Toll-like receptor 4 in pancreatic damage and immune infiltration in acute pancreatitis.

Acute pancreatitis is a complex inflammatory disease resulting in extreme pain and can result in significant morbidity and mortality. It can be caused by several factors ranging from genetics, alcohol use, gall stones, and ductal obstruction caused by calcification or neutrophil extracellular traps. Acute pancreatitis is also characterized by immune cell infiltration of neutrophils and M1 macrophages. Toll-like receptor 4 (TLR4) is a pattern recognition receptor that has been noted to respond to endogenous ligands such as high mobility group box 1 (HMGB1) protein and or exogenous ligands such as lipopolysaccharide both of which can be present during the progression of acute pancreatitis. This receptor can be found on a variety of cell types from endothelial cells to resident and infiltrating immune cells leading to production of pro-inflammatory cytokines as well as immune cell activation and maturation resulting in the furthering of pancreatic damage during acute pancreatitis. In this review we will address the various mechanisms mediated by TLR4 in the advancement of acute pancreatitis and how targeting this receptor could lead to improved outcomes for patients suffering from this condition.

[Effect and mechanism of Maxing Shigan Decoction on reducing inflammatory response in rats with cough variant asthma via TLR4/MyD88/NF-κB and p38 MAPK signaling pathways].

This study aims to investigate the effect and mechanism of Maxingshigan Decoction on inflammation in the rat model of cough variant asthma(CVA). The SPF-grade SD rats of 6-8 weeks were randomized into normal, model, Montelukast sodium, and low-, medium-, and high-dose Maxing Shigan Decoction groups, with 8 rats in each group. The CVA rat model was induced by ovalbumin(OVA) and aluminum hydroxide sensitization and ovalbumin stimulation. The normal group and model group were administrated with equal volume of normal saline by gavage, and other groups with corresponding drugs by gavage. After the experiment, the number of white blood cells in blood and the levels of interleukin-6(IL-6), interleukin-10(IL-10), and tumor necrosis factor-α(TNF-α) in the serum were measured. The lung tissue was stained with hematoxylin-eosin(HE). Western blot was employed to determine the protein levels of nuclear factor-κB(NF-κB), Toll-like receptor 4(TLR4), myeloid differentiation protein(MyD88), and mitogen-activated protein kinase(MAPK) in the lung tissue. Real-time PCR was carried out to measure the mRNA levels of TLR4 and MyD88 in the lung tissue. Compared with the normal group, the model group showed increased white blood cells, elevated IL-6 and TNF-α levels(P<0.01), lowered IL-10 level(P<0.01), up-regulated protein levels of TLR4, MyD88, p-p65/NF-κB p65, and p-p38 MAPK/p38 MAPK(P<0.01) and mRNA levels of TLR4 and MyD88(P<0.01) in the lung tissue. HE staining showed obvious infiltration of inflammatory cells around the airway and cell disarrangement in the model group. Compared with the model group, Montelukast sodium and high-dose Maxing Shigan Decoction reduced the white blood cells, lowered the IL-6 and TNF-α levels(P<0.01), and elevated the IL-10 level(P<0.01). Moreover, they down-regulated the protein levels of TLR4, MyD88, p-p65/NF-κB p65, p-p38 MAPK/p38 MAPK in the lung tissue(P<0.01) and the mRNA levels of TLR4 and MyD88 in the lung tissue(P<0.01). HE staining showed that Montelukast sodium and high-dose Maxing Shigan Decoction reduced inflammatory cell infiltration and cell disarrangement. The number of white blood cells, the levels of IL-10 and TNF-α in the serum, the protein levels of TLR4, MyD88, p-p65/NF-κB p65, and p-p38 MAPK/p38 MAPK, and the mRNA levels of TLR4 and MyD88 in the lung tissue showed no significant differences between the Montelukast sodium group and high-dose Maxing Shigan Decoction group. Maxing Shigan Decoction can inhibit airway inflammation in CVA rats by inhibiting the activation of TLR4/MyD88/NF-κB and p38 MAPK signaling pathways.

[Ligusticum cycloprolactam inhibits IL-1ß-induced apoptosis and inflammation of rat chondrocytes via HMGB1/TLR4/NF-κB signaling pathway].

