Expression Of Toll-like Receptor Research Articles

Glycerol monolaurate improves intestinal morphology and antioxidant status by suppressing inflammatory responses and nuclear factor kappa-B signaling in lipopolysaccharide-exposed chicken embryos

Medium-chain fatty acids and their derivatives are natural ingredients that support immunological functions in animals. The effects of glycerol monolaurate (GML) on intestinal innate immunity and associated molecular mechanisms were investigated using a chicken embryo model. Sixty-four Arbor Acres broiler embryos were randomly allocated into four groups. On embryonic day 17.5, the broiler embryos were administered with 9 mg of GML, which was followed by a 12-h incubation period and a 12-h challenge with 32 μg of lipopolysaccharide (LPS). On embryonic day 18.5, the jejunum and ileum were harvested. Results indicated that GML reversed the LPS-induced decline in villus height and upregulated the expression of mucin 2 (P < 0.05). GML decreased LPS-induced malondialdehyde production and boosted antioxidant enzyme activity (P < 0.05). GML alleviated LPS-stimulated intestinal secretion of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α) (P < 0.05). GML also normalized LPS-induced changes in the gene expression of Toll-like receptor 4, nuclear factor kappa-B p65 (NF-κB p65), cyclooxygenase-2, NOD-like receptor protein 3, IL-18, zonula occludens 1, and occludin (P < 0.05). GML enhanced as well the expression of AMP-activated protein kinase α1 and claudin 1 (P < 0.05). In conclusion, GML improved intestinal morphology and antioxidant status by alleviating inflammatory responses and modulating NF-κB signaling in LPS-challenged broiler embryos.

Arctiin Inhibits Inflammation, Fibrosis, and Tumor Cell Migration in Rats With Ehrlich Solid Carcinoma.

ESC or Ehrlich solid carcinoma is a type of tumor originating from a spontaneous mammary adenocarcinoma in mice. It is a highly aggressive and fast-growing carcinoma that can create a solid mass when inserted under the skin. Its solid, undifferentiated form makes it an ideal model for researching cancer biology, tumor immunology, and testing various anti-cancer treatments. Additionally, arctiin has multiple beneficial properties, such as anti-proliferative, anti-oxidative, anti-adipogenic, and anti-bacterial. This study aimed to explore the potential anti-cancer benefits of arctiin in rats with ESC while also analyzing its effects on cell fibrosis markers, tumor cell migration, and inflammasome pathways. Rats were given a tumor in their left hind limb via an intramuscular injection consisting of 2×106 cells. After eight days, some of the rats received a daily oral dose of 30 mg/kg of arctiin for three weeks. Muscle samples were observed under an electron microscope or stained with hematoxylin/eosin. Additionally, gene expression and protein levels of toll-like receptor 4 (TLR4), NLR family pyrin domain containing 3 (NLRP3), signal transducer and activator of transcription 3 (STAT3), transforming growth factor (TGF)-β, endothelial growth factor (VEGF), and cyclin D1 were assessed in another part of the muscle samples. When ESC rats were given arctiin as a treatment, their mean survival time increased and their tumor volume and weight decreased. Additionally, when tumor tissue was examined under an electron microscope, it showed signs of pleomorphic cells, necrosis, nuclear fragmentation, membrane damage with cytoplasmic content spilling, and loss of cellular junction. The stained sections with hematoxylin/eosin showed a dense cellular mass and compressed, degenerated, and atrophied muscle. However, treatment with arctiin improved all these effects. Finally, the expression of TLR4, NLRP3, STAT3, TGF-β, VEGF, and cyclin D1 was significantly reduced with arctiin treatment. Through the use of arctiin, tumor sizeand weight were effectively reduced, leading to an increase in the average survival time of rats and an improvement in muscle structure. Additional research has shown that arctiin is able to suppress inflammation, fibrosis, and the migration of tumor cells by inhibiting STAT3, TGF-β1, TLR4, NLRP3, VEGF, and cyclin D1.

Effects of dietary chloroquine on fish growth, hepatic intermediary metabolism, antioxidant and inflammatory responses in turbot

