mRNA Expression Of IL-1β Research Articles

Baicalin attenuates PD-1/PD-L1 axis-induced immunosuppression in piglets challenged with Glaesserella parasuis by inhibiting the PI3K/Akt/mTOR and RAS/MEK/ERK signalling pathways

Infection of piglets with Glaesserella parasuis (G. parasuis) induces host immunosuppression. However, the mechanism underlying the immunosuppression of piglets remains unclear. Activation of the PD-1/PD-L1 axis has been shown to trigger host immunosuppression. Baicalin possesses anti-inflammatory and immunomodulatory functions. However, whether baicalin inhibits PD-1/PD-L1 activation and thus alleviates host immunosuppression has not been investigated. In this study, the effect of baicalin on the attenuation of piglet immunosuppression induced by G. parasuis was evaluated. Seventy piglets were randomly divided into the control group, infection group, levamisole group, BMS-1 group, 25 mg/kg baicalin group, 50 mg/kg baicalin group and 100 mg/kg baicalin group. Following pretreatment with levamisole, BMS-1 or baicalin, the piglets were challenged with 1 × 108 CFU of G. parasuis. Our results showed that baicalin, levamisole and BMS-1 modified routine blood indicators and biochemical parameters; downregulated IL-1β, IL-10, IL-18, TNF-α and IFN-γ mRNA expression; and upregulated IL-2 and IL-8 mRNA expression in blood. Baicalin, levamisole and BMS-1 increased the proportions of CD3+ T cells, CD3+CD4+ T cells, CD3+CD8+ T cells and CD3–CD21+ B cells in the splenocyte population, increased the proportions of CD3+ T cells, CD3+CD4+ T cells and CD3+CD8+ T cells in the blood, and inhibited PD-1/PD-L1 and TIM-3 activation. Baicalin, levamisole and BMS-1 reduced p-PI3K, p-Akt, and p-mTOR expression, the p-MEK1/2/MEK1/2 and p-ERK1/2/ERK1/2 ratios and increased RAS expression. Baicalin, levamisole and BMS-1 provided substantial protection against G. parasuis challenge and relieved tissue histopathological damage. Our findings might provide new strategies for controlling G. parasuis infection and other immunosuppressive diseases.

Polysaccharides, the active component of Dendrobiumofficinale flower, ameliorates chronic pharyngitis in rats via TLR4/NF-κb pathway regulation

Ethnopharmacological relevanceChronic pharyngitis persistently afflicts a large population and accounts for approximately one-third of otolaryngology patients. Currently, the treatment of CP remains controversial because of the poor outcomes. Dendrobium officinale is a well-used “Yin-nourishing” traditional Chinese medicinal and edible herb used for thousands of years in China. The flowers of D. officinale are often used in folk of China to make tea for voice protection on and throat clearing. Aim of the studyThis study was to evaluate beneficial effects of polysaccharides from D. officinale flower (DOFP) on CP and its potential mechanisms. MethodsChemical characterization of DOFP, including polysaccharide content and monosaccharide composition, structural characterization using Fourier transform infrared spectroscopy were performed. A CP model was established in rats by administering a mixture of Chinese Baijiu and chili pepper liquid, combined with low-concentration ammonia spraying. The general states, amount of oral secretion, and apparent state of the pharynx of CP rats were observed during the period of DOFP administration. Furthermore, hemorheological parameters were measured using an automatic hematology analyzer. The levels of tumor necrosis factor-α (TNF-α), interleukin 1β (1L-1β), lipopolysaccharide (LPS), and D-lactate (D-LA) in the serum were measured by enzyme-linked immunosorbent assay. Morphological changes in the pharynx and colon were observed by hematoxylin-eosin staining. The expression of nuclear factor-κB p65 (NF-κB p65), p-NF-κB p65, cyclooxygenase-2 (COX-2), interleukin 1β（IL-1β）and mucin 5AC (MUC5AC) in the pharynx,Claudin-1, Occludin, and interleukin 6 (IL-6) in the colon was detected by immunohistochemistry and Western Blot. The mRNA expression of TLR4, COX-2, and IL-1β in the pharynx were determined using reverse transcription quantitative real-time PCR. ResultsIn this study, DOFP with a total polysaccharide content of 71.44% and a composition of D-mannose, galacturonic acid, glucose, galactose, and arabinose in a molar ratio of 3.95:2.19:1.00:0.74:1.30, was isolated from the flowers of D. officinale. DOFP improved the general state and exhibited significant effects on reducing oral secretion, alleviating pharyngeal injury, suppressing inflammatory cell infiltration in the pharynx, decreasing the serum levels of TNF-α and IL-1β, and reducing the number of white blood cells and lymphocytes in the model rats. Moreover, the expressions of TLR4, p-NF-κB p65, COX-2, IL-1β and MUC5AC in the pharynx of model rats were obviously inhibited. In addition, the levels of LPS, D-LA in the serum and the protein expression of IL-6 in the colon were downregulated when the protein expression of Occludin and Claudin-1 in the colon were upregulated. ConclusionsDOFP exerts significant ameliorating effects on CP and it likely acts by inhibiting LPS/TLR4-associated inflammatory mediator activation and reducing excessive secretion of mucus by repairing the intestinal barrier in CP rats.

Assessment of the role of high-density lipoproteins and their immunomodulatory activity in systemic lupus erythematosus immunopathology

ObjectiveTo explore the potential associations between high-density lipoprotein (HDL) levels and inflammasome components in the context of systemic lupus erythematosus (SLE).MethodsA cross-sectional study was conducted. A group of 50 patients...

IL-10 Enhances the Inhibitory Effect of Adipose-Derived Stromal Cells on Insulin Resistance/Liver Gluconeogenesis by Treg Cell Induction.