Chondrocytes are unique resident cells in the articular cartilage, and the pathological changes of them can lead to the occurrence of osteoarthritis(OA). Ligusticum cycloprolactam(LIGc) are derivatives of Z-ligustilide(LIG), a pharmacodynamic marker of Angelica sinensis, which has various biological functions such as anti-inflammation and inhibition of cell apoptosis. However, its protective effect on chondrocytes in the case of OA and the underlying mechanism remain unclear. This study conducted in vitro experiments to explore the molecular mechanism of LIGc in protecting chondrocytes from OA. The inflammation model of rat OA chondrocyte model was established by using interleukin-1ß(IL-1ß) to induce. LIGc alone and combined with glycyrrhizic acid(GA), a blocker of the high mobility group box-1 protein(HMGB1)/Toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB) signaling pathway, were used to intervene in the model, and the therapeutic effects were systematically evaluated. The viability of chondrocytes treated with different concentrations of LIGc was measured by the cell counting kit-8(CCK-8), and the optimal LIGc concentration was screened out. Annexin V-FITC/PI apoptosis detection kit was employed to examine the apoptosis of chondrocytes in each group. The enzyme-linked immunosorbent assay(ELISA) was employed to measure the expression of cyclooxygenase-2(COX-2), prostaglandin-2(PGE2), and tumor necrosis factor-alpha(TNF-α) in the supernatant of chondrocytes in each group. Western blot was employed to determine the protein levels of B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), caspase-3, HMGB1, TLR4, and NF-κB p65. The mRNA levels of HMGB1, TLR4, NF-κB p65, and myeloid differentiation factor 88(MyD88) in chondrocytes were determined by real-time fluorescent quantitative PCR(RT-qPCR). The safe concentration range of LIGc on chondrocytes was determined by CCK-8, and then the optimal concentration of LIGc for exerting the effect was clarified. Under the intervention of IL-1ß, the rat chondrocyte model of OA was successfully established. The modeled chondrocytes showed increased apoptosis rate, promoted expression of COX-2, PGE2, and TNF-α, up-regulated protein levels of Bax, caspase-3, HMGB1, TLR4, and NF-κB p65 and mRNA levels of HMGB1, TLR4, NF-κB p65, and MyD88, and down-regulated protein level of Bcl-2. However, LIGc reversed the IL-1ß-induced changes of the above factors. Moreover, LIGc combined with GA showed more significant reversal effect than LIGc alone. These fin-dings indicate that LIGc extracted and derived from the traditional Chinese medicine A. sinensis can inhibit the inflammatory response of chondrocytes and reduce the apoptosis of chondrocytes, and this effect may be related to the HMGB1/TLR4/NF-κB signaling pathway. The pharmacological effect of LIGc on protecting chondrocytes has potential value in delaying the progression of OA and improving the clinical symptoms of patients, and deserves further study.

Introduction of protein vaccine candidate based on AP65, AP33, and α-actinin proteins against Trichomonas vaginalis parasite: an immunoinformatics design.

BACKGROUND: Trichomonas vaginalis is the most common nonviral sexually transmitted disease (STI) worldwide. Vaccination is generally considered to be one of the most effective methods of preventing infectious diseases. Using AP65, AP33 and α-actinin proteins, this research aims to develop a protein vaccine against Trichomonas vaginalis. METHODS: Based on the B-cell and T-cell epitope prediction servers, the most antigenic epitopes were selected, and with the necessary evaluations, epitope-rich domains of three proteins, AP65, AP33, and α-actinin, were selected and linked. Subsequently, the ability of the vaccine to interact with toll-like receptors 2 and 4 (TLR2 and TLR4) was assessed. The stability of the interactions was also studied by molecular dynamics for a duration of 100 nanoseconds. RESULTS: The designed protein consists of 780 amino acids with a molecular weight of 85247.31 daltons. The results of the interaction of the vaccine candidate with TLR2 and TLR4 of the immune system also showed that there are strong interactions between the vaccine candidate protein with TLR2 (-890.7 kcal mol-1) and TLR4 (-967.3 kcal mol-1). All parameters studied to evaluate the stability of the protein structure and the protein-TLR2 and protein-TLR4 complexes showed that the structure of the vaccine candidate protein is stable alone and in complex with the immune system receptors. Investigation of the ability of the designed protein to induce an immune response using the C-ImmSim web server also showed that the designed protein is capable of stimulating B- and T-cell lymphocytes to produce the necessary cytokines and antibodies against Trichomonas vaginalis. CONCLUSIONS: Overall, our vaccine may have potential protection against Trichomonas vaginalis. However, for experimental in vivo and in vitro studies, it may be a good vaccine candidate.

TLR4 TIR domain and nucleolin GAR domain synergistically mediate RSV infection and induce neuronal inflammatory damage in SH-SY5Y cells.

Previous research results of our group showed that Toll-like receptor 4 (TLR4) and nucleolin synergistically mediate respiratory syncytial virus (RSV) infection in human central neuron cells, but the specific mechanism remains unclear. Here we designed and synthesized lentiviruses with TIR (674-815 aa), TLR4 (del 674-815 aa), GAR (645-707 aa), and NCL (del 645-707 aa) domains, and obtained stable overexpression cell lines by drug screening, and subsequently infected RSV at different time points. Laser confocal microscopy and coimmunoprecipitation were used for the observation of co-localization and interaction of TIR/GAR domains. Western blot analysis was used for the detection of p-NF-κB and LC3 protein expression. Real-time PCR was used for the detection of TLR4/NCL mRNA expression. ELISA assay was used to measure IL-6, IL-1ß, and TNF-α concentrations and flow cytometric analysis was used for the study of apoptosis. Our results suggest that overexpression of TIR and GAR domains can exacerbate apoptosis and autophagy, and that TIR and GAR domains can synergistically mediate RSV infection and activate the NF-κB signaling pathway, which regulates the secretion of downstream inflammatory factors, such as IL-6, IL-1ß, and TNF-α, and ultimately leads to neuronal inflammatory injury.