Autophagy is a conserved cellular self-digestion process and is essential for individual growth, cellular metabolism and inflammatory responses. It was responsive to starvation, pathogens infection and environmental stress. However, the information on the regulation of autophagy in fish hepatic intermediary metabolism, antioxidant system, and immune responses were limited. In the present study, turbot with inhibited autophagy flux was built by dietary chloroquine. The hepatic metabolic response, antioxidant enzymes and immune responses were explored. Results showed that dietary chloroquine induced the expression of Beclin 1, SQSTM and LC-3II, and effectively inhibited autophagy flux. Autophagy dysfunction depressed fish growth and feed utilization, while it induced clusters of liver lipid droplets. The genes involved in lipolysis and fatty acid β-oxidation, as well as the lipogenesis-related genes in chloroquine group were depressed. The phosphorylation of AMPK was activated in chloroquine group, and the genes involved in glycolysis were induced. The hepatic content of malonyldialdehyde and the activities of SOD and CAT were induced when autophagy was inhibited. The content of Complement 3, Complement 4 and Immunoglobulin M, as well as the activity of lysozyme in plasma were depressed in chloroquine group. Dietary chloroquine induced the expression of toll-like receptors and stimulated the expression of myd88 and nf-κb p65, as well as the pro-inflammatory cytokines, such as tnf-α and il-1β. The expression of anti-inflammatory cytokine tgf-β was depressed in the chloroquine group. Our results would extend the knowledge on the role of autophagy in teleost and assist in improving fishery production.

Anti-cytokine storm activity of fraxin and quercetin, alone and in combination, and their possible molecular mechanisms via TLR4 and PPAR-γ signaling pathways in lipopolysaccharide-induced RAW 264.7 cell line

Background: Cytokine storm syndrome (CSS) is a leading cause of morbidity and mortality in patients with late-stage coronavirus disease 2019 (COVID-19), causing multiple organ failure and death. According to prior research, fraxin, and quercetin have anti-inflammatory, antioxidant, antimicrobial, and antiviral properties. Therefore, this study aimed to investigate the anti-cytokine storm activity of fraxin and quercetin, their combination, and the molecular mechanism behind this activity in Lipopolysaccharide (LPS)-induced RAW 264.7 cells. Methods: LPS-induced macrophage cells were treated with fraxin, quercetin, or their combinations at various doses for cytotoxicity, anti-cytokine assays, and gene expression analyses. An enzyme-linked immunosorbent assay was used to quantify the levels of proinflammatory cytokines, interleukin 1 beta (IL1β), interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α), and RT-PCR was used to measure the gene expression of PPAR-γ and Toll-like receptor 4 (TLR-4) concerning GAPDH as a reference gene. Results: The results revealed a slight decrease in cell viability only when higher concentrations were applied to the cells. Fraxin, quercetin, and their combination reduced the generation of proinflammatory cytokines. The combination (fraxin + quercetin (FQ)) reduced the levels of IL-1β, IL-6, and TNF-α by 56.2%, 58.5%, and 70.6% respectively, compared to the LPS-only control; pretreatment of cells with farxin, quercetin, and their combination resulted in significant inhibition of TLR-4 gene expression by 89%, 82%, and 93% respectively, compared to the control (P˂ 0.05); FQ upregulated PPAR-γ expression up to 60-fold compared to the control, while fraxin and quercetin increased PPAR-γ by 17.6 and 8.6-folds, respectively. Conclusions: Based on these findings, fraxin, quercetin and their combination might be considered anti-cytokine storm agents, which could lay the groundwork for future research into other combinations of fraxin and quercetin, as well as the pathways involved in the molecular mechanisms underlying the synergistic anti-cytokine-storm-activity.

Disruption of the primocolonizing microbiota alters epithelial homeostasis and imprints stem cells in the colon of neonatal piglets.

The gut microbiota plays a key role in the postnatal development of the intestinal epithelium. However, the bacterial members of the primocolonizing microbiota driving these effects are not fully identified and the mechanisms underlying their long-term influence on epithelial homeostasis remain poorly described. Here, we used a model of newborn piglets treated during the first week of life with the antibiotic colistin in order to deplete specific gram-negative bacteria that are transiently dominant in the neonatal gut microbiota. Colistin depleted Proteobacteria and Fusobacteriota from the neonatal colon microbiota, reduced the bacterial predicted capacity to synthetize lipopolysaccharide (LPS), and increased the concentration of succinate in the colon. The colistin-induced disruption of the primocolonizing microbiota was associated with altered gene expression in the colon epithelium including a reduction of toll-like receptor 4 (TLR4) and lysozyme (LYZ). Our data obtained in porcine colonic organoid cell monolayers suggested that these effects were not driven by the variation of succinate or LPS levels nor by a direct effect of colistin on epithelial cells. The disruption of the primocolonizing microbiota imprinted colon epithelial stem cells since the expression of TLR4 and LYZ remained lower in organoids derived from colistin-treated piglet colonic crypts after several passages when compared to control piglets. Finally, the stable imprinting of LYZ in colon organoids was independent of the H3K4me3 level in its transcription start site. Altogether, our results show that disruption of the primocolonizing gut microbiota alters epithelial innate immunity in the colon and imprints stem cells, which could have long-term consequences for gut health.