The modulation of cellular phenotypes within adipose tissue provides a potential means for therapeutic intervention for diabetes. Endogenous interleukin-10 (IL-10) protects against diet-induced insulin resistance. We examined the effects and mechanisms of action of IL-10-treated adipose-derived stromal cells on diabetes-induced insulin resistance and liver gluconeogenesis. We harvested stromal vascular fractions (SVFs) from the adipose tissue of diabetic (Leprdb/db) mice and treated them with IL-10 in vitro. SVFs treated with 10 or 100 ng of IL-10 were injected into the inguinal adipose tissue of Leprdb/db mice. IL-10 treatment suppressed the mRNA expression of IL-6, IL-33, CCL2, TNF-α, and IL-1β. Additionally, it suppressed the protein expression of IL-6, pmTOR, pJNK, and pNF-κB but enhanced Foxp3 mRNA expression in SVFs from diabetic mice. Meanwhile, IL-10 treatment repressed CCL2 and PDGFRα expression in adipose tissue macrophages (ATMs) and IL-6 expression in non-ATMs but increased the Foxp3 and IL-10 mRNA expression of ATMs from diabetic mice. Injection of IL-10-treated SVFs decreased the IL-6, IL-33, CCL2, IL-1β, and CCL2 but enhanced the Foxp3 and IL-10 mRNA expression of adipose tissue from Leprdb/db mice. Furthermore, injection of IL-10-treated SVFs increased CD4+ regulatory T cells (Tregs) in SVFs and adipose IL-10 levels and suppressed plasma adiponectin levels and DPP4 activity in diabetic mice. Injection of IL-10-treated SVFs decreased hepatic G6PC and PCK1 mRNA expression and increased Akt activation, STAT3 phosphorylation in the liver, and glucose tolerance in diabetic mice. Our data suggest that IL-10 treatment decreases inflammation in adipose SVFs of diabetic mice. Injection of IL-10-treated SVFs into the adipose tissue decreased diabetes-induced gluconeogenesis gene expression, DPP4 activity, and insulin resistance by enhancing Treg cells in diabetic mice. These data suggest that IL-10-treated adipose stromal vascular cells could be a promising therapeutic strategy for diabetes mellitus.

Astragaloside IV Alleviates Podocyte Injury in Diabetic Nephropathy through Regulating IRE-1α/NF-κ B/NLRP3 Pathway.

To investigate the effects of astragaloside IV (AS-IV) on podocyte injury of diabetic nephropathy (DN) and reveal its potential mechanism. In in vitro experiment, podocytes were divided into 4 groups, normal, high glucose (HG), inositol-requiring enzyme 1 (IRE-1) α activator (HG+thapsigargin 1 µmol/L), and IRE-1α inhibitor (HG+STF-083010, 20 µmol/L) groups. Additionally, podocytes were divided into 4 groups, including normal, HG, AS-IV (HG+AS-IV 20 µmol/L), and IRE-1α inhibitor (HG+STF-083010, 20 µmol/L) groups, respectively. After 24 h treatment, the morphology of podocytes and endoplasmic reticulum (ER) was observed by electron microscopy. The expressions of glucose-regulated protein 78 (GRP78) and IRE-1α were detected by cellular immunofluorescence. In in vivo experiment, DN rat model was established via a consecutive 3-day intraperitoneal streptozotocin (STZ) injections. A total of 40 rats were assigned into the normal, DN, AS-IV [AS-IV 40 mg/(kg·d)], and IRE-1α inhibitor [STF-083010, 10 mg/(kg·d)] groups (n=10), respectively. The general condition, 24-h urine volume, random blood glucose, urinary protein excretion rate (UAER), urea nitrogen (BUN), and serum creatinine (SCr) levels of rats were measured after 8 weeks of intervention. Pathological changes in the renal tissue were observed by hematoxylin and eosin (HE) staining. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to detect the expressions of GRP78, IRE-1α, nuclear factor kappa Bp65 (NF-κBp65), interleukin (IL)-1β, NLR family pyrin domain containing 3 (NLRP3), caspase-1, gasdermin D-N (GSDMD-N), and nephrin at the mRNA and protein levels in vivo and in vitro, respectively. Cytoplasmic vacuolation and ER swelling were observed in the HG and IRE-1α activator groups. Podocyte morphology and ER expansion were improved in AS-IV and IRE-1α inhibitor groups compared with HG group. Cellular immunofluorescence showed that compared with the normal group, the fluorescence intensity of GRP78 and IRE-1α in the HG and IRE-1α activator groups were significantly increased whereas decreased in AS-IV and IRE-1α inhibitor groups (P<0.05). Compared with the normal group, the mRNA and protein expressions of GRP78, IRE-1α, NF-κ Bp65, IL-1β, NLRP3, caspase-1 and GSDMD-N in the HG group was increased (P<0.05). Compared with HG group, the expression of above indices was decreased in the AS-IV and IRE-1α inhibitor groups, and the expression in the IRE-1α activator group was increased (P<0.05). The expression of nephrin was decreased in the HG group, and increased in AS-IV and IRE-1α inhibitor groups (P<0.05). The in vivo experiment results revealed that compared to the normal group, the levels of blood glucose, triglyceride, total cholesterol, BUN, blood creatinine and urinary protein in the DN group were higher (P<0.05). Compared with DN group, the above indices in AS-IV and IRE-1α inhibitor groups were decreased (P<0.05). HE staining revealed glomerular hypertrophy, mesangial widening and mesangial cell proliferation in the renal tissue of the DN group. Compared with the DN group, the above pathological changes in renal tissue of AS-IV and IRE-1α inhibitor groups were alleviated. Quantitative RT-PCR and Western blot results of GRP78, IRE-1α, NF-κ Bp65, IL-1β, NLRP3, caspase-1 and GSDMD-N were consistent with immunofluorescence analysis. AS-IV could reduce ERS and inflammation, improve podocyte pyroptosis, thus exerting a podocyte-protective effect in DN, through regulating IRE-1α/NF-κ B/NLRP3 signaling pathway.