Expression of Toll-like receptors in oral squamous cell carcinoma.

Almost 380,000 new cases of oral cancer were reported worldwide in 2020. Oral squamous cell carcinoma (OSCC) accounts for 90% of all types of oral cancers. Emerging studies have shown association of Toll-like receptors (TLRs) in carcinogenesis. The present study aimed to investigate the expression levels and tissue localization of TRL1 to TRL10 and NF-κB between OSCC and healthy oral mucosa, as well as effect of Candida colonization in TRL expression in OSCC. Full thickness biopsies and microbial samples from 30 newly diagnosed primary OSCC patients and 26 health controls were collected. The expression of TLR1 to TLR10 and NF-κB was analyzed by immunohistochemistry. Microbial samples were collected from oral mucosa to detect Candida. OSCC epithelium showed lower staining intensity of TRL1, TRL2 TRL5, and TRL8 as compared to healthy controls. Similarly, staining intensity of TRL3, TRL4, TRL7, and TRL8 were significantly decreased in basement membrane (BM) zone. Likewise, OSCC endothelium showed lower staining intensity of TLR4, TLR7 and TLR8. Expression of NF-κB was significantly stronger in normal healthy tissue compared to OSCC sample. Positive correlation was found between the expression of NF-κB, TRL9 and TRL10 in basal layer of the infiltrative zone OSCC samples (P = 0.04 and P = 0.002, respectively). Significant increase in TRL4 was seen in BM zone of sample colonized with Candida (P = 0.01). According to the limited number of samples, our data indicates downregulation of TLRs and NF-κB in OSCC, and upregulation of TLR4 expression with presence of Candida.

Renoprotective effect of diacetylrhein on diclofenac-induced acute kidney injury in rats via modulating Nrf2/NF-κB/NLRP3/GSDMD signaling pathways.

Diclofenac (DF)-induced acute kidney injury (AKI) is characterized by glomerular dysfunction and acute tubular necrosis. Due to limited treatment approaches, effective and safe drug therapy to protect against such AKI is still needed. Diacetylrhein (DAR), an anthraquinone derivative, has different antioxidant and anti-inflammatory properties. Therefore, the aim of the current study was to investigate the renoprotective effect of DAR on DF-induced AKI while elucidating the potential underlying mechanism. Our results showed that DAR (50 and 100 mg/kg) markedly abrogated DF-induced kidney dysfunction decreasing SCr, BUN, serum NGAL, and serum KIM1 levels. Moreover, DAR treatment remarkably maintained renal redox balance and reduced the levels of pro-inflammatory biomarkers in the kidney. Mechanistically, DAR boosted Nrf2/HO-1 antioxidant and anti-inflammatory response in the kidney while suppressing renal TLR4/NF-κB and NLRP3/caspase-1 inflammatory signaling pathways. In addition, DAR markedly inhibited renal pyroptosis via targeting of GSDMD activation. Collectively, this study confirmed that the interplay between Nrf2/HO-1 and TLR4/NF-κB/NLRP3/Caspase-1 signaling pathways and pyroptotic cell death mediates DF-induced AKI and reported that DAR has a dose-dependent renoprotective effect on DF-induced AKI in rats. This effect is due to powerful antioxidant, anti-inflammatory, and anti-pyroptotic activities that could provide a promising treatment approach to protect against DF-induced AKI.

DFMG decreases angiogenesis to uphold plaque stability by inhibiting the TLR4/VEGF pathway in mice.

The aim of this study was to elucidate the specific mechanism through which 7-difluoromethoxy-5,4'-dimethoxygenistein (DFMG) inhibits angiogenesis in atherosclerosis (AS) plaques, given its previously observed but poorly understood inhibitory effects. In vitro, a model using Human Umbilical Vein Endothelial (HUVEC-12) cells simulated the initial lesion in the atherosclerotic pathological process, specifically oxidative stress injury, by exposing cells to 30 µmol/L LPC. Additionally, an AS mouse model was developed in ApoE knockout mice through a 16-week period of high-fat feeding. DFMG demonstrated a reduction in tubule quantities in the tube formation assay and neovascularization induced by oxidative stress-damaged endothelial cells in the chicken embryo chorioallantoic membrane assay. Furthermore, DFMG decreased lipid levels in the blood of ApoE knockout mice with AS, along with a decrease in atherosclerotic plaques and neovascularizations in the aortic arch and descending aorta of AS animal models. DFMG treatment upregulated microRNA140 (miR-140) expression and suppressed VEGF secretion in HUVEC-12 cells. These effects were counteracted by Toll-like receptor 4 (TLR4) overexpression in HUVEC-12 cells subjected to oxidative injury or in a mouse model of AS. Dual-luciferase reporter assays demonstrated that miR-140 directly targeted TLR4. Immunohistochemical assay findings indicated a significant inverse relationship between miR-140 expression and TLR4 expression in ApoE knockout mice subjected to a high-fat diet. The study observed a close association between DFMG inhibitory effects on angiogenesis and plaque stability in AS, and the inhibition of the TLR4/NF-κB/VEGF signaling pathway, negatively regulated by miR-140.

