TLR4 Levels Research Articles

Investigation on improving immunologic reconstitution insufficiency using DiwuYanggan capsules in AIDS patients

BackgroundThis study aimed to explore the mechanism of action of DiWuYangGan (DWYG) capsule in improving Immunological non-responder (INR) by analyzing the active ingredients of DWYG.MethodsThe study employed a randomized, controlled, double-blind, single-simulation method. Patients were randomly divided into control and trial groups and treated with the primal highly effective antiretroviral therapy. To demonstrate the effect of DWYG on INR, patients in the control group were administered simulated DWYG, whereas patients in the trial group were administered DWYG capsules (ChiCTR1900024673). The chemical composition of DWYG was analyzed using ultra-performance liquid chromatography-high-resolution mass spectrometry. Potential targets of DWYG in the treatment of INR were identified and predicted using network pharmacology and molecular docking. The molecular mechanisms underlying the effects of DWYG were validated using a peripheral blood monocyte model.ResultsThe CD4:CD8 ratio in the trial group was significantly higher than that in the control group (p < 0.01). A total of 210 DWYG compounds were identified and network pharmacology revealed 182 potential therapeutic targets for DWYG and INR. The results of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses showed that the toll-like receptor signaling pathway is one of the key pathways. This study demonstrated that DWYG reduced the expression level of TLR4 and the levels of IL-2, IL-10, and TNF-α, which are important cytokines involved in the immune response.ConclusionThe efficacy of DWYG in the treatment of INR confirmed the potential practical components of DWYG. Moreover, the results of network pharmacology and experimental validation showed that DWYG could restore the immune function of acquired immune deficiency syndrome patients by inhibiting the expression of TLR4 and related signaling pathways and the overactivation of immune function.Clinical Trial Registration:https://www.chictr.org.cn/index.html, identifier ChiCTR1900024673.

Effect of vaccination against Mycoplasma hyopneumoniae on divergent pig genetic groups

The bacterium Mycoplasma hyopneumoniae (Mhp) causes a chronic infectious respiratory disease in pigs, leading to important economic losses. This study aimed to compare the immune response of the local Piau breed and a commercial line to Mhp vaccination. For this, two phases were carried out. In the first, gene expression of toll-like receptors (TLR2, TLR4, TLR6, and TLR10) and cytokines (IL2, IL6, IL8, IL10, IL12, IL13, TNFα, and TGFβ) was assessed in porcine blood mononuclear cells (PBMC) from the two genetic groups before and after vaccination. In the second experiment, nitric oxide production, specific antibodies, and gene expression of toll-like receptors and cytokines were evaluated in bronchoalveolar lavage fluid (BALF) cells of vaccinated and unvaccinated pigs. After vaccination against Mhp, TLR2, TLR4, TLR6, TLR10, IL6, TNFα, and TGFβ expression levels were elevated in PBMC from commercial animals, and TLR6, TLR10, and TGFβ expression levels were elevated in PBMC from the Piau group. Vaccination also increased the production of Mhp-specific IgG antibodies in BALF cells in the Piau breed. Comparison of the two genetic groups revealed differences in TNFα and IL10 expression in BALF cells. These results show that Piau pigs have different immune responses to vaccination compared with commercial animals. It is worth noting that these genetic differences between both genetic groups may be related to phenotypic differences in Mhp resistance or susceptibility.

Engineering of phosphatidylserine-targeting ROS-responsive polymeric prodrug for the repair of ischemia-reperfusion-induced acute kidney injury

Ischemia-reperfusion-induced acute kidney injury (IR-AKI) commonly occurs in situations such as hemorrhagic shock, kidney transplantation, and cardiovascular surgery. As one of the significant causes of AKI, IR-AKI is characterized by its high incidence and mortality rates. Currently, effective inflammation control is the key for the treatment of IR-AKI. In this study, we developed an ROS-responsive polymeric prodrugs (Zn-D/DTH) which could target the externalized PS of apoptotic cells, and then responsively released HDM (anti-inflammatory peptides) in the presence of intracellular ROS. Zn-D/DTH effectively ameliorated renal function and mitigated pathological alterations such as the loss of the brush border, tubular dilation, and accumulation of cellular debris within the tubular lumens. Furthermore, Zn-D/DTH greatly reduced the generation of pro-inflammatory factors like IL-6, COX-2, and iNOS in renal tissues, suggesting its protective role largely stems from suppression of the inflammatory response. Additional mechanism exploration revealed that Zn-D/DTH markedly decreased the expression levels of TLR4 and MyD88, as well as the phosphorylation of NF-κB in the damaged kidneys. This, in turn, reduced the number of apoptotic tubular cells and the activity of Caspase 9 and Caspase 3 caused by ischemia-reperfusion. Additionally, Zn-D/DTH treatment showed improvement in the long-term renal damage and fibrosis induced by ischemia-reperfusion. The experimental outcomes indicated that Zn-D/DTH attenuated renal ischemia-reperfusion injury and delayed the transition from acute kidney injury to chronic kidney disease by downregulating the TLR4/MyD88/NF-κB signaling pathway and reducing the expression of apoptotic caspases, thereby inhibiting inflammation and reducing cell apoptosis.