CcPTGS2a-like gene-mediated NF-κB/ERK signaling regulation in the common carp (Cyprinus carpio)

Prostaglandin-endoperoxide synthase 2 (PTGS2), a common biological macromolecule, is pivotal for innate immunity and pathogen recognition. In this study, we identified and characterized a CcPTGS2a-like gene in the common carp (Cyprinus carpio) with an open reading frame (ORF) of 1821 bp and epidermal growth factor and peroxidase domains. Our multiple sequence analysis revealed high homology between the amino acid sequence of CcPTGS2a-like and those of its homologs in other fish. CcPTGS2a-like mRNA and protein expressions were significantly upregulated in the spleen, head kidney, liver, and gill tissues upon exposure to Aeromonas hydrophila stimulation. CcPTGS2a-like protein recognized the conserved bacterial surface components and exhibited detectable bacterial binding activity. CcPTGS2a-like overexpression before exposure to A. hydrophila notably enhanced the survival rate of common carp, concomitant with decreased bacterial burden. The NF-κB/ERK signaling pathway initiated the immune response in common carp upon infection with A. hydrophila. CcPTGS2a-like overexpression or interference in the head kidney and Epithelioma papulosum cyprinid cells could modulate the p–NF–κB (p-p-65), p-IκBα, and p-ERK1/2 levels as well as the IL-1β and IL-6 mRNA expression. These results indicated potential CcPTGS2a-like involvement in the immune response of the common carp to bacterial infections through the NF-κB/ERK signaling pathway.

Moderate capsaicin-containing kochujang alleviates memory impairment through the gut-brain axis in rats with scopolamine-induced amnesia

This study investigated the efficacy and mechanism of traditionally made kochujang(TMK) with different capsaicin levels to alleviate memory impairment in rats with scopolamine-induced amnesia. Sprague-Dawley male rats were administered scopolamine (2 mg/kg bw/day) intraperitoneally to suppress the parasympathetic nervous system(PNS) and induce memory impairment. The rats were divided into four experimental groups, each consuming a diet containing 1 % kochujang in a 43-energy% high-fat diet(HFD) for 8 weeks. The TMK samples used for the study were categorized according to their capsaicin(CPS) content as follows: Low-CPS(0.5 mg%), medium-CPS(1.2 mg%), and high-CPS(1.7 mg%). In addition, factory-made kochujang (FMK; 1.1 mg% capsaicin) was also tested. The effects of kochujang were compared with the Control group(scopolamine), Positive-control(scopolamine+donepezil), and Normal-control(saline) fed HFD. Kochujang consumption reduced body weight and fat mass compared to the Control group. Compared to the Control, memory function measured using passive avoidance, water maze, and novel object recognition tests was enhanced in kochujang-fed rats, especially in the Medium-CPS group, similar to Positive-control. The Medium-CPS and Positive-control groups also exhibited inhibition of hippocampal cell death and increased cholesterol and triglyceride contents and mRNA expression of TNF-α and IL-1β in the brain tissue compared to the Control group. Additionally, TMK elevated short-chain fatty acid, particularly, butyrate concentration in the portal vein. Scopolamine disturbed large intestine cell morphology and gut microbiota composition, and kochujang improved them. Kochujang in the medium-CPS (1.2 mg%) had a more significant impact on the gut microbiota in the interaction analysis between gut microbiota and memory function. In conclusion, kochujang, especially with medium-CPS (1.2 mg%), is a potential dietary intervention to mitigate memory impairment and promote overall cognitive health through improving eubiosis, potentially linked to the gut-brain axis in PNS-suppressed rats.

Cytokines and chemokines skin gene expression in correlation with immune cells in blood and severity in equine insect bite hypersensitivity.

Insect bite hypersensitivity (IBH) is the most frequent skin allergy of horses and is highly debilitating, especially in the chronic phase. IBH is caused by IgE-mediated hypersensitivity reactions to culicoides midge bites and an imbalanced immune response that reduces the welfare of affected horses. In the present study, we investigated the pathological mechanisms of IBH, aiming to understand the immune cell modulation in acute allergic skin lesions of IBH horses with the goal of finding possible biomarkers for a diagnostic approach to monitor treatment success. By qPCR, we quantified the gene expression of cytokines, chemokines, and immune receptors in skin punch biopsies of IBH with different severity levels and healthy horses simultaneously in tandem with the analysis of immune cell counts in the blood. Our data show an increase in blood eosinophils, monocytes, and basophils with a concomitant, significant increase in associated cytokine, chemokine, and immune cell receptor mRNA expression levels in the lesional skin of IBH horses. Moreover, IL-5Ra, CCR5, IFN-γ, and IL-31Ra were strongly associated with IBH severity, while IL-31 and IL-33 were rather associated with a milder form of IBH. In addition, our data show a strong correlation of basophil cell count in blood with IL-31Ra, IL-5, IL-5Ra, IFN-γ, HRH2, HRH4, CCR3, CCR5, IL-12b, IL-10, IL-1β, and CCL26 mRNA expression in skin punch biopsies of IBH horses. In summary, several cytokines and chemokines have been found to be associated with disease severity, hence contributing to IBH pathology. These molecules can be used as potential biomarkers to monitor the onset and progression of the disease or even to evaluate and monitor the efficacy of new therapeutic treatments for IBH skin allergy. To our knowledge, this is the first study that investigated immune cells together with a large set of genes related to their biological function, including correlation to disease severity, in a large cohort of healthy and IBH horses.

