viability of the microglia after P.gingivalis treatment was evaluated through CCK-8 and live/dead cell staining. Inflammation in brain tissue after P.gingivalis treatment and the immune response of microglia in vitro were detected by RT‒PCR, Western blotting and IF. Moreover, the RNA sequence was used, and the role of the NF-κB signalling pathway in microglial activation was analysed after P.gingivalis stimulation. ResultsThe mRNA and protein levels of IL-6 and IL-17 were increased, and the expression of IL-10 was decreased in brain tissue after P.gingivalis oral administration. The viability of the HMC3 cells significantly decreased with 5% P.gingivalis after stimulation. The results of live/dead cell staining also showed the inhibitory effect of 5% P.gingivalis supplementation on cell viability. Moreover, 5% P.gingivalis supplementation increased the mRNA and protein levels of IL-6 and IL-17 and decreased IL-10 expression in HMC3 cells. P.gingivalis supplementation increased the mRNA and protein levels of iNOS and CD86 and decreased CD206 expression in HMC3 cells. RNA sequencing revealed that the NF-κB signalling pathway was involved in this process. Furthermore, p-P65 was upregulated and p-IKBα was downregulated in brain tissue and HMC3 cells after P.gingivalis stimulation, and an NF-κB signalling pathway inhibitor (QNZ) reversed the viability, M1 polarization and inflammatory factors of microglia in HMC3 cells in vitro. ConclusionsIn conclusion, P.gingivalis induced neuroinflammation in the brain, possibly through promotion of M1 polarization of microglia via activation of the NF-κB signalling pathway during the progression of AD.

A novel probiotic formula, BLLL, ameliorates chronic stress-induced depression-like behaviors in mice by reducing neuroinflammation and increasing neurotrophic factors.

Introduction: Probiotics have been recognized for their various biological activities, including antioxidant and anti-inflammatory properties. This study investigates the therapeutic effect of a novel probiotic formula, BLLL, consisting of Bifidobacterium breve, Lactobacillus plantarum, Lactobacillus paracasei, and Lactobacillus helveticus, on chronic stress-induced depression-like behaviors in mice. Methods: The BLLL formula or phosphate-buffered saline (PBS) was given orally at a dose of 2, 4, or 8 × 1010 CFU/kg once daily for 10days in mice treated with chronic unpredictable stress (CUS) treated or vehicle. Depression-like behaviors were assessed using the sucrose preference test (SPT), the forced swimming test (FST), and the tail suspension test (TST). The mRNA and/or protein expression of interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), IL-4, IL-10, and chitinase-3-like protein 3 (CHI3L1, also known as Ym-1), as well as the concentration of nitrite, malondialdehyde (MDA), glutathione (GSH), and brain-derived neurotrophic factor (BDNF) in the hippocampus and medial prefrontal cortex were examined. Results: The BLLL formula treatment at a dose of 8 × 1010 CFU/kg, but not at a dose of 2 or 4 × 1010 CFU/kg, improved CUS-induced depression-like behaviors in mice, as shown by the decrease in immobility time in the TST and FST and the increase in sucrose intake in the SPT. Further analysis revealed that BLLL treatment suppressed the CUS-induced increase in IL-1β, IL-6, and TNF-α mRNA and protein levels, as well as the CUS-induced decrease in IL-4, IL-10, and Ym-1 mRNA and/or protein levels in the hippocampus and medial prefrontal cortex. In addition, treatment with the BLLL formula countered the CUS-induced increase in nitrite and MDA levels and the CUS-induced decrease in GSH content and BDNF concentration in the hippocampus and medial prefrontal cortex. Conclusion: These results demonstrate that the novel probiotic formula BLLL ameliorates chronic stress-induced depression-like behavior in mice by suppressing neuroinflammation and oxido-nitrosative stress in the brain.

The Protective Effect of Melatonin on LPS-Induced Myocardial Injury via the Caspase-11/GSDMD Pathway.