The ameliorating effects of adipose-derived stromal vascular fraction cells on blue light-induced rat retinal injury via modulation of TLR4 signaling, apoptosis, and glial cell activity.

Blue light (BL)-induced retinal injury has become a very common problem due to over exposure to blue light-emitting sources. This study aimed to investigate the possible ameliorating impact of stromal vascular fraction cells (SVFCs) on BL-induced retinal injury. Forty male albino rats were randomly allocated into four groups. The control group rats were kept in 12-h light/12-h dark. Rats of SVFC-control as the control group, but rats were intravenously injected once by SVFCs. Rats of both the BL-group and BL-SVFC group were exposed to BL for 2 weeks; then rats of the BL-SVFC group were intravenously injected once by SVFCs. Following the BL exposure, rats were kept for 8 weeks. Physical and physiological studies were performed; then retinal tissues were collected for biochemical and histological studies. The BL-group showed physical and physiological changes indicating affection of the visual function. Biochemical marker assessment showed a significant increase in MDA, TLR4 and MYD88 tissue levels with a significant decrease in TAC levels. Histological and ultrastructural assessment showed disruption of the normal histological architecture with retinal pigment epithelium, photoreceptors, and ganglion cell deterioration. A significant increase in NF-κB, caspase-3, and GFAP immunoreactivity was also detected. BL-SVFC group showed a significant improvement in physical, physiological, and biochemical parameters. Retinal tissues revealed amelioration of retinal structural and ultrastructural deterioration and a significant decrease in NF-κB and caspase-3 immunoreactivity with a significant increase in GFAP immunoreaction. This study concluded that SVFCs could ameliorate the BL-induced retinal injury through TLR-4/MYD-88/NF-κB signaling inhibition, regenerative, anti-oxidative, and anti-apoptotic effects.

Tilianin suppresses NLRP3 inflammasome activation in myocardial ischemia/reperfusion injury via inhibition of TLR4/NF-κB and NEK7/NLRP3.

Tilianin, a flavonoid compound derived from Dracocephalum moldavica L., is recognized for its diverse biological functionalities, in particular alleviating myocardial ischemia-reperfusion injury (MIRI). There is ample evidence suggesting that the NLRP3 inflammasome has a significant impact on the development of MIRI. In this study, rats undergoing the ligation and subsequent release of the left anterior descending (LAD) coronary artery and H9c2 cardiomyocytes subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) were used to investigate the effects of tilianin on NLRP3 inflammasome and its anti-MIRI mechanisms. Upon reperfusion, the rats were intraperitoneally injected with tilianin at doses of 3, 10, 30mg/kg. H9c2 cells were treated with tilianin at concentrations of 10, 30, and 50μg/mL. Echocardiography, TTC staining and TUNEL staining demonstrated that tilianin remarkably improved cardiac function and mitigated myocardial damage in MIRI rats. Additionally, notable inflammatory response reduction by tilianin was evidenced by subsequent hematatoxylin-eosin (HE) staining, inflammatory cytokines assay, and quantitative proteomics. Further western blotting analysis and immunofluorescence staining showed tilianin decreased the levels of TLR4, p-NF-κB, NLRP3, and ASC in MIRI rats and H9c2 cells exposed to OGD/R, alongside a significant reduction in cleaved gasdermin D, mature IL-1β and IL-18. Molecular docking, cellular thermal shift assay (CETSA) and co-immunoprecipitation (co-IP) assay revealed that tilianin impeded the interaction between NLRP3 and NEK7. Taken together, tilianin protects cardiomyocytes from MIRI by suppressing NLRP3 inflammasome through the inhibition of the TLR4/NF-κB signaling pathway and the disruption of the NEK7/NLRP3 interface. These findings underscore the potential of tilianin as a promising therapeutic candidate for MIRI.