Hydralazine inhibits neuroinflammation and oxidative stress in APP/PS1 mice via TLR4/NF-κB and Nrf2 pathways

Alzheimer's disease (AD) is a common chronic progressive neurodegenerative disorder, and curative treatment has not been developed. The objective of this study was to investigate the potential effects of hydralazine (Hyd, a hypertension treatment drug) on the development process of AD and its mechanisms. We treated 6-month-old male APP/PS1 mice with Hyd for 5 weeks, measured changes in behavior and pathological status, and analyzed differences in gene expression by RNA sequencing. The results demonstrated that Hyd improved cognitive deficits and decreased amyloid beta protein deposition in the cortex and hippocampus, while RNA sequencing analysis suggested that the regulation of neuroinflammation and energy metabolism might play pivotal roles for Hyd's beneficial effects. Therefore, we further investigated inflammatory response, redox state, and mitochondrial function, as well as the expression of toll-like receptor 4 (TLR4)/nuclear factor Kappa B (NF-κB)-dependent neuroinflammation gene and nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated antioxidant gene in AD mice. The results showed that Hyd reduced the damage of neuroinflammation and oxidative stress, improved mitochondrial dysfunction, downregulated pro-inflammation gene expression, and upregulated antioxidant gene expression. The results in lipopolysaccharide (LPS)-induced BV2 cell model demonstrated that Hyd suppressed pro-inflammatory response via TLR4/NF-κB signaling pathway. In addition, by silencing the Nrf2 gene expression, it was found that Hyd can reduce LPS-induced reactive oxygen species production by activating the Nrf2 signaling pathway. Therefore, administration of Hyd in the early stage of AD might be beneficial in delaying the pathological development of AD via inhibiting neuroinflammation and oxidative stress.

Xixin Decoction improves learning and memory ability of SAMP8 by enhancing neuroprotective effect and inhibiting neuroinflammation

This study aimed to explore the possible effect of Xixin Decoction(XXD) on the learning and memory ability of Alzheimer's disease(AD) model senescence-accelerated mouse-prone 8(SAMP8) and the related mechanism in enhancing neuroprotective effect and reducing neuroinflammation. Forty SAMP8 were randomly divided into a model group(10 mL·kg~(-1)·d~(-1)), a probiotics group(0.39 g·kg~(-1)·d~(-1)), a high-dose group of XXD granules(H-XXD, 5.07 g·kg~(-1)·d~(-1)), a medium-dose group of XXD granules(M-XXD, 2.535 g·kg~(-1)·d~(-1)), and a low-dose group of XXD granules(L-XXD, 1.267 5 g·kg~(-1)·d~(-1)). Eight senescence-accelerated mouse-resistant 1(SAMR1) of the same age and strain were assigned to the control group(10 mL·kg~(-1)·d~(-1)). After ten weeks of intragastric administration, the Morris water maze was used to test the changes in spatial learning and memory ability of mice after treatment. Meanwhile, immunofluorescence staining was used to detect the positive expression of receptor for advanced glycation end products(AGER), Toll-like receptor 1(TLR1), and Toll-like receptor 2(TLR2) in the hippocampal CA1 region of mice. Western blot was employed to test the protein expression levels of silencing information regulator 2 related enzyme 1(SIRT1), AGER, TLR1, and TLR2 in the hippocampus of mice. Enzyme linked immunosorbent assay(ELISA) was applied to assess the levels of Aβ_(1-42) in the hippocampus of mice and the levels of nuclear factor κB p65(NF-κB p65), NOD-like receptor protein 3(NLRP3), tumor necrosis factor-α(TNF-α), and interleukin-1β(IL-1β) in the serum and hippocampus of mice. Compared with the model group, XXD significantly improved the spatial learning and memory ability of SAMP8, increased the expression of neuroprotective factors in the hippocampus, decreased the levels of neuroinflammatory factors, and inhibited the expression of Aβ_(1-42). In particular, H-XXD significantly increased the expression of SIRT1 in the hippocampus of mice, reduced the expression levels of NF-κB p65, NLRP3, TNF-α, and IL-1β in the serum and hippocampus of mice, and decreased the expression of AGER, TLR1, and TLR2 in the hippocampus of mice(P&lt;0.05 or P&lt;0.01). XXD may improve the spatial learning and memory ability of AD model SAMP8 by enhancing the neuroprotective effect and inhibiting neuroinflammation.