Paricalcitol attenuates oxidative stress and inflammatory response in the liver of NAFLD rats by regulating FOXO3a and NFκB acetylation

The lack of therapeutics along with complex pathophysiology made non-alcoholic fatty liver disease (NAFLD) a research hotspot. Studies showed that the deficiency of Vitamin D plays a vital role in NAFLD pathogenesis. While several research studies focused on vitamin D supplementation in NAFLD, there is still a need to understand the regulatory mechanism of direct vitamin D receptor activation in NAFLD. In the present study, we explored the role of direct Vitamin D receptor activation using paricalcitol in choline-deficient high-fat diet-induced NAFLD rat liver and its modulation on protein acetylation. Our results showed that paricalcitol administration significantly reduced the fat accumulation in HepG2 cells and the liver of NAFLD rats. Paricalcitol attenuated the elevated serum level of alanine transaminase, aspartate transaminase, insulin, low-density lipoprotein, triglyceride, and increased high-density lipoprotein in NAFLD rats. Paricalcitol significantly decreased the increased total protein acetylation by enhancing the SIRT1 and SIRT3 expression in NAFLD liver. Further, the study revealed that paricalcitol reduced the acetylation of NFκB and FOXO3a in NAFLD liver along with a decrease in the mRNA expression of IL1β, NFκB, TNFα, and increased catalase and MnSOD. Moreover, total antioxidant activity, glutathione, and catalase were also elevated, whereas lipid peroxidation, myeloperoxidase, and reactive oxygen species levels were significantly decreased in the liver of NAFLD after paricalcitol administration. The study concludes that the downregulation of SIRT1 and SIRT3 in NAFLD liver was associated with an increased acetylated NFκB and FOXO3a. Paricalcitol effectively reversed hepatic inflammation and oxidative stress in NAFLD rats through transcriptional regulation of NFκB and FOXO3a, respectively, by inhibiting their acetylation.

Andrographis paniculata Ethanolic Extract Improved Doxorubicin-induced Cardiac Inflammation, Alterations in Liver Function Parameters and Anemia

Background: Doxorubicin (DOX), an efficacious chemotherapy drug is compromised by cardiotoxicity, myelosuppression, and hepatotoxicity. Due to the limited success of current treatments for DOX toxicity, there is a pressing need to explore alternative medical interventions, particularly from plant sources. This study was conducted to investigate the potential protective effect of ethanolic extract of Andrographis paniculata leaves (EEAP) against DOX-induced cardiac inflammation, liver toxicity, and anemia.Materials and methods: Sprague-Dawley rats were intraperitoneally injected with DOX at a total dose of 16 mg/kgBW. EEAP was administered orally for 4 weeks at doses of 125, 250, and 500 mg/kgBW/day according to the assigned treatment groups. The mRNA expression levels of interleukin-1β (IL-1β) and nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) in the heart tissue, along with the concentrations of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) and calcium level were examined. Additionally, the hematological parameters (including hematocrit, hemoglobin and red blood cells (RBCs)), aspartate aminotransferase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), and malondialdehyde (MDA) levels in blood were also analyzed.Results: EEAP dose-dependently decreased the mRNA expressions of IL-1β (p&lt;0.05), tended to decrease mRNA expression of NLRP3 and the concentrations of NFκB and calcium in heart tissue compared with the DOX-only group. Additionally, EEAP dose-dependently decreased ALP values (p&lt;0.0001) and tended to improve hematological parameters, as well as AST and MDA levels in serum.Conclusion: This extract may prevent DOX-induced cardiac inflammation, anemia, and hepatotoxicity. However, further studies are needed to confirm these findings, including the efficacy profile of the extract in cancer rats treated with DOX.Keywords: doxorubicin, Andrographis paniculata, inflammation, anemia, hepatotoxicity, herbal medicine

Anti-Inflammatory and Cancer-Preventive Potential of Chamomile (Matricaria chamomilla L.): A Comprehensive In Silico and In Vitro Study.

Chamomile tea, renowned for its exquisite taste, has been appreciated for centuries not only for its flavor but also for its myriad health benefits. In this study, we investigated the preventive potential of chamomile (Matricaria chamomilla L.) towards cancer by focusing on its anti-inflammatory activity. A virtual drug screening of 212 phytochemicals from chamomile revealed β-amyrin, β-eudesmol, β-sitosterol, apigenin, daucosterol, and myricetin as potent NF-κB inhibitors. The in silico results were verified through microscale thermophoresis, reporter cell line experiments, and flow cytometric determination of reactive oxygen species and mitochondrial membrane potential. An oncobiogram generated through comparison of 91 anticancer agents with known modes of action using the NCI tumor cell line panel revealed significant relationships of cytotoxic chamomile compounds, lupeol, and quercetin to microtubule inhibitors. This hypothesis was verified by confocal microscopy using α-tubulin-GFP-transfected U2OS cells and molecular docking of lupeol and quercetin to tubulins. Both compounds induced G2/M cell cycle arrest and necrosis rather than apoptosis. Interestingly, lupeol and quercetin were not involved in major mechanisms of resistance to established anticancer drugs (ABC transporters, TP53, or EGFR). Performing hierarchical cluster analyses of proteomic expression data of the NCI cell line panel identified two sets of 40 proteins determining sensitivity and resistance to lupeol and quercetin, further pointing to the multi-specific nature of chamomile compounds. Furthermore, lupeol, quercetin, and β-amyrin inhibited the mRNA expression of the proinflammatory cytokines IL-1β and IL6 in NF-κB reporter cells (HEK-Blue Null1). Moreover, Kaplan-Meier-based survival analyses with NF-κB as the target protein of these compounds were performed by mining the TCGA-based KM-Plotter repository with 7489 cancer patients. Renal clear cell carcinomas (grade 3, low mutational rate, low neoantigen load) were significantly associated with shorter survival of patients, indicating that these subgroups of tumors might benefit from NF-κB inhibition by chamomile compounds. This study revealed the potential of chamomile, positioning it as a promising preventive agent against inflammation and cancer. Further research and clinical studies are recommended.

