Toll-like Receptor 4 mRNA Levels Research Articles

Signals from intestinal microbiota mediate the crosstalk between the lung-gut axis in an influenza infection mouse model

IntroductionIntroduction: The influenza virus primarily targets the respiratory tract, yet both the respiratory and intestinal systems suffer damage during infection. The connection between lung and intestinal damage remains unclear.MethodsOur experiment employs 16S rRNA technology and Liquid Chromatography-Mass Spectrometry (LC-MS) to detect the impact of influenza virus infection on the fecal content and metabolites in mice. Additionally, it investigates the effect of influenza virus infection on intestinal damage and its underlying mechanisms through HE staining, Western blot, Q-PCR, and flow cytometry.ResultsOur study found that influenza virus infection caused significant damage to both the lungs and intestines, with the virus detected exclusively in the lungs. Antibiotic treatment worsened the severity of lung and intestinal damage. Moreover, mRNA levels of Toll-like receptor 7 (TLR7) and Interferon-b (IFN-b) significantly increased in the lungs post-infection. Analysis of intestinal microbiota revealed notable shifts in composition after influenza infection, including increased Enterobacteriaceae and decreased Lactobacillaceae. Conversely, antibiotic treatment reduced microbial diversity, notably affecting Firmicutes, Proteobacteria, and Bacteroidetes. Metabolomics showed altered amino acid metabolism pathways due to influenza infection and antibiotics. Abnormal expression of indoleamine 2,3-dioxygenase 1 (IDO1) in the colon disrupted the balance between helper T17 cells (Th17) and regulatory T cells (Treg cells) in the intestine. Mice infected with the influenza virus and supplemented with tryptophan and Lactobacillus showed reduced lung and intestinal damage, decreased Enterobacteriaceae levels in the intestine, and decreased IDO1 activity.DiscussionOverall, influenza infection caused damage to lung and intestinal tissues, disrupted intestinal microbiota and metabolites, and affected Th17/Treg balance. Antibiotic treatment exacerbated these effects. Supplementation with tryptophan and Lactobacillus improved lung and intestinal health, highlighting a new understanding of the lung-intestine connection in influenza-induced intestinal disease.

The effects of Lactobacillus plantarum and oxolinic acid on growth, immune-related genes, and gut bacterial community structures in juvenile Japanese eel (Anguilla Japonica)

This research aims to understand the distinct effects of Lactobacillus plantarum and Oxolinic Acid on the growth, immune-related genes, and gut bacterial community structures of juvenile Japanese eel.In this study, the Lactobacillus plantarum strain MH079448 was added to the diet for Japanese eel juveniles as test group (LP), with no bacteria administrated as control group (CK). Meanwhile, the antibiotic Oxolinic Acid immersion treatment was administrated in an extra group as a comparison for alternative evaluation (OA). The mRNA levels of toll-like receptors 2 (TLR-2) and tumor necrosis factor-alpha (TNF-α) in livers and gills of Japanese eel were analyzed by qRT-PCR technology. The intestinal microbiota was analyzed by High-throughput Sequencing (HTS) analysis.The results showed that at the end of experiment, the specific growth rates (SGR) and weight-gain rate (WGR) of Japanese eels in both LP and OA were improved to an approximately equal level, viz., 38.38 ± 15.2%, 1.18 ± 0.39%/d and 43.67 ± 16.52%, 1.29 ± 0.39%/d, respectively, which were significantly higher than the CK (8.73 ± 4.81%, 0.3 ± 0.16%/d) (p < 0.05). At the beginning of the experiment, there were no significant differences in the expression levels of TLR-2 and TNF-α in the gill and liver (p > 0.05). However, on day 28, compared with CK, both LP and OA showed significant upregulation in the expression levels of TLR-2 and TNF-α in the gill. Specifically, there was no significant difference in the expression levels of TLR-2 between LP and OA in the gill (5.97 ± 0.33 and 5.73 ± 0.38 respectively) (p > 0.05), while TNF-α exhibited significant differences in the gill (3.40 ± 0.24 and 4.96 ± 0.17 respectively) (p < 0.05). In the liver, on day 28, LP showed significant upregulation in the expression levels of TLR-2 and TNF-α (0.079 ± 0.006 and 0.116 ± 0.018, respectively) (p < 0.05). Concerning the TLR-2 in the liver of OA, it showed significant upregulation to 0.132 ± 0.009 (p < 0.05), which was significantly higher than that of LP (0.079 ± 0.006) in the liver(p < 0.05). However, on day 28, compared with CK and LP (0.049 ± 0.04 and 0.116 ± 0.018, respectively), the expression level of TNF-α in OA of liver was significantly downregulated (0.039 ± 0.002) (p < 0.05).Further, at the end of the experiment, Gammaproteobacteria was still the dominant class in CK and OA with the relative abundance of 95.09% and 92.49%, correspondingly, whereas in the LP, its value dropped to 82.79%. Compared to CK, the relative abundance of total probiotics in LP were increased by 1.619%, but it decreased by 0.464% in OA. Their total (opportunistic) pathogens in LP and OA were decreased by 4.562% and 16.803%, respectively. Functionally, compared to CK, the predicted functions of intestinal microbiota, such as energy metabolism, nutrient absorption and secondary metabolites were significantly enhanced in both LP and OA(p < 0.05). The results of this study firstly demonstrated that compared to Oxolinic Acid, Lactobacillus plantarum MH079448 approximately equally improved the growth performance, and approximately equally enhanced immune response. However, concerning the optimized community structures of intestinal microbiota in juvenile Japanese eels, Lactobacillus plantarum MH079448 was better than Oxolinic Acid, especially in relative abundance of total probiotics improvement.