Melatonin (MT) has been demonstrated to have cardioprotective effects. Nevertheless, the precise mechanism through which MT provides protection against the etiology of LPS-induced myocardial injury remains uncertain. In this investigation, our objective was to explore the impact of MT on LPS-induced myocardial injury in an in vitro setting. H9C2 cells were categorized into four groups: a control group (H9C2 group), an MT group, an LPS group, and an MT + LPS group. The H9C2 group received treatment with sterile saline solution, the LPS group was exposed to 5 μg/mL LPS for 24 hours, the MT + LPS group underwent pretreatment with 150 μmol/L MT for 2 hours, followed by exposure to 5 μg/mL LPS for 24 hours, and the MT group received only 150 μmol/L MT for 2 hours. Cell viability and lactate dehydrogenase (LDH) release were assessed using the CCK-8 assay and LDH activity assay, respectively. The levels of reactive oxygen species (ROS) were quantified in each group of cells, and the percentage of propidium iodide (PI)-stained apoptotic cells was determined by flow cytometry. The mRNA levels of caspase11, GSDMD, and IL-18 in each group of cells were quantified. MT treatment significantly protected H9C2 cells from LPS-induced damage, as evidenced by decreased LDH release. LPS treatment markedly increased ROS levels in H9C2 cells, which were subsequently reduced by MT. LPS caused a substantial decrease in superoxide dismutase (SOD) activity and a significant increase in malondialdehyde (MDA) levels, while MT treatment significantly reversed these effects. Additionally, MT markedly enhanced the proportion of viable H9C2 cells compared to LPS-treated controls, as evidenced by the PI staining assay. LPS upregulated both mRNA levels and protein levels of IL-18 in H9C2 cells. However, MT treatment effectively mitigated this LPS-induced increase. Furthermore, MT significantly decreased LPS-induced protein levels of cleaved-caspase 11 and GSDMD-N in H9C2 cells. Overall, our findings suggest that MT inhibits the Caspase11-GSDMD signaling pathway via pyroptosis-related proteins (caspase-11 and GSDMD-N) and reduces the expression of inflammation-related cytokines (IL-18), thereby exerting a protective effect on H9C2 cells after LPS injury.

Neuroprotection of the P2X7 receptor antagonist A740003 on retinal ganglion cells in experimental glaucoma.

The aim of this study was to explore the neuroprotective effects of the P2X7 receptor antagonist A740003 on retinal ganglion cells (RGCs) in chronic intraocular hypertension (COH) experimental glaucoma mouse model. Bioinformatics was used to analyze the glaucoma-related genes. Western blot, real-time fluorescence quantitative PCR, and immunofluorescence staining techniques were employed to explore the mechanisms underlying the neuroprotective effects of A740003 on RGCs in COH retinas. Bioinformatic analysis revealed that oxidative stress, neuroinflammation, and cell apoptosis were highly related to the pathogenesis of glaucoma. In COH retinas, intraocular pressure elevation significantly increased the levels of translocator protein, a marker of microglial activation, which could be reversed by intravitreal preinjection of A740003. A740003 also suppressed the increased mRNA levels of proinflammatory cytokines interleukin (IL) 1β and tumor necrosis factor α in COH retinas. In addition, although the mRNA levels of anti-inflammatory cytokine IL-4 and IL-10 were kept unchanged in COH retinas, administration of A740003 could increase their levels. The mRNA and protein levels of Bax and cleaved caspase-3 were increased in COH retinas, which could be partially reversed by A740003, while the levels of Bcl-2 kept unchanged in COH retinas with or without the injections of A740003. Furthermore, A740003 partially attenuated the reduction in the numbers of Brn-3a-positive RGCs in COH mice. A740003 could provide neuroprotective roles on RGCs by inhibiting the microglia activation, attenuating the retinal inflammatory response, reducing the apoptosis of RGCs, and enhancing the survival of RGCs in COH experimental glaucoma.

Naringin from Ganshuang granule inhibits inflammatory to relieve liver fibrosis through TGF-β-Smad signaling pathway.