Anti-Inflammatory Effect of Atorvastatin and Rosuvastatin on Monosodium Urate-Induced Inflammation through IL-37/Smad3-Complex Activation in an In Vitro Study Using THP-1 Macrophages.

Objective: The pleiotropic effect of hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) is responsible for potent defense against inflammatory response. This study evaluated the inhibitory effects of HMG-CoA reductase inhibitors on the monosodium urate (MSU)-induced inflammatory response through the regulation of interleukin-37 (IL-37) expression. Methods: Serum was collected from patients with gout (n = 40) and from healthy controls (n = 30). The mRNA and protein expression of the target molecules IL-1β, IL-37, caspase-1, and Smad3 were measured in THP-1 macrophages stimulated with MSU, atorvastatin, or rosuvastatin using a real-time quantitative polymerase chain reaction and Western blot assay. Transfection with IL-1β or Smad3 siRNA in THP-1 macrophages was used to verify the pharmaceutical effect of statins in uric-acid-induced inflammation. Results: Serum IL-37 levels in gout patients were significantly higher than in controls (p < 0.001) and was associated with the serum uric acid level (r = 0.382, p = 0.008). THP-1 cells stimulated with MSU markedly induced IL-37 mRNA expression and the transition of IL-37 from the cytoplasm to the nucleus. Recombinant IL-37 treatment dose-dependently inhibited activation of caspase-1 and IL-1β in MSU-induced inflammation. Atorvastatin and rosuvastatin attenuated caspase-1 activation and mature IL-1β expression but augmented translocation of IL-37 from the cytoplasm to the nucleus. Atorvastatin and rosuvastatin induced phosphorylation of Smad3 in THP-1 cells treated with MSU crystals. Statins potently attenuated translocation of IL-37 from the cytoplasm to the nucleus in THP-1 macrophages transfected with Smad3 siRNA compared to cells with negative control siRNA. Conclusions: This study revealed that statins inhibit the MSU-induced inflammatory response through phosphorylated Smad3-mediated IL-37 expression in THP-1 macrophages.

Effects of IL-6R Expression on IL-6/STAT3/HIF-1α Signaling Pathway in a Mouse Model of Parkinson's Disease with Type 2 Diabetes Co-Morbidity.

More and more evidence has shown the process of Parkinson's disease (PD). Probably, inflammation exerts a crucial role between them. Therefore, the aim of this study was to analyze the impact of interleukin-6 receptor (IL-6R) expression on the IL-6/signal transducer and activator of transcription 3 (STAT3)/hypoxia-inducible factor-1α (HIF-1α) inflammatory signaling pathway within a mouse model of PD with type 2 diabetes mellitus (T2DM) as co-morbidity. We chose healthy wild-type C57BL/6J male mice at the age of 10 weeks to prepare a mouse model of PD with T2DM co-morbidity. Adeno-associated virus (AAV) overexpressing IL-6R or AAV IL-6R-shRNA genes were injected into the substantia nigra (SN) of the mice. The behavioral indices of the pole test were used for examining the motor function of the mice. Using immunofluorescence analysis, the impacts of IL-6R on the level of tyrosine hydroxylase (TH) and anti-ionized calcium-binding adaptor molecule 1 (IBA-1) on dopaminergic neurons and microglia were examined. Additionally, enzyme-linked immunosorbent assay (ELISA) was adopted for determining the expressions of HIF-1α and inflammatory cytokines like tumor necrosis factor-α (TNF-α), IL-1β, IL-6, and IL-4 in the serum. In this study, the protein expression levels of TH, α-Synuclein (α-Syn), IBA-1, IL-6, IL-6R, phosphorylated and total signal transducer and activator of transcription 3 (p-STAT3 (Tyr705) and STAT3) and HIF-1α in the SN were tested via western blotting. To ascertain the mRNA expressions of TNF-α, IL-1β, IL-6, IL-4, and HIF-1α, we used quantitative Real-Time Polymerase Chain Reaction (RT-qPCR). IL-6R-shRNA treatment could markedly shorten the total time of PD in the T2DM co-morbidity mouse model based on the pole test results, reverse the decrease in TH-positive neurons stimulated by 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP), and lower the activation of microglia (all p < 0.05). Further, IL-6R-shRNA treatment hindered the expression of IL-6, p-STAT3 (Tyr705), and HIF-1α in the SN, lowered the levels of TNF-α, IL-1β, IL-6, IL-4, and HIF-1α in the serum, and mRNA expressions of TNF-α, IL-1β, IL-6, and HIF-1α in the SN (all p < 0.05). In contrast, IL-6R overexpression reduced TH levels, upregulated the level of IBA-1, IL-6, p-STAT3 (Tyr705), and HIF-1α, increased the level of IL-1β, TNF-α, IL-6, IL-4, and HIF-1α (all p < 0.05) in the serum and SN in the PD mouse model with T2DM as a co-morbidity. PD progression with T2DM as a co-morbidity can be boosted by AAV IL-6R-overexpression through upregulation of the IL-6/STAT3/HIF-1α axis. Conversely, AAV IL-6R-shRNA treatment suppressed the IL-6/STAT3/HIF-1α pathway and alleviated neuroinflammation, thus weakening the development of PD with T2DM as a co-morbidity.