Herbal melanin modulates PGE2 and IL-6 gastroprotective markers through COX-2 and TLR4 signaling in the gastric cancer cell line AGS

We reported a gastric anti-ulcerogenic effect of the Nigella sativa (L.)-derived herbal melanin (HM) using rat models. However, the molecular mechanisms underlying this HM gastroprotective effect remain unknown. Cyclooxygenase-2 (COX-2)-catalyzed prostaglandin E2 (PGE2) and toll-like receptor 4 (TLR4)-mediated interleukin-6 (IL-6) production and secretion play major roles in gastric mucosal protection. In the current study, the human gastric carcinoma epithelial cell line AGS was used as a model to investigate the effect of HM on TLR4, COX-2, glycoprotein mucin 4 protein and gene expression using immuno-cyto-fluorescence staining, Western blot technology, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Gastroprotective markers PGE2 and IL-6 production and secretion were also assessed using an enzyme-linked immunosorbent assay (ELISA). Bacterial lipopolysaccharides (LPS), well-known inducers of TLR4, COX-2, PGE2 and IL-6 expression, were used as a positive control. We showed that HM upregulated its main receptor TLR4 gene and protein expression in AGS cells. HM increased, in a dose- and time-dependent manner, the secretion of PGE2 and the expression of COX-2 mRNA and protein, which was detected in the nucleus, cytoplasm and predominantly at the intercellular junctions of the AGS cells. In addition, HM enhanced IL-6 production and secretion, and upregulated the mucin 4 gene expression, the hallmarks of gastroprotection. To check whether HM-induced PGE2 and IL-6 through TLR4 signaling and COX-2 generated, AGS cells were pre-treated with a TLR4 signaling inhibitor TAK242 and the COX-2 inhibitor NS-398. A loss of the stimulatory effects of HM on COX-2, PGE2 and IL-6 production and secretion was observed in TAK242 and NS-398-pre-treated AGS cells, confirming the role of TLR4 signaling and COX-2 generated in the HM gastroprotective effects. In conclusion, our results showed that HM enhances TLR4/COX-2-mediated secretion of gastroprotective markers PGE2 and IL-6, and upregulates mucin 4 gene expression in the human gastric epithelial cell line AGS, which may contribute to the promising beneficial gastroprotective effect of HM for human gastric prevention and treatment.

Gastrin attenuates sepsis-induced myocardial dysfunction by down-regulation of TLR4 expression in macrophages.

Myocardial dysfunction is the most serious complication of sepsis. Sepsis-induced myocardial dysfunction (SMD) is often associated with gastrointestinal dysfunction, but its pathophysiological significance remains unclear. The present study found that patients with SMD had higher plasma gastrin concentrations than those without SMD. In mice, knockdown of the gastrin receptor, cholecystokinin B receptor (Cckbr), aggravated lipopolysaccharide (LPS)-induced cardiac dysfunction and increased inflammation in the heart, whereas the intravenous administration of gastrin ameliorated SMD and cardiac injury. Macrophage infiltration plays a significant role in SMD because depletion of macrophages by the intravenous injection of clodronate liposomes, 48h prior to LPS administration, alleviated LPS-induced cardiac injury in Cckbr-deficient mice. The intravenous injection of bone marrow macrophages (BMMs) overexpressing Cckbr reduced LPS-induced myocardial dysfunction. Furthermore, gastrin treatment inhibited toll-like receptor 4 (TLR4) expression through the peroxisome proliferator-activated receptor α (PPAR-α) signaling pathway in BMMs. Thus, our findings provide insights into the mechanism of the protective role of gastrin/CCKBR in SMD, which could be used to develop new treatment modalities for SMD.

Genomic DNA Extracted from Lactiplantibacillus plantarum Attenuates Porphyromonas gingivalis Lipopolysaccharide (LPS)-Induced Inflammatory Responses via Suppression of Toll-Like Receptor (TLR)-Mediated Mitogen-Activated Protein Kinase (MAPK) and Nuclear Factor-κB (NF-κB) Signaling Pathways.

In the present study, we aimed to examine the inhibition of genomic DNA from Lactiplantibacillus plantarum (LpDNA) on Porphyromonas gingivalis lipopolysaccharide (PgLPS)-induced inflammatory responses in RAW264.7 cells. Pretreatment with LpDNA for 15 h significantly inhibited PgLPS-induced mRNA expression and protein secretion of interleukin (IL)-1β, IL-6, and monocyte chemoattractant protein-1. LpDNA pretreatment also reduced the mRNA expression of Toll-like receptor (TLR)2 and TLR4. Furthermore, LpDNA inhibited the phosphorylation of mitogen-activated protein kinases (MAPKs) and the activation of nuclear factor-κB (NF-κB) induced by PgLPS. Taken together, these findings demonstrate that LpDNA attenuates PgLPS-induced inflammatory responses by regulating MAPKs and NF-κB signaling pathways through the suppression of TLR2 and TLR4 expression.

Lactiplantibacillus plantarum L47 and inulin alleviate enterotoxigenic Escherichia coli induced ileal inflammation in piglets by upregulating the levels of α-linolenic acid and 12,13-epoxyoctadecenoic acid.