Molecular mechanisms of endoplasmic reticulum stress-mediated acute kidney injury in juvenile rats and the protective role of mesencephalic astrocyte-derived neurotrophic factor.

This study examined the role of endoplasmic reticulum stress in pediatric acute kidney injury and the therapeutic effect of midbrain astrocyte-derived neurotrophic factor. Two-week-old Sprague-Dawley rats were divided into: Sham, ischemia-reperfusion injury-induced acute kidney injury (AKI), mesencephalic astrocyte-derived neurotrophic factor (MANF)-treated, tauroursodeoxycholic acid (TUDCA)-treated. Analyses were conducted 24h post-treatment. Serum creatinine, cystatin C, Albumin, MANF levels were measured, cytokine concentrations in serum and renal tissues were determined using a Luminex assay. Histopathology was assessed via light and electron microscopy. Western blotting and RT-qPCR analyzed markers for oxidative stress, apoptosis, endoplasmic reticulum (ER) stress, and autophagy. HK-2 cells underwent hypoxia/reoxygenation (H/R) to simulate AKI and were treated with MANF or TUDCA. AKI rats had increased serum creatinine, cystatin C, and inflammatory cytokines, along with significant renal damage, and showed loose and swollen ER structures, reduced cell proliferation, and elevated levels of IRE1, PERK, ATF6, CHOP, LC3-II/I, KIM-1, TLR4, JNK, and NF-κB. MANF treatment reduced these biomarkers and protein levels, improved ER structure and cell proliferation, alleviated oxidative stress, apoptosis, ER stress, and inhibited JNK/TLR4/NF-κB signaling. In HK-2 cells, MANF reduced ER stress and inflammation post-H/R exposure. MANF treatment alleviates ER stress, oxidative stress, apoptosis, and inflammation in pediatric AKI, improving renal function and morphology.

FuKe QianJin capsule alleviates endometritis via inhibiting inflammation and pyroptosis through modulating TLR4/ NF-κB /NLRP3 pathway

Ethnopharmacological relevanceFuke Qianjin Capsule (FKC), a traditional Chinese medicine commonly employed for treating endometritis, lacks reported treatment mechanisms. Aim of the studyThe aim of the present study was to explore the role and mechanism of FKC in lipopolysaccharide (LPS)-induced endometritis. Materials and methodsThe main active ingredients of FKC were identified via high-performance liquid chromatography (HPLC) in conjunction with standard substances. Prior to endometritis induction, Sprague Dawley female rats received FKC for 7 days. The endometritis model was established through an intrauterine injection of 1 mg/kg LPS. Concurrently, an LPS-induced RAW264.7 cell inflammation model was utilized, in which the cells were treated with serum containing Fuke Qianjin Capsule. Pathological alterations in the endometrium were assessed via H&E staining and transmission electron microscopy (TEM). The contents of MPO in uterine tissues, and NO release in cells, along with the secretion of IL-18, IL-1β, IL-6, and TNF-α in both tissues and cells, were determined via assay kits. The mRNA levels of Nlrp3, Caspase-1, Gsdmd, and Il-1β in uterine tissues and cells were analyzed via qPCR. The protein levels of TLR4, p65, p-P65, NLRP3, Caspase-1, GSDMD, and IL-1β in these samples were evaluated through Western blot analysis. Immunofluorescence was used to assess the protein levels of p-P65 and NLRP3 in uterine tissues and cells. ResultsFive primary active components of FKC were identified. Treatment with FKC in vivo mitigated endometrial pathological damage and significantly decreased the levels of MPO, IL-18, IL-1β, IL-6, and TNF-α, as well as the levels of Nlrp3, Caspase-1, Gsdmd, and Il-1β mRNA in tissue samples. Treatment with FKC inhibited the expression of TLR4, p-P65, NLRP3, Caspase-1, GSDMD, and IL-1β, as well as reduced NLRP3 protein fluorescence intensity, and inhibited P65 phosphorylation. In vitro findings demonstrated that FKC-containing serum reduced IL-18, IL-1β, IL-6, and TNF-α levels, as well as reduced Nlrp3, Caspase-1, Gsdmd, and Il-1β mRNA levels. In addition, FKC-containing serum inhibited the protein expression of TLR4, p-P65, NLRP3, Caspase-1, GSDMD, and IL-1β. FKC-containing serum also reduced NLRP3 protein fluorescence intensity and suppressed P65 phosphorylation. ConclusionFKC reverses the LPS induced NLRP3 inflammasome activation, and mitigates inflammation and pyroptosis through the modulation of the TLR4/NF-κB/NLRP3 pathway, thereby alleviating endometritis.