The present study aims to investigate the specific protective effects and underlying mechanisms of Ganshuang granule (GSG) on dimethylnitrosamine (DMN)-induced hepatic fibrosis in rat models. Hepatic fibrosis was experimentally evoked in rats by DMN administration, and varying dosages of GSG were employed as an intervention. Hepatocellular damage was assessed by measuring serum levels of aminotransferase and bilirubin, accompanied by histopathological examinations of hepatic tissue. The hepatic concentrations of platelet-derived growth factor (PDGF) and transforming growth factor-β1 (TGF-β1) were quantitated via enzyme-linked immunosorbent assay (ELISA). The expression of α-smooth muscle actin (α-SMA) within hepatic tissue was evaluated using immunohistochemical techniques. The levels of hepatic interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and a spectrum of interleukins (IL-2, IL-4, IL-6, IL-10) were quantified by quantitative real-time PCR (qRT-PCR). Additionally, hepatic stellate cells (HSCs) were cultured in vitro and exposed to TNF-α in the presence of naringin, a principal component of GSG. The gene expression levels of tissue inhibitor of metalloproteinase-1 (TIMP-1) and matrix metallopeptidase-1 (MMP-1) in these cells were also quantified by qRT-PCR. Proliferative activity of HSCs was evaluated by the Cell Counting Kit-8 assay. Finally, alterations in Smad protein expression were analyzed through Western blotting. Administration of GSG in rats with fibrosis resulted in reduced levels of serum aminotransferases and bilirubin, along with alleviation of histopathological liver injury. Furthermore, the fibrosis rats treated with GSG exhibited significant downregulation of hepatic TGF-β1, PDGF, and TNF-α levels. Additionally, GSG treatment led to increased mRNA levels of IFN-γ, IL-2, and IL-4, as well as decreased expression of α-SMA in the liver. Furthermore, treatment with naringin, a pivotal extract of GSG, resulted in elevated expression of MMP-1 and decreased levels of TIMP-1 in TNF-α-stimulated HSCs when compared to the control group. Additionally, naringin administration led to a reduction in Smad expression within the HSCs. GSG has the potential to mitigate fibrosis induced by DMN in rat models through the regulation of inflammatory and fibrosis factors. Notably, naringin, the primary extract of GSG, may exert a pivotal role in modulating the TGF-β-Smad signaling pathway.

In vitro study of momordin Ic in improving atopic dermatitis by regulating claudin-1/MyD88 and targeting NLRP3

Atopic dermatitis (AD) is a non-infectious inflammatory skin disease characterized by persistent itching of the skin, and it has become a global health problem with increasing incidence and complex pathogenesis. Momordin Ic (MMI) is a triterpenoid saponin that has anti-inflammatory effect and may play an effective role in the treatment of AD. To explore the molecular mechanism of MMI in relieving AD, and to provide some experimental data for the clinical application of MMI in AD. The mRNA levels of IL-33, IL-4 and NF-κB were detected by RT-qPCR. The protein expressions of Filaggrin, Claudin-1, MyD88 and NLRP3 were detected by western blotting. Transepithelial/transendothelial electrical resistance (TEER) method was used to detect the tight-junction function of HaCaT cells. Molecular docking experiment was performed on CB-DOCK2 to predict the binding of MMI and NLRP3. MMI reversed the decrease of filaggrin protein expression and intercellular resistance in HaCaT cells induced by TNF-α and IFN-γ. In addition, MMI reversed the increase in IL-33 and IL-4 mRNA levels in HaCaT cells treated with TNF-α and IFN-γ. In addition, MMI promoted the expression of Claudin-1 and inhibited the expression of MyD88 and NLRP3. In addition, the molecular docking results showed that MMI and NLRP3 had interaction sites. MMI improved the inflammatory response of HaCaT cells by regulating the expression of Claudin-1/MyD88 and targeting NLRP3, which may act a positive role in relieving AD.

Quercetin through miR-147–5p/Clip3 axis reducing Th17 cell differentiation to alleviate periodontitis

