TLR4 mRNA Levels Research Articles

Interaction of inflammatory factors related to pulmonary infection and TLR4/NF-κB signaling pathway in patients with spontaneous intracerebral hemorrhage.

To investigate the interaction of inflammatory factors related to pulmonary infection and the TLR4/NF-κB signaling pathway in patients with spontaneous intracerebral hemorrhage (ICH). A total of 325 critically ill ICH patients treated in our hospital from May 2021 to February 2024 were selected for this study. Based on whether the patient developed a pulmonary infection during treatment, they were divided into the infection group (n=86) and the non-infection group (n=239). The distribution characteristics of pathogens were observed, and changes in serum and defensin, hs-CRP, IL-4, TLR4 mRNA, and NF-κB mRNA were compared. Pearson correlation analysis was used to analyze the relationship between serum defensin, inflammatory factors, and the TLR4/NF-κB signaling pathway. A logistic regression model was used to construct a combined prediction model with defensin, hs-CRP, IL-4, HMGB1, TLR4 mRNA, and NF-κB mRNA. ROC curves were drawn to analyze the area under the curve (AUC), sensitivity, and specificity of defensin, hs-CRP, IL-4, HMGB1, TLR4 mRNA, NF-κB mRNA, and the six combined parameters for predicting pulmonary infection in critically ill ICH patients. In 86 ICH patients with pulmonary infection, 94 strains of pathogens were isolated, with 28 (29.79%) Gram-positive bacteria, 58 (61.70%) Gram-negative bacteria, and 8 (8.51%) fungi. The levels of defensin, hs-CRP, IL-4, HMGB1, TLR4 mRNA, and NF-κB mRNA in the infection group were significantly higher than those in the non-infection group (P<0.05). Pearson correlation analysis showed that defensin, hs-CRP, IL-4, and HMGB1 were positively correlated with TLR4 mRNA and NF-κB mRNA (P<0.05). ROC curve analysis showed that the AUC values for defensin, hs-CRP, IL-4, HMGB1, TLR4 mRNA, NF-κB mRNA, and the six combined parameters for predicting pulmonary infection were 0.789, 0.778, 0.690, 0.792, 0.741, 0.750, and 0.870, respectively. The main pathogens causing pulmonary infection in critically ill ICH patients are Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Staphylococcus aureus, and Streptococcus hemolyticus. Defensin, hs-CRP, IL-4, HMGB1, and other indicators are influenced by the TLR4/NF-κB signaling pathway in patients with secondary pulmonary infection.

Yeast cell wall polysaccharides accelerate yet in-feed antibiotic delays intestinal development and maturation via modulating gut microbiome in chickens

BackgroundIt is important to promote intestinal development and maturation of chicks for feed digestion and utilization, intestinal health, and disease resistance. This study aimed to investigate the effects of dietary yeast cell wall polysaccharides (YCWP) addition on intestinal development and maturation of chickens and its potential action mechanism.Methods180 one-day-old male Arbor Acres broilers were randomly assigned to three groups containing control (basal diets without any antibiotics or anticoccidial drug), bacitracin methylene disalicylate (BMD)-treated group (50 mg/kg) and YCWP-supplemented group (100 mg/kg).ResultsCompared with control group, in-feed antibiotic BMD continuous administration significantly decreased crypt depth (d 21) and villus height (d 42) along with mucosal maltase activity (d 42) in the ileum (P < 0.05). Also, BMD markedly downregulated gene expression levels of β-catenin, lysozyme, occludin and FABP-2 (d 21) and innate immune related genes CD83 and MHC-I mRNA levels (d 42, P < 0.05), and decreased goblet cell counts in the ileum of chickens (d 21 and d 42, P < 0.05). While, TLR-2, TLR-6 and iNOS mRNA abundances were notably upregulated by BMD treatment (d 42, P < 0.05). Nevertheless, dietary YCWP addition significantly increased the ratio of villus height to crypt depth (d 21), villus surface area (d 21 and d 42), ileal alkaline phosphatase and maltase activities as well as goblet cell (d 21 and d 42) and IgA-producing plasma cell numbers as compared to BMD treatment (d 21, P < 0.05). YCWP addition also upregulated gene expression levels of Lgr5, Wnt/β-catenin signaling pathway related gene (Wnt3, β-catenin, d 21; β-catenin, d 42), intestinal cells proliferation marker Ki-67 and barrier function related genes (occludin, d 21 and d 42, P < 0.05). Moreover, YCWP significantly increased antigen presenting cell marker related genes (MHC-II, d 21; CD83 and MHC-I, d 42), TLR-1, TLR-2 and TLR-6 mRNA levels (d 21, P < 0.05). Cecal microbiome analysis showed that YCWP addition obviously improved cecal microbial composition, as indicated by increasing relative abundance of Fournierella, Psychrobacter and Ruminiclostridium on d 21, and Alistipes and Lactobacillus on d 42, which were positively related with gut development and maturation related indexes (P < 0.05).ConclusionCollectively, YCWP promoted yet antibiotic BMD delayed intestinal morphological and immunological development linked with modulating gut microbiome in chickens.