Iron Oxide Nanoparticles Inhibit Tumor Progression and Suppress Lung Metastases in Mouse Models of Breast Cancer.

Systemic exposure to starch-coated iron oxide nanoparticles (IONPs) can stimulate antitumor T cell responses, even when little IONP is retained within the tumor. Here, we demonstrate in mouse models of metastatic breast cancer that IONPs can alter the host immune landscape, leading to systemic immune-mediated disease suppression. We report that a single intravenous injection of IONPs can inhibit primary tumor growth, suppress metastases, and extend survival. Gene expression analysis revealed the activation of Toll-like receptor (TLR) pathways involving signaling via Toll/Interleukin-1 receptor domain-containing adaptor-inducing IFN-ß (TRIF), a TLR pathway adaptor protein. Requisite participation of TRIF in suppressing tumor progression was demonstrated with histopathologic evidence of upregulated IFN-regulatory factor 3 (IRF3), a downstream protein, and confirmed in a TRIF knockout syngeneic mouse model of metastatic breast cancer. Neither starch-coated polystyrene nanoparticles lacking iron, nor iron-containing dextran-coated parenteral iron replacement agent, induced significant antitumor effects, suggesting a dependence on the type of IONP formulation. Analysis of multiple independent clinical databases supports a hypothesis that upregulation of TLR3 and IRF3 correlates with increased overall survival among breast cancer patients. Taken together, these data support a compelling rationale to re-examine IONP formulations as harboring anticancer immune (nano)adjuvant properties to generate a therapeutic benefit without requiring uptake by cancer cells.

Jiedu Xiaozheng Yin Inhibits the Progression of Colitis Associated Colorectal Cancer by Stimulating Macrophage Polarization Towards an M1 Phenotype via the TLR4 Pathway.

To investigate the effect of Jiedu Xiaozheng Yin (JXY) on the polarization of macrophages in colitis-associated colon cancer (CAC). An orthotopic model of CAC was established to monitor changes in the pathological state of mice. Colon length, number of colon tumors were recorded, and indices for liver, spleen, and thymus were calculated. Hematoxylin and eosin (H&E) staining was employed to observe intestinal mucosal injury and tumor formation. Immunohistochemistry (IHC) staining was utilized to investigate the effect of JXY on M1 and M2 polarization of macrophages in the colonic mucosa of CAC mice. For in vitro experiments, RT-qPCR (Reverse Transcription-quantitative PCR) and flow cytometry were used to observe the effect of JXY on various M1-related molecules such as IL-1ß, TNF-α, iNOS, CD80, CD86, and its phagocytic function as well as M2-related molecules including Arg-1, CD206, and IL-10. Subsequently, after antagonizing the TLR4 pathway with antagonists (TAK242, PDTC, KG501, SR11302, LY294002), the expression of IL-6, TNF-α, iNOS, and IL-1ß mRNA were detected by RT-qPCR. In vivo experiments, the results showed that JXY improved the pathological condition of mice in general. And JXY treatment decreased the shortening of colon length and number of tumors as compared to non-treated CAC mice. Additionally, JXY treatment improved the lesions in the colonic tissue and induced a polarization of intestinal mucosal macrophages towards the M1 phenotype, while inhibiting polarization towards the M2 phenotype. In vitro experiments further confirmed that JXY treatment promoted the activation of macrophages towards the M1 phenotype, leading to increased expression of IL-1ß, TNF-α, iNOS, CD80, CD86, as well as enhanced phagocytic function. JXY treatment concomitantly inhibited the expression of M2-phenotype related molecules Arginase-1 (Arg-1), CD206, and IL-10. Furthermore, JXY inhibited M1-related molecules such as IL-6, TNF-α, iNOS, and IL-1ß after antagonizing the TLR4 pathway. Obviously, JXY could exhibit inhibitory effects on the development of colon tumors in mice with CAC by promoting M1 polarization through TLR4-mediated signaling and impeding M2 polarization of macrophages.

Toll-like receptor activation regulates the paracrine effect of adipose-derived mesenchymal stem cells on reversing osteoarthritic phenotype of chondrocytes.