Study of gene polymorphisms in Toll-like receptor 2 in patients with acute lymphoblastic leukemia

ObjectivesAcute Lymphoblastic Leukemia (ALL) is a multifactorial disease that results from the interaction between multiple genetic factors. ALL is characterized by uncontrolled production of hematopoietic precursor cells of the lymphoid progenitors within the bone marrow. The development of hematological malignancies has been associated with malignant-like cells that express low levels of immunogenic surface molecules, thus, facilitating their escape from cellular antineoplastic immune responses. This risk may be partly influenced by variations in polymorphic genes that control immune function and regulation. Toll-like receptors (TLRs) are well known pattern recognition receptors playing key role in innate immune response. Abnormal expression and dysregulation of TLRs will provide an opportunity for cancer cells to escape from the immune system and enhance their proliferation and angiogenesis. Toll-like receptor 2 (TLR2) play an essential role in innate immunity. Single nucleotide polymorphisms (SNPs) are present in a number of TLR genes and have been associated with various disorders. MethodsIn this study, 265 subjects have been divided into two groups included 150 patients with ALL and115 healthy volunteers. All subjects were genotyped using TaqMan PCR techniques. In total, Five SNPs were statistically evaluated in the TLR2 (rs1898830 A/G, rs3804099 T/C, rs3804100 T/C, rs1339 T/C, and rs1337 C/G), which may influence the susceptibility of ALL. Minor allele frequency and genotype distribution were compared across the study groups, and the relative risk and differences between patients and controls were estimated. Moreover, the mRNA expression level was evaluated in patients with ALL and the matched healthy individuals by Real-Time Quantitative Reverse Transcription PCR (qRT-PCR). ResultsTLR2 rs1898830 A/G; rs3804099 T/C; rs3804100 T/C; rs1339 T/C, were significantly decrease the risk in our population, overall and for certain subtypes and ALL samples exhibited significant increase in the mRNA levels of TLR2. ConclusionsThis study shows that TLR2 could be an independent prognostic factor of ALL risks in the Saudi population. Suggesting that genetic variation in genes associated with an immune response may be important in the etiology of ALL. In addition, the results herein revealed that TLR2 overexpression is associated with ALL and has diverse biological significance in the context of the complex relationship between inflammation and cancer development. Therefore, these data could open further studies to explore the possible clinical relevance of TLRs as pathological markers for Leukemia and enhance the strategies regarding hematological malignancies prevention based on their gene expression.