Alternatives to antibiotics for preventing bacteria-induced inflammation in early-weaned farm animals are sorely needed. Our previous study showed that Lactiplantibacillus plantarum L47 and inulin could alleviate dextran sulfate sodium (DSS)-induced colitis in mice. To explore the protective effects of L.plantarum L47 and inulin on the ileal inflammatory response in weaned piglets challenged with enterotoxigenic Escherichia coli (ETEC), 28 weaned piglets were assigned into four groups, namely, CON group-orally given 10mL/d phosphate buffer saline (PBS), LI47 group-orally given a mixture of 10mL/d L.plantarum L47 and inulin, ECON group-orally given 10mL/d PBS and challenged by ETEC, and ELI47 group-orally given 10mL/d L.plantarum L47 and inulin mixture and challenged by ETEC. The results demonstrated that the combination of L.plantarum L47 and inulin reduced inflammatory responses and relieved the inflammatory damage caused by ETEC, including ileal morphological damage, reduced protein expression of ileal tight junction, decreased antioxidant capacity, and decreased anti-inflammatory factors. Transcriptome analysis revealed that L.plantarum L47 and inulin up-regulated the gene expression of phospholipase A2 group IIA (PLA2G2A) (P<0.05) as well as affected alpha-linolenic acid (ALA) metabolism and linoleic acid metabolism. Moreover, L.plantarum L47 and inulin increased the levels of ALA (P<0.05), lipoteichoic acid (LTA) (P<0.05), and 12,13-epoxyoctadecenoic acid (12,13-EpOME) (P<0.05) and the protein expression of Toll-like receptor 2 (TLR2) (P=0.05) in the ileal mucosa. In conclusion, L.plantarum L47 and inulin together alleviated ETEC-induced ileal inflammation in piglets by up-regulating the levels of ALA and 12,13-EpOME via the LTA/TLR2/PLA2G2A pathway.

The role of mediator suppressor of cytokine signaling (SOCS), toll-like receptor 3 (TLR-3) and nuclear factor kappa B (NFκB) on cytokine production during dengue virus infection.

Inability to understand the pathogenesis of severe dengue, in particular the control mechanism of immune responses, has led to high mortality rate for patients with dengue shock syndrome (DSS). The aim of this study was to determine the control mechanism of cytokine production by mediator suppressor of cytokine signaling (SOCS), toll-like receptor 3 (TLR-3) and nuclear factor kappa B (NFκB) during DENV infection. Peripheral blood mononuclear blood cells (PBMC), isolated from healthy individuals, were infected with dengue virus (DENV)-2 strain SJN-006 Cosmopolitan genotype (isolated from Bali, Indonesia). The relative gene expression of SOCS-3, TLR-3, NFκB, and the cytokine genes (interleukin (IL)-6, IL-8, interferon inducible protein 10 (IP-10), and macrophage inflammatory protein-1 beta (MIP-1β)) were measured using qRT-PCR at 6, 12 and 24 hours post infection (hpi). Student t-test and Mann-Whitney test were used to compare the gene expressions while causal correlations were analyzed using regression test and path analyses. DENV-2 infection increased the gene expression of SOCS-3, TLR-3, and NFκB after 12 and 24 hpi. The expression of IL-6, IL-8, IP-10, and MIP-1β genes was increased and peaked at different times post-infection. NFκB and SOCS-3 genes likely have role in the upregulation of IL-8 and IL-6 gene expression, respectively. MIP-1β gene expression was significantly induced by both NFκB and SOCS-3. In conclusion, our study suggested that SOCS-3, TLR-3, and NFκB are important in regulating the production of IL-6, IL-8, IP-10, MIP-1β during early phase of DENV-2 infection. This enriches our understanding on pathogenesis pathway of DENV-associated cytokine storm.

Impacts of Eimeria coinfection on growth performance, intestinal health and immune responses of broiler chickens

Coccidiosis caused by Eimeria is one of the most severe chicken diseases and imposes huge economic losses to the poultry industry globally. Multi-Eimeria species coinfections are common with the most prevalent combination being mixtures of Eimeria acervulina and Eimeria tenella. Although detrimental impacts of either E. acervulina or E. tenella on chicken health are well recognized, no information is available regarding their coinfection effects so far. This study was designed to investigate the influence of coinfection with E. acervulina and E. tenella on broiler chickens. 144 one-day-old broiler chickens within each of trials (trial I or II) were divided into four groups, namely, control group (CG), E. acervulina infection group (EAIG), E. tenella infection group (ETIG) and dual (E. acervulina and E. tenella) infection group (DIG). Then, chickens were measured for weight loss, lesion scores, oocyst outputs, histological changes and expressions of pro-inflammatory (interleukin [IL]−6, IL-8 and IL-18), regulatory (IL-10 and IL-22) cytokines and Toll-like receptors (TLR; TLR2 and TLR4) as well as intestinal barrier (mucin 2 [MUC2] and fattey acid-bingding proteins 2 and 6 [FABP2 and FABP6])- and tight junction (TJ; zonula occluden-1 [ZO-1], occludin [OCLN], and claudins 1 and 5 [CLDN1 and CLDN5])-related proteins at 3, 5, 7, 10, 14 and 21 days post-infection, respectively. Our results consistently showed that although ETIG and DIG exhibited a higher level of weight loss and a more amount of oocyst excretion than EAIG, DIG had lighter lesions than EAIG in the early phase because of coinfection with E. tenella. A higher (P < 0.05) ratio of duodenal villous height to crypt depth was also observed in DIG than EAIG. Moreover, histological changes in the duodenum and cecum varied by single and dual Eimeria infections. Expressions of the intestinal barrier- and TJ-related genes of EAIG, ETIG and DIG were significantly (P < 0.05) upregulated but their levels exhibited differential changes among infected chickens. Similarly, the infected chickens showed significant (P < 0.05) inflammatory responses and higher (P < 0.05) expressions of TLRs in the intestines in comparison to CG. These results presented a comprehensive physiological, pathological and immunological characterization of E. acervulina and E. tenella coinfection in broiler chickens and also shed insights into pathogenesis of multi-coccidia coinfections.