Vitamin D3 mediates autophagy to alleviate inflammatory responses in bovine endometrial epithelial cells and organoids via the PI3K/AKT/mTOR pathway

As a natural anti-inflammatory agent, it remains unclear whether the anti-inflammatory effects of VD3 (1,25 dihydroxyvitamin D3) are related to autophagy. This study investigates the impact of VD3 on inflammatory injury, autophagy, oxidative stress, and apoptosis in bovine endometrial epithelial cells (BEECs) and bovine endometrial organoids (BEOs). BEECs and BEOs were treated with LPS (1 μg/ml) for 24 hours, followed by treatment with LPS+VD3 (50 ng/ml) for 6 hours. Cell viability was assessed using the CCK8 assay. The expression levels of inflammatory factors (IL-1β, IL-6, TLR4, NF-κB), autophagy markers (Beclin-1, ATG5, ATG7, p62), and components of the PI3K/AKT/mTOR pathway (PI3K, AKT, and mTOR) were quantified using qRT-PCR and Western blot analyses. LC3B expression was detected by immunofluorescence, and the apoptosis rate was assessed using Annexin V. The results demonstrated a significant decrease in the expression levels of IL-1β, IL-6, TLR4, and NF-κB, along with a notable increase in the activity of CAT and SOD2 in the LPS+VD3 group (P < 0.05). The expression of autophagy-related factors was significantly increased, whereas the expression of signaling pathway factors was decreased in the LPS+VD3 group (P < 0.05). Additionally, apoptosis was significantly alleviated in the LPS+VD3 group (P < 0.05). Collectively, these findings indicate that VD3 modulates autophagy, attenuates oxidative stress and inflammatory damage in BEECs and BEOs, and inhibits LPS-induced apoptosis via the PI3K/AKT/mTOR pathway.

Mechanism research of Tollip negative feedback regulation in TLR4 signaling pathways based on spinal tuberculosis: Detection of Tollip and NF-κB expression levels

The emergence of drug-resistant mycobacterium tuberculosis (MTB, or TB) strains has led to an increasing incidence of TB. Spinal tuberculosis is the most common extrapulmonary tuberculosis. In the present study, tollip, a negative feedback regulatory factor in TLR4 signaling pathway was chosen based on previous studies on osteoarticular tuberculosis. U937 cells were transfected with recombinant lentivirus containing shRNA (RNA interference, RNAi) or overexpression vector containing Tollip gene and tested in vitro. The expression levels of Tollip and TLR4 were detected by Real-time PCR and immunofluorescence techniques, and the cell morphology and infection effect were observed by DAPI staining. The results suggested that Tollip gene could negatively inhibit the expression of related factors in TLR4 signaling pathway, and thus is a potential biomarker for early diagnosis.

Assessment of immunological factors in COVID-19 patients treated by convalescent plasma.

Following the outbreak of COVID-19, several immunotherapy methods were used to modulate the immune responses of patients. In this study, we aimed to evaluate the immune response to COVID-19 in patients receiving convalescent plasma. In this regard, this randomized controlled trial included 30 patients who were divided into two groups according to receiving convalescent plasma or normal control plasma. Samples from both groups were collected on days 0, 1, 3, 5 and 7 after plasma infusion. We measured the expression level of TLR7/8, IRF3/7, CTLA-4, PD-1 and T cell transcription factors by Real-time PCR in the mentioned groups. Thirteen cytokines were also evaluated using flow cytometry method. Results showed that compared to the normal control plasma group, the expression levels of TLR7, 8, IRF3, 7 and PD-1 and CTLA-4, on days 3, 5 and 7 after convalescent plasma infusion, were significantly decreased. On the other hand, Gene expression results showed that the expression levels of Tbet, RORγ3 and Foxp3 on days 3, 5 and 7 after convalescent plasma infusion were significantly increased compared to the normal control plasma group. After convalescent plasma infusion, the viral load was significantly decreased compared to the normal control plasma group. Convalescent plasma infusion also reduced the plasma cytokines levels, including IL-6, IL-10, and IL-4, and enhanced the level of IL-2, IFN- γ and perforin comparing the normal control plasma group. According to the results, the convalescent plasma infusion led to a decrease in the expression of innate immunity receptors and an increase in the expression of transcription factors of adaptive immunity. Therefore, it may be concluded that convalescent plasma infusion can modulate the immune response. To achieve a reliable consequence, further studies are required.