BackgroundQuercetin (QU) plays an important role in treating periodontitis; however, the mechanism through which microRNAs regulate Th17 cell differentiation has not been determined. MethodsQU was administered intragastrically to periodontitis rats once a day for one month. The morphology of alveolar bone was observed by micro-CT, gingival tissue structure was observed by HE staining, IL-6, TNF-α, IL-17A, RORγt, FOXP3 and IL-10 were detected by immunohistochemical staining, and Th17 and Treg cells in the peripheral blood were detected by flow cytometry. CD4+T cells were induced to differentiate into Th17 cells in vitro. Cell viability was determined by CCK8, and IL-17A and RORγt were detected by qPCR. Th17 cells were detected by flow cytometry, microRNA sequencing and bioinformatics analysis were used to screen key microRNAs, the phenotypic changes of Th17 cells were observed after overexpressed microRNAs via mimics. TargetScan database, in situ hybridization, and dual-luciferase reporter experiment were used to predict and prove target genes of microRNAs. The phenotype of Th17 cells was observed after overexpression of microRNA and target gene. ResultsCompared with periodontitis group, the distance from cementoenamel junction(CEJ) to alveolar bone(AB) was decreased, the structure of gingival papilla was improved, IL-6, TNF-α, IL-17, and RORγt were downregulated, FOXP3 and IL-10 were upregulated, the proportion of Th17 decreased and Treg increased in peripheral blood after QU treatment. Compared with Th17 cell group, mRNA levels of IL-17A and RORγt were decreased, and proportion of Th17 cells was significantly lower in the coculture group. MiR-147–5p was low in control group, upregulated in Th17 cell group, and downregulated after QU intervention, it's eight bases were inversely related to 3′UTR of Clip3, miR-147–5p with Clip3 were co-located in cells of periodontal tissue. Compared with those in Th17-mimicsNC + QU cells, the mRNA levels of RORγt and IL-17A upregulated, and proportion of Th17 cells increased in Th17-miR-147–5p + QU cells. The miR-147–5p mimics inhibited the luciferase activity of the WT Clip3 3′UTR but had no effect on the Mut Clip3 3′UTR. Clip3 was significantly downregulated after the overexpression of miR-147–5p. Mimics transfected with miR-147–5p reversed the decrease in the proportion of Th17 cells induced by QU, while the overexpression of Clip3 antagonized the effect of miR-147–5p and further reduced the proportion of Th17 cells. Moreover, the overexpression of miR-147–5p reversed the decreases in the mRNA levels of IL-17 and RORγt induced by QU treatment, while pcDNA3.1 Clip3 treatment further decreased the mRNA levels of IL-17 and RORγt. ConclusionQU reducing inflammatory response and promoting alveolar bone injury and repair, which closely relative to inhibit the differentiation of CD4+T cells into Th17 cells by downregulating miR-147–5p to promote the activation of Clip3.

The Role of miR-150-5p/SOCS1 Pathway in Arsenic-Induced Pyroptosis of LX-2 Cells.

This study aims to explore the mechanism of pyroptosis of human hepatocyte LX-2 cells induced by NaAsO2 through the miR-150-5p/SOCS1 pathway. LX-2 cells were transfected with different concentrations of NaAsO2, miR-150-5p inhibitor, and SOCS1 agonist. Cell activity, cell pyroptosis, and the expression of related genes and proteins were detected by scanning electron microscopy, CCK-8, qRT-PCR, western blot, and immunofluorescence. Compared with the control group, 10 µmol/L and 20 µmol/L NaAsO2 significantly elevated the protein expression levels of the pyroptosis-related proteins NLRP3, GSDMD, GSDMD-N, caspase1, and cleaved caspase1 as well as the mRNA levels of NLRP3, GSDMD, caspase1, IL-18, and IL-1β. The typical pyroptosis with swelling and rupture of the plasma membrane was observed through scanning electron microscopy. The expression of miR-150-5p of the NaAsO2 intervention group increased, while the expression of SOCS1 decreased; then the level of NF-κB p65 elevated. With co-treatment of miR-150-5p inhibitor, SOCS1 agonist, and NaAsO2, the cell pyroptosis was attenuated, and the expressions of NLRP3, caspase1, GSDMD, GSDMD-N, IL-18, IL-1β, p65 of the group of miR-150-5p inhibitor and NaAsO2 group, and of the group of SOCS1 agonist and NaAsO2 reduced compared with the NaAsO2 group. Arsenic exposure promotes miR-150-5p, inhibits the expression of SOCS1, and activates the NF-κB/NLRP3 pathway in LX-2 cell pyroptosis.

Immunologic Profiling of Immune-Related Cutaneous Adverse Events with Checkpoint Inhibitors Reveals Polarized Actionable Pathways.