Protective effect of low-dose lactulose in dextran sulfate sodium induced ulcerative colitis model of rats

Although low-dose lactulose has shown a good theoretical foundation for the treatment of ulcerative colitis (UC) in previous studies, the exact effects and mechanism remain unclear. The rats were randomly distributed into 5 groups, i.e., normal drinking water was provided for an initial 14 days in blank control group, 4% dextran sulfate sodium was used for modeling in the remaining 4 groups. During the 15-24th day, rats in the blank control group were administered with 0.9% saline (0.5 ml/d) by gavage. In the rest 4 groups, rats were administered 0.9% saline (0.5 ml/d, UC model), mesalazine (400 mg/kg/d), lactulose (1000 mg/kg/d), and lactulose + mesalazine (two-drug combination) by gavage. In addition to symptoms and pathological changes, serum IL-6, TNF-α, and High-sensitivity C-reactive protein(Hs-CRP) by ELISA analysis, mRNA and protein expression levels of TLR-2, TLR-4, Nuclear factor-κB(NF-κB), IL-6, and TNF-α in colon tissues by RT-qPCR and WB analyses respectively. Meanwhile, short-chain fatty acid(SCFAs) and intestinal flora were analyzed. Low-dose lactulose improved symptoms (diarrhea, blood in stool, weight loss) and pathological inflammation. In addition to serum IL-6, TNF-α, and Hs-CRP, the mRNA and protein expression levels of TLR-2, TLR-4, NF-κB, IL-6 and TNF-α in the colon were down-regulated with the intervention of lactulose.Meanwhile, lactulose decreased the ileocecal PH, increased SCFAs and altered the intestinal flora. Low-dose lactulose may be beneficial to UC by regulating TLRs/NF-κB pathway, reducing ileocecal PH, increasing SCFAs, regulating intestinal flora and improving the intestinal mucosal barrier. Meanwhile, low-dose lactulose and mesalazine may have additive effects upon combination.

Short-term exposure to low doses of aflatoxin B1 aggravates nonalcoholic steatohepatitis by TLR4-mediated necroptosis.

Aflatoxin B1 (AFB1), a worldwide mycotoxin found in food and foodstuffs, is a potent hepatotoxin in humans and animals. Non-alcoholic fatty liver disease (NAFLD), a widespread disease, could progress from simple steatosis to non-alcoholic steatohepatitis (NASH), hepatic cirrhosis, and even hepatocellular carcinoma (HCC). To date, little is known concerning the relationship between AFB1 and the progression of NAFLD. The effects of low doses of AFB1 on the development of NASH and their mechanism were investigated in vivo and in vitro. The results in vivo showed that AFB1 at 20 and 40 μg/kg.bw aggravated CDAHFD-induced NASH in mice as demonstrated by increasing the serum and liver lipid accumulation, liver inflammation and injury. The results in vitro showed that AFB1 at 1.0 μM aggravated FFA-induced lipid accumulation, inflammation and cell damage in HepG2 cells. In addition, RNA-seq indicated that necroptosis, Toll like receptor signaling and TNF-α signaling showed a significant change in KEGG pathway enrichment in AFB1 at 40 μg/kg.bw. AFB1 significantly upregulated the mRNA and protein levels of TLR4, RIPK3, p-RIPK3, MLKL and p-MLKL, and increased TUNEL positive cells. Also, immunofluorescence results showed that TUNEL, TLR4, TNF-α had co-localized with RIPK3, respectively. Necroptosis inhibitor (GSK-872) attenuated the aggravating effects of AFB1 on NASH. Knockout of TLR4 inhibited necroptosis and rescued the aggravating effects of AFB1 on NASH. These data indicate that low dose of AFB1 aggravated NASH via TLR4-mediated necroptosis. This suggests that low dose of AFB1 is potentially harmful to animals and humans, as they exacerbate NASH.