Exploring the hub Genes and Potential Mechanisms of Complement system-related Genes in Parkinson Disease: Based on Transcriptome Sequencing and Mendelian Randomization.

An accurate diagnosis of Parkinson's disease (PD) remains challenging and the exact cause of the disease is unclean. The aims are to identify hub genes associated with the complement system in PD and to explore their underlying molecular mechanisms. Initially, differentially expressed genes (DEGs) and key module genes related to PD were mined through differential expression analysis and WGCNA. Then, differentially expressed CSRGs (DE-CSRGs) were obtained by intersecting the DEGs, key module genes and CSRGs. Subsequently, MR analysis was executed to identify genes causally associated with PD. Based on genes with significant MR results, the expression level and diagnostic performance verification were achieved to yield hub genes. Functional enrichment and immune infiltration analyses were accomplished to insight into the pathogenesis of PD. qRT-PCR was employed to evaluate the expression levels of hub genes. After MR analysis and related verification, CD93, CTSS, PRKCD and TLR2 were finally identified as hub genes. Enrichment analysis indicated that the main enriched pathways for hub genes. Immune infiltration analysis found that the hub genes showed significant correlation with a variety of immune cells (such as myeloid-derived suppressor cell and macrophage). In the qRT-PCR results, the expression levels of CTSS, PRKCD and TLR2 were consistent with those we obtained from public databases. Hence, we mined four hub genes associated with complement system in PD which provided novel perspectives for the diagnosis and treatment of PD.

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.

Electroacupuncture attenuates depressive-like behaviors in poststroke depression mice through promoting hippocampal neurogenesis and inhibiting TLR4/NF-κB/NLRP3 signaling pathway.

The aim of this study was to investigate the impact and underlying molecular mechanisms of electroacupuncture on mice with poststroke depression (PSD). Mice were randomly allocated into sham, PSD, and electroacupuncture groups. Mice in the PSD and electroacupuncture groups underwent middle cerebral artery occlusion (MCAO) surgery following with sedentary behavior. Electroacupuncture targeting Zusanli (ST36) acupoint was performed 24 h after MCAO for 4 weeks in electroacupuncture group. The sucrose preference test, forced swimming test, open field test, tail suspension test, elevated plus maze, Catwalk analysis, RNA sequencing, Nissl staining, Golgi staining, TUNEL staining, Edu labeling, and doublecortin staining were performed. Lymphocyte subsets in peripheral blood and the levels of IL-1β, IL-6, TNF-α, and expression of Iba1/CD86, Iba1/NLRP3, TLR4/p38/NF-κB/NLRP3 pathways in the hippocampus were detected. Electroacupuncture effectively protected against the development of depression-like symptoms. The number of granulosa cells and doublecortin-positive cells in the dentate gyrus (DG) were significantly decreased in PSD group, which were significantly upregulated ( P < 0.01) by electroacupuncture. Electroacupuncture also significantly reduced ( P < 0.05) TUNEL-positive cells in the DG and CA1. RNA-seq revealed that electroacupuncture may exert antidepressant effect by regulating the inflammation mediated by TLR4/NF-κB/NLRP3 pathway in hippocampus. Electroacupuncture remarkably elevated ( P < 0.01) the ratio of CD4+ to CD8+ T cells and percentage of CD3-CD49b+ cells in CD45+CD49b+ cells in the peripheral blood. Electroacupuncture significantly reduced ( P < 0.05) the high levels of IL-1β, IL-6, TNF-α, iba1, TLR4, p-p38, p-NF-κB, and NLRP3 and sedentary behavior. Electroacupuncture was observed to mitigate depression symptoms and increase hippocampal neurogenesis in mice with PSD, possibly by inhibiting TLR4/p38/NF-κB/NLRP3 pathways and improving the microglia-mediated inflammatory microenvironment in the hippocampus.

Taxus chinensis (Pilg.) Rehder fruit attenuates aging behaviors and neuroinflammation by inhibiting microglia activation via TLR4/NF-κB/NLRP3 pathway