BACKGROUND: The therapeutic efficacy of intra-articular mesenchymal stem cells (MSCs) injection for patients with osteoarthritis (OA) currently exhibits inconsistency, and the underlying mechanism remains elusive. It has been postulated that the immunomodulatory properties and paracrine activity of MSCs might be influenced by the inflammatory micro-environment within osteoarthritic joints, potentially contributing to this observed inconsistency. METHODS: Adipose-derived MSCs (ADSCs) were isolated from SD rats and pre-treated with Toll-like receptor 3 (TLR3) agonist Poly I:C or Toll-like receptor 4 (TLR4) agonist LPS. The pre-treated ADSCs were then co-cultured with IL-1ß-induced osteoarthritic chondrocytes using a Transwell system to analyze the paracrine effect of ADSCs on reversing the osteoarthritic phenotype of chondrocytes. RESULTS: RT-PCR and Western blot analysis revealed that Poly I:C and LPS pre-treatments up-regulated the expression of IL-10 and IL-6 in ADSCs, respectively. Furthermore, only Poly I:C-preconditioned ADSCs significantly promoted proliferation while inhibiting apoptosis in IL-1ß-treated chondrocytes. Additionally, Poly I:C-preconditioned ADSCs downregulated MMP13 expression while upregulating aggrecan and collagen II expression levels in IL-1ß-treated chondrocytes. CONCLUSIONS: TLR3 activation polarizes ADSCs into an immunomodulatory phenotype distinct from TLR4 activation, exerting differential effects on reversing the osteoarthritic phenotype of chondrocytes; thus indicating that MSCs' paracrine effect regulated by TLRs signaling impacts the efficacy of intra-articular MSCs injection.

Extracellular mixed histones are neurotoxic and modulate select neuroimmune responses of glial cells.

Although histone proteins are widely known for their intranuclear functions where they organize DNA, all five histone types can also be released into the extracellular space from damaged cells. Extracellular histones can interact with pattern recognition receptors of peripheral immune cells, including toll-like receptor 4 (TLR4), causing pro-inflammatory activation, which indicates they may act as damage-associated molecular patterns (DAMPs) in peripheral tissues. Very limited information is available about functions of extracellular histones in the central nervous system (CNS). To address this knowledge gap, we applied mixed histones (MH) to cultured cells modeling neurons, microglia, and astrocytes. Microglia are the professional CNS immunocytes, while astrocytes are the main support cells for neurons. Both these cell types are critical for neuroimmune responses and their dysregulated activity contributes to neurodegenerative diseases. We measured effects of extracellular MH on cell viability and select neuroimmune functions of microglia and astrocytes. MH were toxic to cultured primary murine neurons and also reduced viability of NSC-34 murine and SH-SY5Y human neuron-like cells in TLR4-dependent manner. MH did not affect the viability of resting or immune-stimulated BV-2 murine microglia or U118 MG human astrocytic cells. When applied to BV-2 cells, MH enhanced secretion of the potential neurotoxin glutamate, but did not modulate the release of nitric oxide (NO), tumor necrosis factor-α (TNF), C-X-C motif chemokine ligand 10 (CXCL10), or the overall cytotoxicity of lipopolysaccharide (LPS)- and/or interferon (IFN)-γ-stimulated BV-2 microglial cells towards NSC-34 neuron-like cells. We demonstrated, for the first time, that MH downregulated phagocytic activity of LPS-stimulated BV-2 microglia. However, MH also exhibited protective effect by ameliorating the cytotoxicity of LPS-stimulated U118 MG astrocytic cells towards SH-SY5Y neuron-like cells. Our data demonstrate extracellular MH could both damage neurons and alter neuroimmune functions of glial cells. These actions of MH could be targeted for treatment of neurodegenerative diseases.

[Mechanism of Fushen Granules treat peritoneal dialysis-related peritonitis by regulating TLR4/NF-κB pathway:based on network pharmacology and animal experiments].