Analysis of gene expression difference and biological process in chorionic villi of unexplained recurrent spontaneous abortion

ObjectiveTo explore the biological relationship between the regulatory signal pathways involved in differentially expressed genes and recurrent spontaneous abortion (RSA) by analyzing the gene expression microarray data of unexplained RSA.MethodsThe gene expression profile data of chorionic villi from unexplained recurrent abortion with normal karyotype and selective induced abortion were compared. Differentially expressed genes were analyzed by the “Limma” package in R Studio, and Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis were carried out with “Cluster Profiler” and “org.hs.eg.db” packages. Finally, hub genes were identified through constructing the protein-protein interaction (PPI) network from the differentially expressed gene dataset in the STRING database. And the hub genes were verified by RT-PCR. The expression of TH1 and TH2 cytokines representing IL-2, IL-10 and their receptors related to hub gene immune regulation were detected by enzyme-linked immunosorbent assay (ELISA) and western blot (WB), respectively.ResultsA total of 295 differentially expressed genes were identified in the dataset GSE22490, with a significance level of P < 0.05 and an absolute log-fold change > 1.0, which included 166 up-regulated genes and 129 down-regulated genes. Go and KEGG enrichment analysis of these differentially expressed genes (P < 0.05,FDR < 0.05) revealed significant involvement in the regulation of inflammatory and immune responses. The PPI analysis revealed that the hub genes FCGR3A, TLR2, BTK, CLEC7A and CD163 were centrally located in the network cluster which were composed of the proteins encoded by differentially expressed genes associated with RSA. The mRNA levels of FCGR3A, TLR2 and CLEC7A in the RSA group were significantly higher than those in the NC group (P < 0.05). The protein expression level of TLR2 was also significantly increased in the RSA group (P < 0.05). The level of IL-2 in the RSA group was significantly higher than that in the NC group (P < 0.05), while the protein expression level of its receptor was not different(P > 0.05). There was no significant difference in the expression levels of IL-10 and its receptor between the two groups (P > 0.05).ConclusionAbnormal immune response plays an important role in unexplained RSA. The imbalance in immune regulation may be one of the most important reasons behind this phenomenon. These findings provide a foundation for further research into the mechanisms underlying RSA.

Protective effect of Streptococcus salivarius K12 against Mycoplasma pneumoniae infection in mice

To investigate the protective effect of the probiotic bacterium Streptococcus salivarius K12 (K12) against Mycoplasma pneumoniae (Mp) infection in mice. Forty male BALB/c mice were randomized into normal control group, K12 treatment group, Mp infection group, and K12 pretreatment prior to Mp infection group. The probiotic K12 was administered daily by gavage for 14 days before Mp infection induced by intranasal instillation of Mp. Three days after Mp infection, the mice were euthanized for analysis of bronchoalveolar lavage fluid (BALF) cell counts and serum levels of secretory immunoglobulin A (sIgA), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6). RT-qPCR was performed to detect the P1 and community-acquired respiratory distress syndrome ( CARDS ) toxin of Mp in the lung tissues and the mRNA expressions of TNF-α, IL-6, chemokine 1 (CXCL1), matrix metalloproteinase 9 (MMP9), mucin 5ac (MUC5ac), collagen 3a1 (Col3a1), Toll-like receptor 2 (TLR2) and TLR4; the protein expressions of TLR2 and TLR4 in the lung tissue were detected using Western blotting. Pathological changes in the lung tissue and airway remodeling were examined with HE staining and AB/PAS staining. Compared with the Mp-infected mice with PBS treatment, the infected mice with K12 treatment showed significantly lowered mRNA levels of P1 and CARDS in the lung tissue and reduced white blood cell counts in the BALF (P<0.05). In spite of the absence of significant differences in serum levels of inflammatory factors between the two groups, the mRNA expressions of TNF‑α, IL-6, CXCL1, MMP9, MUC5ac and COL3A1 and the mRNA and protein levels of TLR2 and TLR4 in the lung tissues were significantly lower in K12-treated mice, in which AB/PAS staining showed obviously decreased mucus secretion. K12 pretreatment can effectively reduce pulmonary inflammatory responses, improve airway remodeling and alleviate lung injury in Mp-infected mice.

Immunomodulatory Effects of Lactiplantibacillus plantarum Strain RW1 During Salmonella Infection in Murine Intestinal Epithelial Cells and Dextran Sulfate Sodium-Induced Murine Colitis.