Ethnopharmacological relevanceAs one of the important by-products of Taxus chinensis (Pilg.) Rehder, its fruit (TCF) has a sweet taste, which is commonly used in folklore to make health care wine reputed for enhancing immune function and promoting anti-aging effects, especially popular in the longevity villages of China for a long history. Evidences had showed that Taxus chinensis fruit contained polysaccharides, flavonoids, amino acids and terpenoids, which all were free of toxic compounds , but its medicinal value has not been fully recognized. Our previous studies have found that TCF extract may reverse many biological events, including oxidative stress, inflammatory response, neuronal apoptosis, etc. by in silico methods, suggesting potential avenues for future pharmaceutical exploration in aging and age-related diseases. Aim of the studyYet, the anti-aging properties of TCF have not been specifically studied, this study aims to fill this gap by investigating the effects of TCF extract (TCFE) in an aging mouse model, particularly focusing on its role in inhibiting microglial activation and elucidating its underlying anti-aging mechanisms. Materials and methodsAn aging mouse model was induced using D-galactose, with interventions involving high, medium, and low doses of TCFE compared to a positive control (2mg/kg rapamycin combined with 100mg/kg metformin). The methodology involved evaluating behavioral changes, serum oxidative and antioxidative markers, hypothalamic β-galactosidase activity, expression of the aging-related protein P63, serum inflammatory factors, and the TLR4/NF-κB/NLRP3 inflammatory pathway in hypothalamic tissues. Additionally, to strengthen our in vivo findings, we conducted in vitro experiments on LPS-stimulated BV2 microglial cells. Finally, UPLC-MS/MS for precise component analysis using compound standards, coupled with molecular docking analyses, were employed to discern and elucidate the anti-inflammatory mechanisms of TCF. ResultsIn vivo results revealed TCFE significantly ameliorated behavioral deficits, reduced oxidative stress markers (MDA) and pro-inflammatory cytokines (IL1-β, IL-6, IFNg, TNFα, IL-17), and increased in antioxidants (SOD, T-AOC) and anti-inflammatory factors (IL-10). TCFE also reduced hypothalamic senescence, improved cellular integrity, lowered p63, and inhibited microglia activation and inflammatory pathways (TLR4, NFKB, NLRP3). The overall effect of TCFE was better than that of the positive drug group (rapamycin combined with metformin). In vitro results further revealed that TCFE markedly decreased IL1-β, NFKB, and TLR4 levels in BV2 microglial cells, showing comparable efficacy to a TLR4 classic positive inhibitor C34, supporting its anti-inflammatory role. Through UPLC-MS/MS analysis coupled with compound standards, we identified ten bioactive compounds, including gallocatechin, epigallocatechin, catechin, procyanidin B2, kaempferol, quercetin, rutin, naringin, apigenin, ginkgetin. All these compounds showed strong binding affinity to TLR4, notably procyanidin B2 and rutin, potentially through hydrogen bonds, aromatic cation-π interactions, and hydrophobic interactions, suggesting a molecular basis for their anti-inflammatory action. ConclusionTCFE showed strong anti-aging effects by inhibiting microglia activation and lessening oxidative stress and modulating inflammatory pathways. This research supports TCF's use in anti-aging and sets a base for future drug development in the realms of neuroinflammation and aging.

Mycoplasma pneumoniae regulates the expression of GP130 in lung epithelial cells through apoptosis and TLR4/ NF-κB pathway during infection

In previous study, lower levels of serum GP130 were reported in children with MPP. GP130 is an important signal transducer, the down regulation of which may influence host immune responses. In this study, we aimed to analyze the regulatory mechanism of GP130 during MP infection. Firstly, the mRNA and protein levels of GP130 both decrease and then increase with increasing multiplicity of infection (MOI: 1 to 40) of MP. The lowest levels of GP130 were detected at MOI of 5. Then, heat treated MP but not trypsin treated MP or MP extracted proteins show regulatory effect to the expression of GP130. These indicate that the down regulation of GP130 is related to protein mediate adhesion process of MP. Gene expression analysis revealed that MP affected apoptosis and the TLR4 pathway in infected cells, and the mRNA level of IL-6 was correlated with that of GP130. Further, Z-VAD-FMK (pan-caspase inhibitor) can suppress the apoptosis induced by MP infection and restore GP130 at protein level. Further studies revealed that MP infection promoted TLR4 internalization but did not activate the NF-κB pathway. The levels of surface TLR4 showed correlation with the transcription of IL-6 and GP130. TAK242 (TLR4 inhibitor) and PS341 (proteasome inhibitor) can restore the decreased transcription of GP130, both of which were able to promote NF-κB pathway activation in MP-infected cells. These suggested that the regulation of TLR4/NF-κB pathway and induced apoptosis post MP infection are involved in the down-regulation of GP130 at transcription and protein levels, respectively.

Sivelestat Sodium Alleviates Ischemia-Reperfusion-Induced Acute Kidney Injury via Suppressing TLR4/Myd88/NF-κB Signaling Pathway in Mice.