Immune-related cutaneous adverse events (ircAE) occur in ≥50% of patients treated with checkpoint inhibitors, but the underlying mechanisms for ircAEs are poorly understood. Phenotyping/biomarker analyses were conducted in 200 patients on checkpoint inhibitors [139 with ircAEs and 61 without (control group)] to characterize their clinical presentation and immunologic endotypes. Cytokines were evaluated in skin biopsies, skin tape strip extracts, and plasma using real-time PCR and Meso Scale Discovery multiplex cytokine assays. Eight ircAE phenotypes were identified: pruritus (26%), maculopapular rash (MPR; 21%), eczema (19%), lichenoid (11%), urticaria (8%), psoriasiform (6%), vitiligo (5%), and bullous dermatitis (4%). All phenotypes showed skin lymphocyte and eosinophil infiltrates. Skin biopsy PCR revealed the highest increase in IFNγ mRNA in patients with lichenoid (P < 0.0001) and psoriasiform dermatitis (P < 0.01) as compared with patients without ircAEs, whereas the highest IL13 mRNA levels were detected in patients with eczema (P < 0.0001, compared with control). IL17A mRNA was selectively increased in psoriasiform (P < 0.001), lichenoid (P < 0.0001), bullous dermatitis (P < 0.05), and MPR (P < 0.001) compared with control. Distinct cytokine profiles were confirmed in skin tape strip and plasma. Analysis determined increased skin/plasma IL4 cytokine in pruritus, skin IL13 in eczema, plasma IL5 and IL31 in eczema and urticaria, and mixed-cytokine pathways in MPR. Broad inhibition via corticosteroids or type 2 cytokine-targeted inhibition resulted in clinical benefit in these ircAEs. In contrast, significant skin upregulation of type 1/type 17 pathways was found in psoriasiform, lichenoid, bullous dermatitis, and type 1 activation in vitiligo. Distinct immunologic ircAE endotypes suggest actionable targets for precision medicine-based interventions.

Betulinic Acid Potentiates Mast Cell Degranulation by Compromising Cell Membrane Integrity and Without Involving Fcεri Receptors

ABSTRACT Mast cells play important role in acquired and natural immunity making these favorable therapeutic targets in various inflammatory diseases. Here we observed that, pentacyclic tri terpenoid betulinic acid (BA) treatment resulted in a significantly high number (9%) of cells positive for Hoechst and negative for annexin-V indicating that BA could interfere with plasma membrane integrity. The degranulation of both activated and non-activated mast cells was enhanced upon treatment with BA. The pre-treatment of BA had remarkable effect on calcium response in activated mast cells which showed increased calcium influx relative compared to untreated cells. The results also showed potentially less migration of BA treated mast cells signifying the possible effect of BA on cell membrane. BA treatment resulted in a significant increase in mRNA levels of IL-13 while as mRNA levels of other target cytokines, IL-6 and TNF-α seem to be not affected. Moreover, there was global Increase in phosphorylation of signaling proteins and no significant change in phosphorylation of FcεRI receptors indicating that the effect of BA was independent of signaling cascade or FcεRI receptor mediated mast cell aggregation. Overall, these results portray BA potentiates mast cell effector functions by compromising the membrane integrity and independent of FcεRI involvement.

Effect of chronic stress on gel-forming mucins in the small intestine of BALB/c mice.

Intestinal homeostasis involves the collaboration of gut barrier components, such as goblet cells and IgA-microbiota complexes, that are under the control of stress that promotes inflammatory responses addressed primarily in the colon. The aim of this study was to evaluate the effect of stress on mucins, goblet cells, and proinflammatory parameters in the proximal and distal regions of the small intestine. A group (n = 6) of female 8-week-old BALB/c mice underwent board immobilization stress (2 h per day for 4 days) and were sacrificed with isoflurane. Samples from proximal and distal small segments were collected to analyze the following: 1) goblet cells stained with periodic acid-Schiff (PAS) and with alcian blue (AB) to visualize histologically neutral and acidic mucins, respectively; 2) IgA-microbiota complexes identified by flow cytometry in intestinal lavages; and 3) MUC2, MUC5AC, and IL-18 mRNA levels in whole mucosal scrapings by reverse transcription-qPCR. Regarding the unstressed group, in the proximal region of small intestine both PAS+ and AB+ goblet cells were unchanged; however, MUC5AC and IL-18 mRNA levels were increased, and the percentage of IgA-microbiota complexes was reduced. In the distal segment, the number of PAS+ goblet cells was increased, whereas the number of AB+ goblet cells was reduced and did not affect the remaining parameters. The data suggest that stress induces inflammation in the proximal small intestine; these findings may provide an experimental reference for human diseases that may affect the proximal small intestine, such as Crohn's disease, in which stress contributes to the progression of intestinal inflammation or relapse.