Inflammatory diseases resulting from bacterial infections or inflammatory bowel disease pose significant threats to the health of both animals and humans. Although probiotics have emerged as a crucial preventive and adjunctive therapy for these conditions, the precise mechanisms through which probiotics regulate inflammatory diseases remain incompletely understood. In our previous study, animal-derived Lactiplantibacillus plantarum strain RW1 (L. plantarum RW1) with probiotic potential was isolated and characterized. In this study, the signaling pathway of L. plantarum RW1 inhibiting the inflammatory response of mouse intestinal epithelial cells caused by Salmonella infection was studied. Our results revealed that infection of Salmonella enterica subsp. enterica serovar Typhimurium strain ATCC14028 (S. Typhimurium ATCC14028) and Salmonella enterica subsp. enterica serovar Typhimurium strain SL1344 (S. Typhimurium SL1344) significantly increased NF-κB/p65 and TLR4 mRNA levels while decreasing IκB and TLR2 mRNA levels. Whereas L. plantarum RW1 treatment significantly reversed these changes. Western blotting confirmed these findings. Additionally, we explored the protective effects of L. plantarum RW1 in a murine colitis model induced by dextran sulfate sodium (DSS). Treatment with L. plantarum RW1 significantly increased both intestinal length and body weight compared to DSS-treated mice. 16S rRNA sequencing analysis demonstrated that L. plantarum RW1 restored the dysbiosis caused by DSS. Flow cytometry analyses further revealed that L. plantarum RW1 specifically increased regulatory T-cell proportions in Peyer's patches while reducing macrophage and neutrophil proportions, indicating the modulatory effects of L. plantarum RW1 on immune responses in gut-associated lymphatic tissue in the context of colitis. This study sheds light on the intricate interaction between probiotics and hosts, offering valuable insights into their potential application for treating inflammatory diseases in animals and humans.

Alterations in Circular RNAs circOprm1 and circSerpini in the Striatum are Associated with Changes in Spatial Working Memory Performance after Morphine Dependence and Withdrawal in Rats.

Modulating role of circRNAs and microRNAs in neurobiological changes induced by drug exposure remains unclear. We examined alterations in some circRNAs and microRNAs in the striatum after morphine dependence and withdrawal and their associations with the changes in spatial working memory performance. Male Wistar rats were used in which 10 days morphine exposure induced dependence. Withdrawal effects were assessed 30 days after stopping morphine exposure. Spatial working memory was assessed using a Y maze test on days 1 and 10 of the drug exposure and 30 days after withdrawal. The gene and protein expression were assessed after dependence and withdrawal. The results revealed that 10 days morphine exposure impaired working memory, which partially reinstated after withdrawal. After 10 days morphine exposure, significant increases in Oprm1 gene and OPRM1 protein levels were detected, which persisted even after withdrawal. The expression of circOprm1 and miR-339-3p decreased in the morphine-dependent group, but they returned to normal levels after withdrawal. The expression of Tlr4 gene and TLR4 protein levels decreased after dependence. While Tlr4 mRNA levels returned to normal after withdrawal, TLR4 protein levels remained lower than the control group. In the morphine-dependent group, both Serpini1 and circSerpini expression significantly increased, but they restored after withdrawal. Expression of miR-181b-3p, miR-181b-5p, miR-181c-3p, and miR-181c-5p decreased after dependence, but they reinstated after withdrawal. It can be concluded that circOprm1 and circSerpini via regulating the OPRM1 and TLR4 expression in the striatum are associated with the neuroadaptation underlying spatial working memory after both morphine dependence and withdrawal.

Study of Neuroinflammation in the Rat Hippocampus during Ethanol Exposure and Pharmacological Correction with Azithromycin: New Data and Future Perspectives.