We aim to detect the effects of sivelestat on renal ischemia-reperfusion associated with AKI and also explore the underlying mechanism. Mice, aged between 8 and 12weeks, were randomly allocated among four distinct groups, respectively normal saline sham group(C), normal saline surgery group(I), sivelestat (50 mg/kg) sham group(S), sivelestat (50 mg/kg) surgery group(SI) (n=6, each group). In the surgical groups, the renal pedicles of mice were clamped withnon-traumatic micro-aneurysm clamps, resulting in ischemia of the kidneys for 45minutes. This was followed by a period of reperfusion lasting 24hours. Sham group mice underwent the identical surgery produced without clamping renal pedicles. Mice blood was obtained from eyeballs, and Serum creatinine and blood urea nitrogen levels were measured. After a 24-hour period of reperfusion, the mice were euthanized, and their kidneys were gathered for various analyses, including Western Blot (WB) analysis, RT-PCR, immunofluorescence (IF), hematoxylin and eosin (H&E) staining, and Tunel assay. Pretreatments with sivelestat decreased renal Neutrophil elastase (NE), serum creatinine, and blood urea nitrogen levels after renal ischemia-reperfusion. Sivelestat also reduced histological damage and cell apoptosis in kidneys following ischemia-reperfusion injury (IRI). In addition, thesivelestat administrationdiminished the levels of mRNA expression of interleukin 6 (IL-6), Macrophage inflammatory protein-2 (MIP-2), monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor (TNF)-α in the kidneys during IRI.The kidney tissues of the SI group had significantly mitigated TLR4, Myd88, and NF-κB p-p65 protein expression levels compared to the I group (all P<0.05). We demonstrated a previously unidentified mechanism that sivelestat effectively attenuates AKI-induced renal dysfunction, possibly through suppressing the TLR4/Myd88/ NF-κB pathway.

Mechanism of Yi-Qi-Bu-Shen Recipe for the Treatment of Diabetic Nephropathy Complicated with Cognitive Dysfunction Based on Network Pharmacology and Experimental Validation.

Diabetic nephropathy (DN) and cognitive dysfunction (CD) are common complications of diabetes. Yi-Qi-Bu-Shen Recipe (YQBS) can effectively reduce blood glucose, improve insulin resistance, and delay the progression of diabetic complications. The underlying mechanisms of its effects need to be further studied. This study elucidates the mechanism of YQBS in DN with CD through network pharmacology and experimental validation. Protein-protein interaction, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed. Male Sprague-Dawley (SD) rats were divided into 6 groups: model, YQBS (2, 4, 8 g/kg), positive control (metformin, 200 mg/kg), and control; the DN model was established by high sugar and high fat diet combined with intraperitoneal streptozotocin injection. After the DN model was established, the rats were gavaged for 10weeks. Serum, kidneys, and hippocampus tissues were collected to measure the expression levels of TLR4, NF-κB, TNF-α, and IL-6. The network pharmacology analysis showed that quercetin and kaempferol were the main active components of YQBS. TNF and IL-6 were the key targets, and TLR4/NF-κB pathway was crucial to YQBS in treating DN complicated with CD. Experimental validation showed that the intervention of YQBS can reduce TNF-α and IL-6 in serum, and also significantly decreases the protein expression of TLR4 and NF-κB. YQBS exerts anti-inflammatory effects on DN with CD through TLR4/NF-κB pathway. This study provides a biological basis for the scientific usage of YQBS in inflammation diseases and supplies experimental evidence for future traditional Chinese medicine development.

Diagnoses Bacterial Wound Infection and Detection of Some Immune Parameters in its

This study aims to diagnoses bacterial wound infection and to estimate the serum levels of TLR-3, CXCL8, TGF-β1, IL-8, IL-17 and IL-22 in wound patients. This study had been included 140 samples (blood and swab) obtained from patients found in Baquba-Teaching Hospital from 15/ 6 /2023 to 20 /3 /2024. Swab samples taken from wound were culture on the blood agar and MacConkey agar, and then Bacteria diagnoses was done by using an automatic VITEK 2 system. The serum levels of immune parameters done by ELISA. The results shown that sixty isolates were grow in culture, the results indicated of 38(63.3%) isolates of gram-negative bacteria, represented by: 12 (20%) isolates E. coli, 18 (30%) Pseudomonas aeruginosa, 6 (10%) Protus spp., 2 (3.3%) Acinetobacter spp. the results indicated of 22 (36.6%) isolates of gram-positive bacteria, represented by: 16 (26.6%) isolates Staphylococcus aureus, 4 (6.6 %) another Staphylococcus spp., 2 (3.3%) Enterococcus spp. The study also shown the a increase in the concentration of TLR-3 , CXCL8 , IL-2 , IL-17 and IL-22 in the serum of bacterial patients compared with the healthy, Where the results of the current study show decreases in the concentration of TGF-β1 among compared with healthy. In conclusion, it was discovered that the infection of bacteria in wound infection was lower in females than males. Pseudomonas aeruginosa and S. aureus were found to be the highly isolated bacteria wound infection, with increases levels of TLR-3, CXCL8, IL-17 and IL-22 in serum in patients and decreases level of TGF-β1 in serum of patients.