Effects and mechanisms of Gegen Qinlian Decoction in inhibiting M1 polarization of macrophages under inflammatory hypoxia microenvironment

To explore the effect and mechanism of Gegen Qinlian Decoction(GQD) in inhibiting M1 polarization of macrophages under inflammatory hypoxia by simulating intestinal hypoxia microenvironment in vitro. A tri-gas incubator was used to simulate normal physiological hypoxia of the colon and inflammatory hypoxia microenvironments of ulcerative colitis(UC). RAW264.7 macrophages were divided into 18.5% O_(2 )(normoxia group), 4% O_2(physiological hypoxia group), and 1% O_2(inflammatory hypoxia group), and they were induced by lipopolysaccharide(LPS) for 24 h. M1 polarization was detected by flow cytometry. Under the condition of 1% inflammatory hypoxia, they were divided into blank group, model group, and GQD-containing serum low, medium, and high dose groups. Flow cytometry was used to detect M1 polarization marker CD86, and ELISA was used to detect the expression of tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β) in cell supernatant. The mRNA expression of hypoxia-inducible factor-1α(HIF-1α), TNF-α, and IL-1β was detected by qRT-PCR. Western blot was used to detect the expression of HIF-1α/nuclear transcription factor-κB(NF-κB) signaling pathway-related proteins. The nuclear translocation of NF-κB p65 was detected by immunofluorescence. The results showed that the positive rate of CD86 in the 1% O_2 group was the highest. Under the condition of 1% inflammatory hypoxia, compared with the blank group, the expression of CD86, TNF-α, IL-1β, and HIF-1α in the model group increased. Compared with the model group, each group of GQD could reduce the expression of CD86, TNF-α, IL-1β, and HIF-1α. Compared with the blank group, the protein expression of HIF-1α, NF-κB p65, p-IKKα/β, and p-IκBα in the model group increased. Compared with the model group, the protein expression of HIF-1α, NF-κB p65, p-IKKα/β, and p-IκBα in GQD groups was significantly decreased. Compared with the blank group, NF-κB p65 in the model group entered the nucleus significantly. Compared with the model group, the nuclear expression of NF-κB p65 was decreased in each GQD group. Studies have shown that GQD may protect the intestine by down-regulating the HIF-1α/NF-κB signaling pathway to inhibit M1 polarization of macrophages and secretion of related inflammatory factors under 1% inflammatory hypoxia.

Evaluation test and analysis of a microneedle and iontophoresis based medical device "CELLADEEP Patch" in skin improvement on ex vivo human-derived skin tissue models.

Microneedles are tiny needles, typically ranging from tens to hundreds of micrometers in length, used in various medical procedures and treatments. The tested medical device named "CELLADEEP Patch" a dissolvable microneedle therapy system (MTS), made of hyaluronic acid and collagen. And the iontophoresis technique is also applied in the system. The study aimed to evaluate the effectiveness of the "CELLADEEP Patch" in skin improvement. Ex vivo human-derived skin tissue models were used in this study and they were divided into three different groups, namely, the Untreated Group, the Negative Control Group, and the Test Group respectively. The Untreated Group received no treatment measures, the Negative Control Group was exposed to ultraviolet B radiation (UVB) irradiation, and the Test Group was exposed to UVB irradiation and treated with "CELLADEEP Patch". Skin moisture content, transdermal water loss, and skin elasticity were evaluated by three clinical devices. Additionally, histological staining and related mRNA expression levels were also analyzed. The results of skin moisture content, transdermal water loss, and skin elasticity evaluation consistently illustrated that the application of "CELLADEEP Patch" led to remarkable skin improvement. And the analysis of histological staining images also confirmed the effectiveness of the "CELLADEEP Patch", especially for increasing collagen density. Moreover, the upregulation of Collagen type 1 a (COL1A1) and hyaluronan synthase 3 mRNA expression and the decrease of Matrix metalloproteinase 1 (MMP-1) and Interleukin-1 beta (IL-1β) mRNA expression reflected its wrinkle improvement, moisturizing and anti-inflammation function. "CELLADEPP Patch", the MTS combined with the iontophoresis technique, exhibits its effectiveness in moisturizing, skin elasticity improvement, and anti-inflammatory function when applied to ex vivo human-derived skin tissue models in experiments. The study has contributed to the understanding of the "CELLADEPP Patch" and laid the foundation for subsequent animal experiments and clinical trials.

The effect of the HMGB1/RAGE/TLR4/NF-κB signalling pathway in patients with idiopathic epilepsy and its relationship with toxoplasmosis.

This study aims to investigate the relationship between toxoplasmosis and this pathway, which may be effective in the formation of epilepsy by acting through the HMGB1/RAGE/TLR4/NF-κB signalling pathway in patients with idiopathic epilepsy. In the study, four different experimental groups were formed by selecting Toxoplasma gondii IgG positive and negative patients with idiopathic epilepsy and healthy controls. Experimental groups were as follows: Group 1: Epilepsy+/Toxo- (E+, T-) (n = 10), Group 2: Epilepsy-/Toxo- (E-, T-) (n = 10), Group 3: Epilepsy-/Toxo+ (E-, T+) (n = 10), Group 4: Epilepsy+/Toxo+ (E+, T+) (n = 10). HMGB1, RAGE, TLR4, TLR1, TLR2, TLR3, IRAK1, IRAK2, IKBKB, IKBKG, BCL3, IL1β, IL10, 1 L8 and TNFα mRNA expression levels in the HMGB/RAGE/TLR4/NF-κB signalling pathway were determined by quantitative simultaneous PCR (qRT-PCR) after collecting blood samples from all patients in the groups. Statistical analysis was performed by one-way ANOVA followed by LSD post-hoc tests, and p < 0.05 was considered to denote statistical significance. The gene expression levels of HMGB1, TLR4, IL10, IL1B, IL8, and TLR2 were significantly higher in the G1 group than in the other groups (p < 0.05). In the G3 group, RAGE and BCL3 gene expression levels were significantly higher than in the other groups (p < 0.05). In the G4 group, however, IRAK2, IKBKB, and IKBKG gene expression levels were significantly higher than in the other groups (p < 0.05). HMGB1, TLR4, IRAK2, IKBKB, IL10, IL1B, IL1B, and IL8 in this signalling pathway are highly expressed in epilepsy patients in G1 and seizures occur with the stimulation of excitatory mechanisms by acting through this pathway. The signalling pathway in epilepsy may be activated by HMGB1, TLR4, and TLR2, which are considered to increase the level of proinflammatory cytokines. In T. gondii, this pathway is activated by RAGE and BCL3.