With prolonged ethanol ingestion, disturbances in the emotional spectrum develop, and memory problems are noted. These symptoms could be mediated by the development of neurochemical changes in the hippocampus of the brain. Although there is evidence that hippocampus is vulnerable to chronic alcohol intoxication and that neuroinflammation and neurodegeneration develop in this brain region, the key molecular mechanisms have not been identified. The aim of the study was to investigate changes in the immune system in the periphery as well as in the hippocampus of rat brain during ethanol exposure and during pharmacological correction with azithromycin (AZM). Long-term ethanol exposure was modeled by injecting rats with a 20%ethanol solution (4g/kg) for 4 weeks. General biochemical and clinical blood analysis was performed in animals. Expression levels of the cytokine genes (Il1β, Ccl2, Il6, Il11, Il13, Tnfα, Tgfβ), Toll-like receptor system genes (Tlr3, Tl4, Tlr7, Nfkb1, Hmgb1), and TLR system-related microRNA molecules (miR-182, miR-155-5p, miR-96-5p, miR-let-7b) were evaluated in the hippocampus. IL-1β protein content was also assessed in the hippocampus. Prolonged exposure to alcohol caused increase in the mRNA and protein levels of IL-1β, and decrease in the mRNA levels of Tnfα, Il11, Tlr3, and Tlr7. The contents of miRlet7b, miR96, and miR155 were downregulated in the hippocampus after long-term alcohol exposure. Elevated levels of THE Il1β mRNA and protein and Hmgb1 mRNA were maintained under conditions of ethanol abstinence. The Tlr3 mRNA levels were decreased after abstinence. Administration of AZM reduced the IL1β, TLR3, and HMGB1 mRNA levels under conditions of ethanol abstinence; and at higher doses of the drug decrease in the IL-1β protein levels in the hippocampus of rat brain was observed. Thus, the study provided new insights into the mechanisms of neuroinflammation in the hippocampus during prolonged exposure to ethanol and upon abstinence. The obtained results allowed us to suggest a number of tasks for further studies in this direction.

Chlorogenic Acid Plays an Important Role in Improving the Growth and Antioxidant Status and Weakening the Inflammatory Response of Largemouth Bass (Micropterus salmoides).

This experiment evaluated the function of chlorogenic acid (CGA) in the growth, health status, and inflammation of largemouth bass (Micropterus salmoides). Over eight weeks, CGA supplementation was designed at five levels: 0, 60, 120, 180, and 240 mg/kg. The 180 and 240 mg/kg CGA-supplemented groups showed significant improvements in the FBW, SGR, and WGR compared to the control group (0 mg/kg) (p < 0.05). All the CGA-supplemented groups exhibited a significant reduction in the FCR (p < 0.05), with the 180 mg/kg CGA group showing the lowest FCR. Nonetheless, there were no appreciable differences in the plasma concentrations of TP, ALT, or AST among the treatments (p > 0.05). Compared to the control group, the 180 mg/kg CGA group exhibited significantly lower TC and TG levels (p < 0.05). The ALP levels showed no significant differences from the control group (p > 0.05). In terms of antioxidant parameters, CGA supplementation considerably reduced the MDA content (p < 0.05) and increased the GSH levels, while decreasing the CAT, SOD, and GPx activity levels Meanwhile, CGA supplementation resulted in reduced mRNA levels of SOD, CAT, Nrf2, Keap1, and NF-κB compared to the control group. In contrast, the mRNA levels of GPx, IL-8, TLR2, and RelA were elevated in the liver. Our findings indicated that CGA supplementation improved the growth performance and antioxidant status and weakened the inflammatory response of largemouth bass. These findings suggest that CGA could be a valuable dietary supplement for enhancing the health and growth of this species.

Deltamethrin increased susceptibility to Aeromonas hydrophila in crucian carp through compromising gill barrier

Pyrethroids serve as a significant method for managing and preventing parasitic diseases in fish. Among these, deltamethrin (DEL) is used extensively in aquatic environments. Our previous work has been confirmed that DEL exposure can induce oxidative stress and immunosuppression on the gill mucosal barrier of crucian carp (Carassius auratus). However, it is not clear whether DEL affects the susceptibility of farmed fish to bacterial infection. In this study, fish was pre-exposed to different DEL concentration (0, 0.3 and 0.6 μg L−1) and then challenged by immersion with Aeromonas hydrophila (1.0 × 10^8 CFU mL−1). After immersion challenge, fish pre-exposed to DEL developed prominent lipopolysaccharides level in gill and serum and had a significantly lower survival rate compared to the control group. In DEL pre-exposure fish after immersion, the gill apoptosis levels were significantly higher and disrupted the tight junction barrier by downregulating the zo1 and claudin12. Furthermore, fish pre-exposed to DEL exhibited increased activities of superoxide dismutase (SOD), total antioxidant capacity (T-AOC), and malonaldehyde (MDA) levels in the early stage after immersion but experiencing decreased activities of glutathione peroxidase (GPx) and lysozyme (LZM) in the later stage after immersion. And this process was regulated by the NRF2 pathway. Additionally, fish pre-exposed to DEL after immersion had significantly lower mRNA levels of immune-related genes tlr4, myd88, tnfα, and il-1β. Overall, these findings indicate that DEL damaged the gill barrier, weakened the immune response, raised LPS levels, and heightened vulnerability to A. hydrophila infection in crucian carp, resulting in mortality. Thus, this work will help social groups and aquaculture workers to understand the potential risk of DEL exposure for bacterial secondary infection in cultured fish.