THIOSTREPTON MODULATES TLR4 EXPRESSION AND INDUCES APOPTOSIS IN MDA MB 231 CELLS: AN IN VITRO AND IN SILICO ANALYSIS

Objective: Toll-like receptors (TLRs) are key pattern recognition receptors involved in tumorigenesis, apoptosis, and metastasis. Triple-negative breast cancer (TNBC) is a highly aggressive malignancy with a poor prognosis. Although the role of TLRs in breast cancer remains underexplored, recent studies suggest targeting TLRs in TNBC could be beneficial. In this study Thiostrepton, an antibiotic and novel inhibitor of TLR7-9 in psoriatic inflammation, was investigated for its effects on TLR3, TLR4, and TLR9 expression in TNBC cells (MDA-MB-231). Materials and Methods: The cytotoxicity of thiostrepton was assessed using the MTT assay. RT-PCR was used to measure gene expression levels of TLR3, TLR4, TLR9, Bax, Bcl-2, Nf-κB, and E-cadherin. Cell morphology changes were analyzed with Acridine Orange/Ethidium Bromide (AO/EtBr) staining. Molecular docking and dynamics simulations examined interactions between thiostrepton and the TLR4-MD-2 complex. Results: Thiostrepton led to a concentration- and time-dependent decrease in cell viability. It significantly inhibited TLR4, Bcl-2 gene expression and increased TLR3, Bax, and Nf-κB levels. The changes in Bax and Bcl-2 gene expression, along with alterations in cell morphology, demonstrated that thiostrepton promoted apoptosis in MDA-MB-231 cells. While TLR9 expression reduction was not significant, thiostrepton notably increased TLR3 expression and decreased TLR4 expression. The three independent molecular dynamics simulations demonstrated that thiostrepton binds stably to the TLR4-MD2 domain, exhibiting a high binding affinity as indicated by the binding free energy calculations. Conclusion: Thiostrepton effectively induces apoptosis and reduces cell viability in TNBC cells. In silico analysis suggest thiostrepton could modulate TLR4, highlighting its potential as a candidate for further research and therapeutic development.

Evaluation of Gene Expression Levels of TLRs 2, 4, and 5 Genes in Patients with Chronic Suppurative Otitis Media (CSOM) and Relationship with Bacterial Infection

Toll-like receptors (TLRs), the building blocks of the innate immune response, are largely responsible for the development of otitis media. The illness may worsen due to mistakes or faults in TLR expression in immune cells found in the blood or middle ear tissue. People with various bacterial infections and chronic otitis media were evaluated for their TLR (TLR2, TLR4, and TLR5) gene expression. The results showed that higher expression levels of TLR2 were associated with Staphylococcus aureus, Streptococcus pneumoniae, and Pseudomonas aeruginosa in mucosal cells, whereas lower expression levels were associated with Moraxella catarrhalis and Haemophilus influenzae. The study found that TLR4 gene expression was higher in the mucosal cells of patients infected with Moraxella catarrhalis, Pseudomonas aeruginosa, Haemophilus influenzae and Streptococcus pneumoniae, compared to healthy controls, while Staphylococcus aureus was associated with lower expression. The study found that patients with infections caused by Haemophilus influenzae, Moraxella catarrhalis, and Staphylococcus aureus had no significant expression levels of the TLR5 gene in their mucosa cells, while patients with infections caused by Pseudomonas aeruginosa and Streptococcus pneumoniae had higher expression levels. TLRs2 mRNA expression levels increased in patients with chronic otitis media infected with Gram-positive bacteria (Staphylococcus aureus and Streptococcus pneumoniae), while TLR-4 and TLR-5 levels increased in patients infected with Gram-negative bacteria (Pseudomonas aeruginosa and Streptococcus pneumoniae).