Exogenous autoinducer-2 alleviates intestinal damage in necrotizing enterocolitis via PAR2/MMP3 signaling pathway

BackgroundImbalanced intestinal microbiota and damage to the intestinal barrier contribute to the development of necrotizing enterocolitis (NEC). Autoinducer-2 (AI-2) plays a crucial role in repairing intestinal damage and reducing inflammation. ObjectiveThis study aimed to investigate the impact of AI-2 on the expression of intestinal zonula occludens-1 (ZO-1) and occludin proteins in NEC. We evaluated its effects in vivo using NEC mice and in vitro using lipopolysaccharide (LPS)-stimulated intestinal cells. MethodsPathological changes in the intestines of neonatal mice were assessed using histological staining and scoring. Cell proliferation was measured using the cell counting kit-8 (CCK-8) assay to determine the optimal conditions for LPS and AI-2 interventions. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to analyze the mRNA levels of matrix metalloproteinase-3 (MMP3), protease activated receptor-2 (PAR2), interleukin-1β (IL-1β), and IL-6. Protein levels of MMP3, PAR2, ZO-1, and occludin were evaluated using western blot, immunohistochemistry, or immunofluorescence. ResultsAI-2 alleviated NEC-induced intestinal damage (P < 0.05) and enhanced the proliferation of damaged IEC-6 cells (P < 0.05). AI-2 intervention reduced the mRNA and protein expressions of MMP3 and PAR2 in intestinal tissue and cells (P < 0.05). Additionally, it increased the protein levels of ZO-1 and occludin (P < 0.05), while reducing IL-1β and IL-6 mRNA expression (P < 0.05). ConclusionAI-2 intervention enhances the expression of tight junction proteins (ZO-1 and occludin), mitigates intestinal damage in NEC neonatal mice and IEC-6 cells, potentially by modulating PAR2 and MMP3 signaling. AI-2 holds promise as a protective intervention for NEC. AI-2 plays a crucial role in repairing intestinal damage and reducing inflammation.

Maresin 1 alleviates neuroinflammation and cognitive decline in a mouse model of cecal ligation and puncture.

Inflammation in the central nervous system plays a crucial role in the occurrence and development of sepsis-associated encephalopathy. This study aims to explore the effects of maresin 1 (MaR1), an anti-inflammatory and pro-resolving lipid mediator, on sepsis-induced neuroinflammation and cognitive impairment. Mice were randomly assigned to 4 groups: A sham group (sham operation+vehicle), a cecal ligation and puncture (CLP) group (CLP operation+vehicle), a MaR1-LD group (CLP operation+1 ng MaR1), and a MaR1-HD group (CLP operation+10 ng MaR1). MaR1 or vehicle was intraperitoneally administered starting 1 h before CLP operation, then every other day for 7 days. Survival rates were monitored, and serum inflammatory cytokines [tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, and IL-6] were measured 24 h after operation using enzyme-linked immunosorbent assay (ELISA). Cognitive function was assessed 7 days after operation using the Morris water maze (MWM) test and novel object recognition (NOR) task. The mRNA expression of TNF-α, IL-1β, IL-6, inducible nitric oxide synthase (iNOS), IL-4, IL-10, and arginase 1 (Arg1) in cortical and hippocampal tissues was determined by real-time reverse transcription PCR (RT-PCR). Western blotting was used to determine the protein expression of iNOS, Arg1, signal transducer and activator of transcription 6 (STAT6), peroxisome proliferator-activated receptor gamma (PPARγ), and phosphorylated STAT6 (p-STAT6) in hippocampal tissue. Microglia activation was visualized via immunofluorescence. Mice were also treated with the PPARγ antagonist GW9662 to confirm the involvement of this pathway in MaR1's effects. CLP increased serum levels of TNF-α, IL-1β, and IL-6, and reduced body weight and survival rates (all P<0.05). Both 1 ng and 10 ng doses of MaR1 significantly reduced serum TNF-α, IL-1β, and IL-6 levels, improved body weight, and increased survival rates (all P<0.05). No significant difference in efficacy was observed between the 2 doses (all P>0.05). MWM test and NOR task indicated that CLP impaired spatial learning, which MaR1 mitigated. However, GW9662 partially reversed MaR1's protective effects. Real-time RT-PCR results demonstrated that, compared to the sham group, mRNA expression of TNF-α, IL-1β, and iNOS significantly increased in hippocampal tissues following CLP (all P<0.05), while IL-4, IL-10, and Arg1 showed a slight decrease, though the differences were not statistically significant (all P>0.05). Compared to the CLP group, both 1 ng and 10 ng MaR1 decreased TNF-α, IL-1β, and iNOS mRNA expression in hippocampal tissues and increased IL-4, IL-10, and Arg1 mRNA expression (all P<0.05). Immunofluorescence results indicated a significant increase in Iba1-positive microglia in the hippocampus after CLP compared to the sham group (P<0.05). Administration of 1 ng and 10 ng MaR1 reduced the percentage area of Iba1-positive cells in the hippocampus compared to the CLP group (both P<0.05). Western blotting results showed that, compared to the CLP group, both 1 ng and 10 ng MaR1 down-regulated the iNOS expression, while up-regulated the expression of Arg1, PPARγ, and p-STAT6 (all P<0.05). However, the inclusion of GW9662 counteracted the MaR1-induced upregulation of Arg1 and PPARγ compared to the MaR1-LD group (all P<0.05). MaR1 inhibits the classical activation of hippocampal microglia, promotes alternative activation, reduces sepsis-induced neuroinflammation, and improves cognitive decline.