Network pharmacology was employed to probe into the mechanism of Fushen Granules in treating peritoneal dialysis-rela-ted peritonitis(PDRP) in rats. The main active components of Fushen Granules were searched against the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, and their targets were predicted. PDRP-related targets were retrieved from DisGeNET and other databases. The common targets shared by the drug and the disease were identified by the online tool, and protein-protein interaction(PPI) network of the common targets. The obtained 276 common targets were imported into DAVID for GO function enrichment and KEGG pathway enrichment. The main signaling pathway of Fushen Granules in the treatment of PDRP was predicted as Toll-like receptor 4(TLR4)/nuclear factor(NF)-κB. The rat model of uremia was induced by 5/6 nephrectomy. From two weeks after operation, the rat model of peritoneal dialysis(PD) was established by intraperitoneal injection of 20 mL dialysate with 1.25% glucose every day. The sham operation group and model group received 2 mL normal saline by gavage every day. The rats in Fushen Gra-nules groups were administrated with 2 mL solutions of low-(0.54 g·kg~(-1)), medium-(1.08 g·kg~(-1)) and high-dose(2.16 g·kg~(-1)) Fushen Granules every day. The bifico group received 2 mL(113.4 mg·kg~(-1)) of bifico solution every day. At the end of the 8th week, the levels of serum creatinine(Scr) and blood urea nitrogen(BUN) in each group were measured. The serum levels of hypersensitive C reactive protein(hs-CRP), tumor necrosis factor(TNF)-α, and interleukin(IL)-6 were measured, and the pathological changes in the colon tissue were observed by hematoxylin-eosin(HE) staining. The serum levels of lipopolysaccharide(LPS) and lipopolysaccharide-binding protein(LBP) of rats were measured, and the expression levels of LBP, TLR4, NF-κB p65, inhibitor of κB kinase α(IκBα), TNF-α, and IL-1ß in the colon tissue were determined. Compared with sham operation group, the model group had abnormal structure of all layers of colon tissue, sparse and shorter intestinal villi, visible edema in mucosal layer, wider gap, obvious local inflammatory cell infiltration, significantly decreased body weight(P<0.01), and significantly increased kidney function index(Scr, BUN) content(P<0.01). Serum levels of inflammatory cytokines(hs-CRP, TNF-α, IL-6), LPS and LBP were significantly increased(P<0.01), protein expressions of LBP, TLR4, NF-κB p65, TNF-α and IL-1ß were significantly increased(P<0.01), and protein expressions of IκBα were significantly decreased(P<0.01). Compared with model group, intestinal villi damage in colonic tissue of rats in low-, medium-and high-dose Fushen Granules groups and bifico group were alleviated to different degrees, edema in submucosa was alleviated, space was narrowed, and inflammatory cell infiltration in lamina propria was reduced. The contents of renal function index(Scr, BUN) and serum inflammatory factors(hs-CRP, TNF-α, IL-6) were significantly decreased(P<0.05 or P<0.01) in medium-and high-dose Fushen Granules groups and bifico group(P<0.05 or P<0.01). Serum LPS and LBP contents in Fushen Granules group and bifico group were significantly decreased(P<0.01), protein expressions of LBP, TLR4, NF-κB p65, TNF-α and IL-1ß in Fushen Granules group were significantly decreased(P<0.05 or P<0.01), and protein expressions of IκBα were significantly increased(P<0.01). The expression of LBP protein in bifico group was significantly decreased(P<0.01). The results suggest that Fushen Granules can protect the residual renal function of PD rats, reduce the inflammatory response, and protect the colon tissue. Based on network pharmacology, TLR4/NF-κB pathway may be the main signaling pathway of Fushen granule in the treatment of PDRP. The results showed that Fushen Granules could improve intestinal inflammation and protect intestinal barrier to prevent PDRP by regulating the expression of key factors in TLR4/NF-κB pathway in colon of PD rats.

[Mechanism of Shenling Baizhu Powder on treatment of alcoholic liver disease by regulating TLR4/NLRP3 pathway].

This study aims to investigate the regulatory effects of Shenling Baizhu Powder(SBP) on cellular autophagy in alcoholic liver disease(ALD) and its intervention effect through the TLR4/NLRP3 pathway. A rat model of chronic ALD was established by gavage of spirits. An ALD cell model was established by stimulating BRL3A cells with alcohol. High-performance liquid chromatography(HPLC) was utilized for the compositional analysis of SBP. Liver tissue from ALD rats underwent hematoxylin-eosin(HE) and oil red O staining for pathological evaluation. Enzyme-linked immunosorbent assay(ELISA) was applied to quantify lipopolysaccharides(LPS), tumor necrosis factor-alpha(TNF-α), interleukin-1 beta(IL-1ß), and interleukin-18(IL-18) levels. Quantitative reverse transcription polymerase chain reaction(qRT-PCR) was conducted to evaluate the mRNA expression of myeloid differentiation factor 88(MyD88) and Toll-like receptor 4(TLR4). The effect of different drugs on BRL3A cell proliferation activity was assessed through CCK-8 analysis. Western blot analysis was performed to examine the protein expression of NOD-like receptor pyrin domain-containing 3(NLRP3), nuclear factor-kappa B P65(NF-κB P65), phosphorylated nuclear factor-kappa B P65(p-P65), caspase-1, P62, Beclin1, and microtubule-associated protein 1 light chain 3(LC3Ⅱ). The results showed that SBP effectively ameliorated hepatic lipid accumulation, reduced liver function, mitigated hepatic tissue inflammation, and reduced levels of LPS, TNF-α, IL-1ß, and IL-18. Moreover, SBP exhibited the capacity to modulate hepatic autophagy induced by prolonged alcohol intake through the TLR4/NLRP3 signaling pathway. This modulation resulted in decreased expression of LC3Ⅱ and Beclin1, an elevation in P62 expression, and the promotion of autolysosome formation. These research findings imply that SBP can substantially enhance liver function and mitigate lipid irregularities in the context of chronic ALD. It achieves this by regulating excessive autophagic responses caused by prolonged spirit consumption, primarily through the inhibition of the TLR4/NLRP3 pathway.

Dishevelled Segment Polarity Protein 3 (DVL3) Induced by Bacterial LPS Promotes the Proliferation and Migration of Prostate Cancer Cells through the TLR4 Pathway.