Curcumin attenuated neuroinflammation via the TLR4/MyD88/NF-κB signaling way in the juvenile rat hippocampus following kainic acid-induced epileptic seizures.

The study examined curcumin's impart on relieving neuroinflammation of juvenile rats in kainic acid (KA) induced epileptic seizures by inhibiting the TLR4/MyD88/NF-κB pathway. There were five groups: control, KA, KA + curcumin (KC), KA + oxcarbazepine (OXC) (KO), KA + curcumin + OXC (KCO) groups. KA was stereotactically injected into right hippocampus following intraperitoneal injection of curcumin or (and) OXC for seven days. The rats in the above groups were randomly divided into three subgroups (at 6h, 24h, and 72h of KA administration) following the seizure degree assessed. The number of NeuN (+) neurons and GFAP (+) astrocytes was counted. The gene and protein levels of TLR4, MyD88, and NF-κB were detected. Compared with the KA group, the seizure latency was longer, and the incidence of status epilepticus (SE) was lower in the KC, KO, and KCO groups. The most significant changes were in the KCO group. At 72h following KA injected, the number of neurons was the least, and the number of astrocytes was the most in the KA group. The number of neurons was the most and the number of astrocytes was the least in the KCO group. At 24h, the mRNA and protein levels of TLR4, MyD88, and NF-κB in the KA group were the most. The above valves were the least in the KCO group. Therefore, curcumin could enhance anti-epileptic effect of OXC, protect injured neurons and reduce proliferated glial cells of the hippocampus of epileptic rats by inhibiting inflammation via the TLR4/MyD88/NF-κB pathway.

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.

Protopine-Type Alkaloids Alleviate Lipopolysaccharide-Induced Intestinal Inflammation and Modulate the Gut Microbiota in Mice.

In this study, we assessed the therapeutic effects of Macleaya cordata (Willd). R. Br.-derived protopine-type alkaloids (MPTAs) in a mouse model of lipopolysaccharide (LPS)-induced intestinal inflammation. The experimental design involved the allocation of mice into distinct groups, including a control group, a model group treated with 6 mg/kg LPS, a berberine group treated with 50 mg/kg berberine hydrochloride and low-, medium- and high-dose MPTA groups treated with 6, 12 and 24 mg/kg MPTAs, respectively. Histological analysis of the ileum, jejunum and duodenum was performed using Hematoxylin and Eosin (H&E) staining. Moreover, the quantification of intestinal goblet cells (GCs) was performed based on PAS staining. The serum levels of IL-1β, IL-6, IL-8 and TNF-α were quantified using an enzyme-linked immunosorbent assay (ELISA), while the mRNA levels of TLR4, NF-κB p65, NLRP3, IL-6 and IL-1β were assessed using quantitative PCR (qPCR). The protein levels of TLR4, Md-2, MyD88, NF-κB p65 and NLRP3 were determined using Western blotting. Furthermore, the 16S rDNA sequences of bacterial taxa were amplified and analysed to determine alterations in the gut microbiota of the mice following MPTA treatment. Different doses of MPTAs were found to elicit distinct therapeutic effects, leading to enhanced intestinal morphology and an increased abundance of intestinal GCs. A significant decrease was noted in the levels of pro-inflammatory cytokines (IL-1β, IL-6, IL-8 and TNF-α). Additionally, the protein levels of TLR4, MyD88, NLRP3 and p-p65/p65 were markedly reduced by MPTA treatment. Furthermore, 16S rDNA sequencing analysis revealed that the administration of 24 mg/kg MPTAs facilitated the restoration of microbial composition.

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.

Dietary Anthocyanins Mitigate High-Fat Diet-Induced Hippocampal Inflammation in Mice