The Role of Toll-like Receptor-4 in Macrophage Imbalance in Lethal COVID-19 Lung Disease, and Its Correlation with Galectin-3.

To the current data, there have been 6,955,141 COVID-19-related deaths worldwide, reported to WHO. Toll-like receptors (TLRs) implicated in bacterial and virus sensing could be a crosstalk between activation of persistent innate-immune inflammation, and macrophage's sub-population alterations, implicated in cytokine storm, macrophage over-activation syndrome, unresolved Acute Respiratory Disease Syndrome (ARDS), and death. The aim of this study is to demonstrate the association between Toll-like-receptor-4 (TLR-4)-induced inflammation and macrophage imbalance in the lung inflammatory infiltrate of lethal COVID-19 disease. Twenty-five cases of autopsy lung tissues were studied by digital pathology-based immunohistochemistry to evaluate expression levels of TLR-4 (CD 284), pan-macrophage marker CD68 (clone KP1), sub-population marker related to alveolar macrophage Galectin-3 (GAL-3) (clone 9C4), and myeloid derived CD163 (clone MRQ-26), respectively. SARS-CoV-2 viral persistence has been evaluated by in situ hybridation (ISH) method. This study showed TLR-4 up-regulation in a subgroup of patients, increased macrophage infiltration in both Spike-1(+) and Spike-1(-) lungs (p < 0.0001), and a macrophage shift with important down-regulation of GAL-3(+) alveolar macrophages associated with Spike-1 persistence (p < 0.05), in favor of CD163(+) myeloid derived monocyte-macrophages. Data show that TLR-4 expression induces a persistent activation of the inflammation, with inefficient resolution, and pathological macrophage shift, thus explaining one of the mechanisms of lethal COVID-19.

Triad3A-Mediated K48-Linked ubiquitination and degradation of TLR9 impairs mitochondrial bioenergetics and exacerbates diabetic cardiomyopathy

IntroductionTargeted protein degradation represents a promising therapeutic approach, while diabetic cardiomyopathy (DCM) arises as a consequence of aberrant insulin secretion and impaired glucose and lipid metabolism in the heart.. ObjectivesConsidering that the Toll-like receptor 9 (TLR9) signaling pathway plays a pivotal role in regulating energy metabolism, safeguarding cardiomyocytes, and influencing glucose uptake, the primary objective of this study was to investigate the impact of TLR9 on diabetic cardiomyopathy (DCM) and elucidate its underlying mechanism. MethodsMouse model of DCM was established using intraperitoneal injection of STZ, and mice were transfected with adeno-associated virus serotype 9-TLR9 (AAV9-TLR9) to assess the role of TLR9 in DCM. To explore the mechanism of TLR9 in regulating DCM disease progression, we conducted interactome analysis and employed multiple molecular approaches. ResultsOur study revealed a significant correlation between TLR9 expression and mouse DCM. TLR9 overexpression markedly mitigated cardiac dysfunction, myocardial fibrosis, oxidative stress, and apoptosis in DCM, while inflammation levels remained relatively unaffected. Mechanistically, TLR9 overexpression positively modulated mitochondrial bioenergetics and activated the AMPK-PGC1a signaling pathway. Furthermore, we identified Triad3A as an interacting protein that facilitated TLR9′s proteasomal degradation through K48-linked ubiquitination. Inhibiting Triad3A expression improved cardiac function and pathological changes in DCM by enhancing TLR9 activity. ConclusionsThe findings of this study highlight the critical role of TLR9 in maintaining cardiac function and mitigating pathological alterations in diabetic cardiomyopathy. Triad3A-mediated regulation of TLR9 expression and function has significant implications for understanding the pathogenesis of DCM. Targeting TLR9 and its interactions with Triad3A may hold promise for the development of novel therapeutic strategies for diabetic cardiomyopathy. Further research is warranted to fully explore the therapeutic potential of TLR9 modulation in the context of cardiovascular diseases.