BACKGROUND: Chronic inflammation is associated with various malignant tumors. Bacterial lipopolysaccharides (LPSs) play a significant part in the event and development of prostate cancer. Dishevelled segment polarity protein 3 (DVL3) is a shared component of the Wnt/ß-catenin and Notch signaling pathways, which are involved in tumor progression, chemoresistance, and maintenance of stem cell-like properties. According to reports, prostatic cancer cell invasion and proliferation are mediated by toll-like receptor 4 (TLR4). However, the role and regulation of DVL3 in prostate cancer and its relationship with TLR4 remain unclear. METHODS: Survival curves were plotted to evaluate the relationship between DVL3 expression and prognosis in patients with prostate cancer. DVL3 was silenced in PC3 and DU145 cells using small interfering RNAs (siRNAs). Subsequently, cell counting kit-8 (CCK-8) assay, colony formation assay, transwell migration assay, and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) were performed to investigate the role of DVL3 in cell proliferation and migration in vitro. The protein markers of potential pathways were analyzed via western blotting. RESULTS: DVL3 expression was linked to prognosis in patients with prostate cancer; In particular, patients with high DVL3 expression had a poor prognosis. LPS stimulation increased (p < 0.01) the expression of DVL3 in PC3 cells. DVL3 regulated tumor cell proliferation and migration by mediating the increase (p < 0.01) in TLR4 expression. Knockout of TLR4 validated that TLR4 played a crucial role in LPS-induced DVL3 expression. Silencing of DVL3 decreased (p < 0.01) the LPS-induced proliferation and migration of PC3 cells. CONCLUSIONS: Bacterial LPS-induced DVL3 promoted the multiplication and migration of prostate cancer cells through the TLR4 pathway. This study offers a valuable reference for the development and clinical application of targeted drugs for prostate cancer.

Exosomal miR-122, miR-128, miR-200, miR-298, and miR-342 as novel diagnostic biomarkers in NAFL/NASH: Impact of LPS/TLR-4/FoxO3 pathway.

Nonalcoholic fatty liver disease (NAFLD) is a common liver disorder affecting a quarter of the global residents. Progression of NAFL into nonalcoholic steatohepatitis (NASH) may cause cirrhosis, liver cancer, and failure. Gut microbiota imbalance causes microbial components translocation into the circulation, triggering liver inflammation and NASH-related fibrosis. MicroRNAs (miRNAs) regulate gene expression via repressing target genes. Exosomal miRNAs are diagnostic and prognostic biomarkers for NAFL and NASH liver damage. Our work investigated the role of the gut microbiota in NAFLD pathogenesis via the lipopolysaccharide/toll-like receptor 4/Forkhead box protein O3 (LPS/TLR-4/FoxO3) pathway and certain miRNAs as noninvasive biomarkers for NAFL or its development to NASH. miRNA expression levels were measured using quantitative reverse transcription polymerase chain reaction (qRT-PCR) in 50 NAFL patients, 50 NASH patients, and 50 normal controls. Plasma LPS, TLR-4, adiponectin, peroxisome proliferator-activated receptor γ (PPAR-γ), and FoxO3 concentrations were measured using enzyme-linked immunosorbent assay (ELISA). In NAFL and NASH patients, miR-122, miR-128, FoxO3, TLR-4, LPS, and PPAR-γ were upregulated while miR-200, miR-298, miR-342, and adiponectin were downregulated compared with the normal control. The examined miRNAs might distinguish NAFL and NASH patients from the normal control using receiver operating characteristic analysis. Our study is the first to examine these miRNAs in NAFLD. Our findings imply that these are potentially promising biomarkers for noninvasive early NAFL diagnosis and NASH progression. Understanding the LPS/TLR-4/FoxO3 pathway involvement in NAFL/NASH pathogenesis may aid disease management.

TAK-242 improves sepsis-associated acute kidney injury in rats by inhibiting the TLR4/NF-κB signaling pathway.

OBJECTIVE: This study was designed to observe the effect of toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB) pathway activity on sepsis-associated acute kidney injury (SA-AKI), thereby providing new considerations for the prevention and treatment of SA-AKI. METHODS: The rats were divided into Sham, cecal ligation and puncture (CLP), CLP + vehicle, and CLP + TAK-242 groups. Except the Sham group, a model of CLP-induced sepsis was established in other groups. After 24 h, the indicators related to kidney injury in blood samples were detected. The pathological changes in the kidneys were observed by hematoxylin-eosin staining, and tubular damage was scored. Oxidative stress-related factors, mitochondrial dysfunction-related indicators in each group were measured; the levels of inflammatory factors in serum and kidney tissue of rats were examined. Finally, the expression of proteins related to the TLR4/NF-κB signaling pathway was observed by western blot. RESULTS: Compared with the CLP + vehicle and CLP + TAK-242 groups, the CLP + TAK-242 group reduced blood urea nitrogen (BUN), creatinine (Cr), cystatin-C (Cys-C), reactive oxygen species (ROS), malondialdehyde (MDA), and inflammatory factors levels (p < 0.01), as well as increased superoxide dismutase (SOD) activity of CLP rats (p < 0.01). Additionally, TAK-242 treatment improved the condition of CLP rats that had glomerular and tubular injuries and mitochondrial disorders (p < 0.01). Further mechanism research revealed that TAK-242 can inhibit the TLR4/NF-κB signaling pathway activated by CLP (p < 0.01). Above indicators after TAK-242 treatment were close to those of the Sham group. CONCLUSION: TAK-242 can improve oxidative stress, mitochondrial dysfunction, and inflammatory response by inhibiting the activity of TLR4/NF-κB signaling pathway, thereby preventing rats from SA-AKI.