BackgroundObesity and consumption of high-fat diets (HFD) are associated with intestinal permeabilization and increased paracellular transport of endotoxins, which can promote neuroinflammation. Inflammation can affect the hypothalamic pituitary adrenal (HPA) axis, which controls responses to stress and downregulates the brain-derived neurotrophic factor (BDNF), which can promote anxiety and depression, conditions frequently found in obesity. We previously showed that consumption of anthocyanins (AC) mitigate HFD-induced insulin resistance, intestinal permeability, and inflammation. ObjectivesThis study investigated if a dietary supplementation with a cyanidin- and delphinidin-rich extract (CDRE) could counteract HFD/obesity-induced hippocampal inflammation in mice. MethodsC57BL/6J male mice were fed for 14 wk on one of the following diets: 1) a control diet containing 10% total calories from fat (C), 2) a control diet supplemented with 40 mg AC/kg body weight (BW) (CAC), 3) a HFD containing 60% total calories from fat (lard) (HF), or 4) the HFD supplemented with 2, 20, or 40 mg AC/kg BW (HFA2, HFA20, and HFA40, respectively). In plasma and in the hippocampus, parameters of neuroinflammation and the underlying cause (endotoxemia) and consequences (alterations to the HPA and BDNF downregulation) were measured. ResultsConsumption of the HFD caused endotoxemia. Accordingly, hippocampal Tlr4 mRNA levels were 110% higher in the HF group, which were both prevented by CDRE supplementation. Consumption of the HFD also caused: 1) microgliosis and increased expression of genes involved in neuroinflammation, that is, Iba-1, Nox4, Tnfα, and Il-1β, 2) alterations of HPA axis regulation, that is, with low expression of mineralocorticoid (MR) and glucocorticoid (GR) receptors; and 3) decreased Bdnf expression. Supplementation of HFD-fed mice with CDRE mitigated neuroinflammation, microgliosis, and MR and BDNF decreases. ConclusionsCDRE supplementation mitigates the negative effects associated with HFD consumption and obesity in mouse hippocampus, in part by decreasing inflammation, improving glucocorticoid metabolism, and upregulating BDNF.

Mechanism of Weiwei granules in the treatment of chronic active Helicobacter pylori gastritis with atrophy based on the TLR4/NF-κB/COX-2 inflammatory signaling pathway.

Our paper aimed to elucidate the mechanism of Weiwei granules in the treatment of Helicobacter pylori (Hp)-positive chronic atrophic gastritis (CAG) based on the TLR4/NF-κB/COX-2 inflammatory signaling pathway. Hp-positive CAG patients were randomized into the control group (treated with quadruple therapy) or the observation group (treated with Weiwei granules based on the control group). The clinical efficacy, Hp clearance rate, and efficacy of traditional Chinese medicine (TCM) symptoms were compared between the two groups after six months of treatment. The scores of various histopathology variables, serum levels of inflammatory factors (interleukin-6 [IL-6], interleukin-8 [IL-8], and tumor necrosis factor-alpha [TNF-α]), gastrin-17 (G-17) and motilin (MTL), pepsinogen (PG) I and PG II, as well as serum levels of gastrointestinal hormone endothelin (ET), epidermal growth factor (EGF), and calcitonin gene-related peptide (CGRP), were compared between the two groups before and after treatment. TLR4, NF-κB, and COX-2 mRNA levels were compared in gastric mucosal tissues before and after treatment in the two groups. After treatment, the clinical efficacy, Hp clearance rate, and efficacy of TCM symptoms of patients in the observation group were higher than those in the control group. After treatment, the scores of various histopathology variables, serum levels of inflammatory factors (IL-6, IL-8, and TNF-α), gastrointestinal hormones (ET and EGF), and the expression levels of TLR4, NF-κB, and COX-2 mRNA in the gastric mucosal tissues were lower and G-17, MTL, CGRP, and PG I levels were higher in the observation group than in the control group. Weiwei granules can effectively improve Hp-positive CAG patients and reduce the expression levels of TLR4, NF-κB, and COX-2.

Essential oil from Cinnamomum cassia Presl bark regulates macrophage polarization and ameliorates lipopolysaccharide-induced acute lung injury through TLR4/MyD88/NF-κB pathway