Huoxin Pill Reduces Myocardial Ischemia Reperfusion Injury in Rats via TLR4/NFκB/NLRP3 Signaling Pathway.

To explore the protective effect of Huoxin Pill (HXP) on acute myocardial ischemia-reperfusion (MIRI) injury in rats. Seventy-five adult SD rats were divided into the sham-operated group, model group, positive drug group (diltiazem hydrochloride, DH), high dose group (24 mg/kg, HXP-H) and low dose group (12 mg/kg, HXP-L) of Huoxin Pill (n=15 for every group) according to the complete randomization method. After 1 week of intragastric administration, the left anterior descending coronary artery of the rat's heart was ligated for 45 min and reperfused for 3 h. Serum was separated and the levels of creatine kinase (CK), creatine kinase isoenzyme (CK-MB) and lactate dehydrogenase (LDH), superoxide dismutase (SOD), and malondialdehyde (MDA), hypersensitive C-reactive protein (hs-CRP) and interleukin-1β (IL-1β) were measured. Myocardial ischemia rate, myocardial infarction rate and myocardial no-reflow rate were determined by staining with Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC). Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine (BATMAN) databases were used to screen for possible active compounds of HXP and their potential therapeutic targets; the results of anti-inflammatory genes associated with MIRI were obtained from GeneCards, Drugbank, Online Mendelian Inheritance in Man (OMIM), and Therapeutic Target Datebase (TTD) databases was performed; Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were used to analyze the intersected targets; molecular docking was performed using AutoDock Tools. Western blot was used to detect the protein expression of Toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NFκB)/NOD-like receptor protein 3 (NLRP3). Compared with the model group, all doses of HXP significantly reduced the levels of LDH, CK and CK-MB (P<0.05, P<0.01); HXP significantly increased serum activity of SOD (P<0.05, P<0.01); all doses of HXP significantly reduced the levels of hs-CRP and IL-1β (P<0.05, P<0.01) and the myocardial infarction rate and myocardial no-reflow rate (P<0.01). GO enrichment analysis mainly involved positive regulation of gene expression, extracellular space and identical protein binding, KEGG pathway enrichment mainly involved PI3K-Akt signaling pathway and lipid and atherosclerosis. Molecular docking results showed that kaempferol and luteolin had a better affinity with TLR4, NFκB and NLRP3 molecules. The protein expressions of TLR4, NFκB and NLRP3 were reduced in the HXP group (P<0.01). HXP has a significant protective effect on myocardial ischemia-reperfusion injury in rats, and its effect may be related to the inhibition of redox response and reduction of the inflammatory response by inhibiting the TLR4NFκB/NLRP3 signaling pathway.

New Insight into the Potential Protective Function of Sulforaphene against ROS-Mediated Oxidative Stress Damage In Vitro and In Vivo.

Sulforaphene (SFE) is a kind of isothiocyanate isolated from radish seeds that can prevent free-radical-induced diseases. In this study, we investigated the protective effect of SFE on oxidative-stress-induced damage and its molecular mechanism in vitro and in vivo. The results of cell experiments show that SFE can alleviate D-gal-induced cytotoxicity, promote cell cycle transformation by inhibiting the production of reactive oxygen species (ROS) and cell apoptosis, and show a protective effect on cells with H2O2-induced oxidative damage. Furthermore, the results of mice experiments show that SFE can alleviate D-galactose-induced kidney damage by inhibiting ROS, malondialdehyde (MDA), and 4-hydroxyalkenals (4-HNE) production; protect the kidney against oxidative stress-induced damage by increasing antioxidant enzyme activity and upregulating the Nrf2 signaling pathway; and inhibit the activity of pro-inflammatory factors by downregulating the expression of Toll-like receptor 4 (TLR4)-mediated inflammatory response. In conclusion, this research shows that SFE has antioxidant effects, providing a new perspective for studying the anti-aging properties of natural compounds.

Hepatitis B Virus Envelope Antigen and Hepatitis B Virus Surface Antigen Both Contribute to the Innate Immune Response During Persistent Hepatitis B Virus Infection.