Microbiome and metabolome analyses reveal significant alterations of gut microbiota and bile acid metabolism in ETEC-challenged weaned piglets by dietary berberine supplementation.

This study mainly investigated the effects of berberine (BBR) on the bile acid metabolism in gut-liver axis and the microbial community in large intestine of weaned piglets challenged with enterotoxigenic Escherichia coli (ETEC) by microbiome and metabolome analyses. Sixty-four piglets were randomly assigned to four groups including Control group, BBR group, ETEC group, and BBR + ETEC group. Dietary BBR supplementation upregulated the colonic mRNA expression of Occludin, Claudin-5, trefoil factor 3 (TFF3), and interleukin (IL)-10, and downregulated colonic IL-1β and IL-8 mRNA expression in piglets challenged with ETEC K88 (p < 0.05). The hepatic non-targeted metabolome results showed that dietary BBR supplementation enriched the metabolic pathways of primary bile acid biosynthesis, tricarboxylic acid cycle, and taurine metabolism. The hepatic targeted metabolome analyses showed that BBR treatment increased the hepatic concentrations of taurocholic acid (TCA) and taurochenodeoxycholic acid (TDCA), but decreased the hepatic cholic acid (CA) concentration (p < 0.05). Further intestinal targeted metabolome analyses indicated that the deoxycholic acid (DCA), hyocholic acid (HCA), 7-ketodeoxycholic acid (7-KDCA), and the unconjugated bile acid concentrations in ileal mucosa was decreased by dietary BBR treatment (p < 0.05). Additionally, BBR treatment significantly upregulated the hepatic holesterol 7 α-hydroxylase (CYP7A1) and sterol 27-hydroxylase (CYP27A1) mRNA expression, and upregulated the ileal mRNA expression of farnesoid X receptor (FXR) and apical sodium-dependent bile acid transporter (ASBT) as well as the colonic mRNA expression of FXR, fibroblast growth factor19 (FGF19), takeda G protein-coupled receptor 5 (TGR5) and organic solute transporters beta (OST-β) in piglets (p < 0.05). Moreover, the microbiome analysis showed that BBR significantly altered the composition and diversity of colonic and cecal microbiota community, with the abundances of Firmicutes (phylum), and Lactobacillus and Megasphaera (genus) significantly increased in the large intestine of piglets (p < 0.05). Spearman correlation analysis showed that the relative abundances of Megasphaera (genus) were positively correlated with Claudin-5, Occludin, TFF3, and hepatic TCDCA concentration, but negatively correlated with hepatic CA and glycocholic acid (GCA) concentration (p < 0.05). Moreover, the relative abundances of Firmicute (phylum) and Lactobacillus (genus) were positively correlated with hepatic TCDCA concentration (p < 0.05). Collectively, dietary BBR supplementation could regulate the gut microbiota and bile acid metabolism through modulation of gut-liver axis, and attenuate the decreased intestinal tight junction expression caused by ETEC, which might help maintain intestinal homeostasis in weaned piglets.

Cyclophilin D induces necrotic core formation by mediating mitochondria-associated macrophage death in advanced atherosclerotic lesions

Background and aimsIn advanced atherosclerotic lesions, macrophage deaths result in necrotic core formation and plaque vulnerability. Cyclophilin D (CypD) is a mitochondria-specific cyclophilin involved in the process of cell death after organ ischemia-reperfusion. However, the role of CypD in atherosclerosis, especially in necrotic core formation, is unknown. Therefore, this experiment aims to clarify the role of CypD in necrotic core formation. MethodsTo clarify the specific role of CypD, encoded by Ppif in mice, apolipoprotein-E/CypD-double knockout (Apoe−/−Ppif−/−) mice were generated. These mice were fed a high-fat diet containing 0.15 % cholesterol for 24 weeks to accelerate atherosclerotic lesion development. ResultsDeletion of CypD decreased the necrotic core size, accompanied by a reduction of macrophage apoptosis compared to control Apoe−/− mice. In RAW264.7 cells, siRNA-mediated knockdown of CypD attenuated the release of cytochrome c from the mitochondria to the cytosol induced by endoplasmic reticulum stress inducer thapsigargin. In addition, necroptosis, induced by TNF-α and caspase inhibitor, was attenuated by knockdown of CypD. Ly-6Chigh inflammatory monocytes in peripheral blood leukocytes and mRNA expression of Il1b in the aorta were decreased by deletion of CypD. In contrast, siRNA-mediated knockdown of CypD did not significantly decrease Il1b nor Ccl2 mRNA expression in RAW264.7 cells treated with LPS and IFN-γ, suggesting that inhibition of inflammation in vivo is likely due to decreased cell death in the atherosclerotic lesions rather than a direct action of CypD deletion on the macrophage. ConclusionsThese results indicate that CypD induces macrophage death and mediates necrotic core formation in advanced atherosclerotic lesions. CypD could be a novel therapeutic target for treating atherosclerotic vascular diseases.