Advances in molecular agents targeting toll-like receptor 4 signaling pathways for potential treatment of sepsis.

Sepsis is a systemic inflammatory response syndrome caused by an infection. Toll-like receptor 4 (TLR4) is activated by endogenous molecules released by injured or necrotic tissues. Additionally, TLR4 is remarkably sensitive to infection of various bacteria and can rapidly stimulate host defense responses. The TLR4 signaling pathway plays an important role in sepsis by activating the inflammatory response. Accordingly, as part of efforts to improve the inflammatory response and survival rate of patients with sepsis, several drugs have been developed to regulate the inflammatory signaling pathways mediated by TLR4. Inhibition of TLR4 signal transduction can be directed toward either TLR4 directly or other proteins in the TLR4 signaling pathway. Here, we review the advances in the development of small-molecule agents and peptides targeting regulation of the TLR4 signaling pathway, which are characterized according to their structural characteristics as polyphenols, terpenoids, steroids, antibiotics, anthraquinones, inorganic compounds, and others. Therefore, regulating the expression of the TLR4 signaling pathway and modulating its effects has broad prospects as a target for the treatment of lung, liver, kidneys, and other important organs injury in sepsis.

CM1, a Chrysin Derivative, Protects from Endotoxin-Induced Lethal Shock by Regulating the Excessive Activation of Inflammatory Responses.

Sepsis, a leading cause of death worldwide, is a harmful inflammatory condition that is primarily caused by an endotoxin released by Gram-negative bacteria. Effective targeted therapeutic strategies for sepsis are lacking. In this study, using an in vitro and in vivo mouse model, we demonstrated that CM1, a derivative of the natural polyphenol chrysin, exerts an anti-inflammatory effect by inducing the expression of the ubiquitin-editing protein TNFAIP3 and the NAD-dependent deacetylase sirtuin 1 (SIRT1). Interestingly, CM1 attenuated the Toll-like receptor 4 (TLR4)-induced production of inflammatory cytokines by inhibiting the extracellular-signal-regulated kinase (ERK)/MAPK and nuclear factor kappa B (NF-κB) signalling pathways. In addition, CM1 induced the expression of TNFAIP3 and SIRT1 on TLR4-stimulated primary macrophages; however, the anti-inflammatory effect of CM1 was abolished by the siRNA-mediated silencing of TNFAPI3 or by the genetic or pharmacologic inhibition of SIRT1. Importantly, intravenous administration of CM1 resulted in decreased susceptibility to endotoxin-induced sepsis, thereby attenuating the production of pro-inflammatory cytokines and neutrophil infiltration into the lung compared to control mice. Collectively, these findings demonstrate that CM1 has therapeutic potential for diverse inflammatory diseases, including sepsis.

Effects of chronic Cr and Ni co-exposure on liver inflammation and autophagy in mice by regulating the TLR4/mTOR pathway.

Exposure to Cr and/or Ni can have widespread implications on the environment and health. However, the specific toxic effects of chronic Cr and Ni co-exposure on mice liver have not been reported. To ascertain the combined toxic effects of chronic Cr and Ni co-exposure on liver damage in mice, 80 6-week-old female C57BL/6 J mice were randomly divided into 4 groups: the Con group, Cr group (Cr+6 50 mg/L), Ni group (Ni+2 110 mg/L), and Cr + Ni group (Cr+6 50 mg/L + Ni+2 110 mg/L). The trial period lasted for 16 weeks. The results showed that Cr+6 and/or Ni+2 increased liver weight and liver index (P < 0.05) in mice, caused histological abnormality and ultrastructural damage, and micronutrients imbalance in mice liver. These findings serve as the basis for subsequent experiments. Compared with the individual exposure group, chronic Cr and Ni co-exposure resulted in decreased levels and activities of ALT, AST, MDA, T-AOC, and T-SOD (P < 0.05) in liver tissue, and decreased the mRNA expression levels of the TLR4/mTOR pathway related factors (TLR4, TRAM, TRIF, TBK-1, IRF-3, MyD88, IRAK-4, TRAF6, TAK-1, IKKß, NF-κB, IL-1ß, IL-6, TNFα, ULK1, Beclin 1, LC3) (P < 0.05) and decreased the protein expression levels of the factors (TLR4, MyD88, TRAF6, NF-κB p50, IL-6, TNFα, ULK1, LC3II/LC3I) (P < 0.05). Moreover, factorial analysis revealed the interaction between Cr and Ni, which was manifested as antagonistic effects on Cr concentration, Ni concentration, and TLR4, MyD88, NF-κB, mTOR, LC3, and p62 mRNA expression levels. In conclusion, the TLR4/mTOR pathway as a mechanism through which chronic Cr and Ni co-exposure induce liver inflammation and autophagy in mice, and there was an antagonistic effect between Cr and Ni. The above results provided a theoretical basis for understanding the underlying processes.