This study aimed to investigate the changes of toll-like receptor 4 (TLR4), proinflammatory cytokine expression, hepatitis B virus surface antigen (HBsAg), and hepatitis B virus envelope antigen (HBeAg) expression as well as innate immune cell percentages in a mouse model of persistent hepatitis B virus (HBV) infection to better understand the innate immune response. Mouse models of persistent HBV infection, HBsAg expression, and HBeAg expression were developed using high-pressure tail-vein injection of recombinant adeno-associated viruses. Enzyme-linked immunosorbent assays (ELISAs) were used to determine the serum proinflammatory cytokine levels. Immunohistochemistry and western blot assays were used to detect TLR4 expression. Flow cytometric analysis was used to assess the percentage of innate immune cells in the whole blood. Persistent HBV infection, HBsAg expression, and HBeAg expression each significantly decreased the expression of TLR4. Persistent HBV infection significantly increased the percentages of T cells and monocytes, whereas it decreased the percentage of natural killer (NK) cells. Persistent HBeAg expression also decreased the percentage of NK cells, whereas persistent HBsAg expression increased the percentage of NK cells. Both persistent HBsAg and HBeAg expression increased the percentage of monocytes. However, both persistent HBsAg and HBeAg expression decreased the percentage of T cells. HBV as well as HBsAg and HBeAg showed similar effects on the expression of TLR4 and proinflammatory cytokines as well as the percentage of monocytes. Persistent HBV infection increased the percentage of T cells and decreased the percentage of NK cells, whereas only persistent HBeAg expression contributed to a decreased percentage of NK cells.

B cell-intrinsic TLR7 expression drives severe lupus in TLR9-deficient mice.

The endosomal Toll-like receptor 7 (TLR7) is a major driver of murine and human systemic lupus erythematosus (SLE). The role of TLR7 in lupus pathogenesis is enhanced when the regulatory role of TLR9 is absent. TLR7 signaling in plasmacytoid DCs (pDC) is generally thought to be a major driver of the IFN response and disease pathology; however, the cell types in which TLR7 acts to mediate disease have not been distinguished. To address this, we selectively deleted TLR7 in either CD11c+ cells or CD19+ cells; using a TLR7-floxed allele, we created on the lupus-prone MRL/lpr background, along with a BM chimera strategy. Unexpectedly, TLR7 deficiency in CD11c+ cells had no impact on disease, while TLR7 deficiency in CD19+ B cells yielded mild suppression of proteinuria and a trend toward reduced glomerular disease. However, in TLR9-deficient MRL/lpr mice with accelerated SLE, B cell-specific TLR7 deficiency greatly improved disease. These results support revision of the mechanism by which TLR7 drives lupus and highlight a cis regulatory interaction between the protective TLR9 and the pathogenic TLR7 within the B cell compartment. They suggest B cell-directed, dual TLR7 antagonism/TLR9 agonism or dual TLR7/9 antagonism as a potential future therapeutic strategy to treat SLE.

Identifying hepatoprotective mechanism and effective components of Yinchenzhufu decoction in chronic cholestatic liver injury using a comprehensive strategy based on metabolomics, molecular biology, pharmacokinetics, and cytology

Ethnopharmacological relevanceIn Traditional Chinese Medicine (TCM), cholestasis liver disease belongs to jaundice. Yinchenzhufu decoction (YCZFD) is a classic formula used for treating jaundice. Aim of the studyThis study was aimed to investigate the potential mechanism and effective components of YCZFD in chronic cholestatic liver injury (CCLI). Materials and methodsA chronic cholestatic mouse model induced by 3, 5-diethoxycarbonyl-1, 4-dihydroxychollidine was used to investigate the effect of YCZFD. Then, metabolomics was used to investigate the metabolites influenced by YCZFD. Serum and liver bile acid (BA) levels were measured using liquid chromatography coupled with triple quadruple mass spectrometry (LC-MS/MS), and the gene and protein expressions of BA transporters and metabolic enzymes were detected. Additionally, the pharmacokinetics of multiple components of YCZFD was explored to clarify the potential effective components. The effects of absorbed components of YCZFD on BA metabolism and transporter function, inflammation, and farnesoid X receptor (FXR) and pregnane X receptor (PXR) activation were analyzed using sandwich cultured rat hepatocytes, AML12 cells, and dual-luciferase receptor systems, respectively. ResultsYCZFD decreased the liver damage in chronic cholestatic mice. Serum metabolomics results indicated that the main pathways influenced by YCZFD involved primary BA biosynthesis and arachidonic acid metabolism. YCZFD upregulated the expression of FXR, PXR, and BA efflux transporters and the metabolic enzymes of liver tissues, promoting BA excretion and metabolism in cholestatic mice. Additionally, YCZFD downregulated the expression of genes and proteins of the toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB) pathway and decreased liver inflammation. The pharmacokinetic study indicated that multiple components showed different pharmacokinetic properties. Among the absorbed components of YCZFD, multiple components activated the transcription of FXR and PXR, regulated BA transporters and metabolic enzyme function, and reduced the gene expression of TLR4 and NF-κB1. ConclusionYCZFD can ameliorate CCLI by promoting the excretion and metabolism of BAs and inhibiting inflammation via the TLR4/NF-κB signaling pathway. The multiple components of YCZFD could act on BA homeostasis regulation and anti-inflammation, exhibiting a combined effect against CCLI.