Cinnamomum cassia Presl, a traditional Chinese medicine recorded in "Shennong's Herbal Classic," has been historically used to treat respiratory diseases and is employed to address inflammation. The essential oil derived from Cinnamomum cassia bark is a primary anti-inflammatory agent. However, there remains ambiguity regarding the chemical composition of cinnamon bark essential oil (BCEO), its principal anti-inflammatory components, and their potential efficacy in typical inflammatory respiratory conditions, such as acute lung injury (ALI). This study aimed to unveil the chemical composition of BCEO. In addition, the mechanism of action of BCEO in ameliorating ALI and regulating macrophage polarization through the TLR4/MyD88/NF-κB pathway was elucidated. BCEO was extracted using supercritical fluid extraction (SFE) and characterized through gas chromatography-mass spectrometry (GC-MS) analysis. Acute oral toxicity was observed in C57BL/6 J mice. The pharmacological effects and underlying mechanisms of BCEO were evaluated in a mouse model of ALI, which was induced by administering 5 mg/kg of lipopolysaccharide (LPS) through intratracheal instillation. GC-MS analysis revealed 99.08% of the constituents of BCEO. The primary components of BCEO were trans-cinnamaldehyde, o-methoxycinnamaldehyde, (+)-α-muurolene, δ-cadinene, and copaene. Oral acute toxicity tests indicated that the maximum tolerated dose of BCEO was 12 g/kg/day. BCEO treatment significantly reduced lung W/D ratio, total protein concentration in BALF, levels of TNF-α, IL-6, and IL-1β in BALF, WBC count and NEU% in peripheral blood, and lung histological damage. Pulmonary function, IL-10 levels, and LYM% in peripheral blood also showed improvement. BCEO effectively decreased the proportion of M1 phenotype macrophages in BALF, M1/M2 ratio, and apoptotic cells in the lung tissue while increasing the proportion of M2 phenotype macrophages in BALF. Furthermore, BCEO treatment led to reduced protein and mRNA levels of TLR4, MyD88, and p-p65, alongside increased p65 expression, suggesting its potential to impede the TLR4/MyD88/NF-κB signaling pathway. SFE-extracted BCEO or its major constituents could serve as a viable treatment for ALI by reducing lung inflammation, improving pulmonary function, and protecting against LPS-induced ALI in mice. This therapeutic effect is achieved by inhibiting M1 macrophage polarization, promoting M2 macrophage polarization, and suppressing the TLR4/MyD88/NF-κB signaling pathway.

Functional comparison of Rab3aa and Rab3ab in grass carp (Ctenopharyngodon idella) immune response and GCRV replication

Rab GTPases not only play a crucial role in the exocytosis and membrane transport of all eukaryotic cells, but are also gradually being found to be involved in innate immunity in mammals. However, the immunological function of Rab3a in fish remains unclear. In this study, two members of Rab3a, CiRab3aa and CiRab3ab, were cloned from grass carp (Ctenopharyngodon idella) for the first time. It was observed that CiRab3aa exhibited a prompt response to grass carp reovirus (GCRV) infection, and suppressing the expression of the CiRab3aa effectively inhibited GCRV replication, whereas the expression level of the CiRab3ab gene did not affect GCRV replication. Furthermore, overexpression of CiRab3aa significantly suppressed the mRNA levels of TLR9, TNF-α, IL-1β, IL-6, IL-10, ERK, and IFN-I following stimulation with CpG ODN 1670 A, suggesting that CiRab3aa is a negative regulator of TLR9 signaling pathway. In conclusion, this study not only revealed functional differences between CiRab3aa and CiRab3ab in grass carp immunity, providing new sights into understanding their function in teleost fish, but also provided a new target for the treatment strategy of grass carp hemorrhagic disease. These results will help address the harm and economic losses caused by GCRV infection in grass carp breeding industry.

Investigation the mechanism of iron overload-induced colonic inflammation following ferric citrate exposure

Iron overload occurs due to excessive iron intake compared to the body’s demand, leading to iron deposition and impairment of multiple organ functions. Our previous study demonstrated that chronic oral administration of ferric citrate (FC) caused colonic inflammatory injury. However, the precise mechanism underlying this inflammatory response remains unclear. The current study aims to investigate the mechanism by which iron overload induced by FC exposure leads to colonic inflammation. To accomplish this, mice were orally exposed to three different concentrations of FC (71 mg/kg/bw (L), 143 mg/kg/bw (M) and 286 mg/kg/bw (H)) for continuous 16 weeks, with the control group receiving ultrapure water (C). Exposure to FC caused disturbances in the excretory system, altered colonic flora alpha diversity, and enriched pathogenic bacteria, such as Mucispirillum, Helicobacter, Desulfovibrio, and Shigella. These changes led to structural disorders of the colonic flora and an inflammatory response phenotype characterized by inflammatory cells infiltration, atrophy of intestinal glands, and irregular thickening of the intestinal wall. Mechanistic studies revealed that FC-exposure activated the NF-κB signaling pathway by up-regulating TLR4, MyD88, and NF-κB mRNA levels and protein expression. This activation resulted in increased production of pro-inflammatory cytokines, further contributing to the colonic inflammation. Additionally, in vitro experiments in SW480 cells confirmed the activation of NF-κB signaling pathway by FC exposure, consistent with the in vivo findings. The significance of this study lies in its elucidation of the mechanism by which iron overload caused by FC exposure leads to colonic inflammation. By identifying the role of pathogenic bacteria and the NF-κB signaling pathway, this study could potentially offer a crucial theoretical foundation for the research on iron overload, as well as provide valuable insights for clinical iron supplementation.