Role of OPG/RANKL/RANK/TLR4 signaling pathway in sepsis-associated acute kidney injury

BackgroundSepsis-associated acute kidney injury (SA-AKI) has high mortality rates. The osteoprotegerin (OPG)/receptor activator of nuclear factor-κB ligand (RANKL)/receptor activator of nuclear factor-κB (RANK)/Toll-like receptor 4 (TLR4) pathway and its potential role in SA-AKI pathogenesis remain to be fully understood. Herein, we addressed this issue using mouse models.MethodsAn SA-AKI mouse model was established using the cecal ligation and puncture method (CLP). Mice were grouped into sham, CLP model, CLP + recombinant RANKL, and CLP + anti-RANKL groups. Serum creatinine (Scr) and blood urea nitrogen (BUN) levels were measured to assess kidney function. ELISA was used to detect serum IL-1β, TNF-α, and IL-6 levels. Real-time quantitative PCR and Western blot were used to detect the mRNA and protein expression levels of OPG, RANKL, RANK, and TLR4 in kidney tissues. HE staining was performed to evaluate the pathological changes.ResultsThe CLP model group showed higher levels of Scr and BUN, indicating impaired kidney function in SA-AKI, compared to the sham group. Treatment with recombinant RANKL in the CLP + recombinant RANKL group reduced Scr and BUN levels, while anti-RANKL treatment in the CLP + anti-RANKL group elevated their levels. Moreover, the CLP model group had significantly increased IL-1β, TNF-α, and IL-6 than the sham group, indicating elevated inflammation in SA-AKI. The CLP + recombinant RANKL group demonstrated decreased cytokine levels, whereas the CLP + anti-RANKL group showed an increase. Additionally, the histopathological evaluation revealed distinct kidney tissue damage in the CLP model group. Recombinant RANKL treatment reduced this damage, while anti-RANKL treatment exacerbated it. Mechanically, the mRNA and protein expression of RANKL were significantly decreased, while those of OPG, RANK, and TLR4 were significantly increased in the CLP model group and the CLP + anti-RANKL group. Interestingly, treatment with recombinant RANKL reversed these changes, as evidenced by significantly increased RANKL but decreased OPG, RANK, and TLR4.ConclusionThe OPG/RANKL/RANK/TLR4 pathway is involved in SA-AKI pathogenesis. Recombinant RANKL treatment attenuates the inflammatory response and kidney tissue damage in SA-AKI, possibly via regulating this pathway. This pathway shows promise as a therapeutic target for SA-AKI.

Growth inhibition of subcutaneous tumor with glioma cells in nude mice by silencing TLR4.

This study investigated the regulatory impact of Toll-like receptor 4 (TLR4) gene on glioma cell proliferation and apoptosis, elucidating the molecular mechanisms underlying TLR4-induced growth inhibition in vivo. U-87MG-Sh and U-87MG-NC cells, with silenced TLR4 and negative control plasmid respectively, were established. Eighteen nude mice, divided into transfection, negative control, and blank control groups, were inoculated with corresponding cells. Over four weeks, the transfection group exhibited significantly reduced tumor growth rates, smaller mass and volume, and lower growth activity compared to controls. Histological analysis revealed sparse tumor cells, increased fibrous connective tissue, and slower angiogenesis in the transfection group. Flow cytometry demonstrated a lower proliferation index and increased G0/1 cell count in the transfection group. mRNA levels of TLR4, NF-κB, and CyclinD1 were significantly lower in the transfection group. TLR4 silencing correlated with U-87MG cell proliferation regulation, growth inhibition, NF-κB and CyclinD1 modulation, and induction of cell cycle arrest and apoptosis. These findings suggest TLR4 as a potential gene therapy target for glioma.

Protective effect of modified Huangqi Chifeng decoction on immunoglobulin A nephropathy through toll-like receptor 4/myeloid differentiation factor 88/nuclear factor-kappa B signaling pathway.

To examine the nephroprotective mechanism of modified Huangqi Chifeng decoction (, MHCD) in immunoglobulin A nephropathy (IgAN) rats. To establish the IgAN rat model, the bovine serum albumin, lipopolysaccharide, and carbon tetrachloride 4 method was employed. The rats were then randomly assigned to the control, model, telmisartan, and high-, medium-, and low-dose MHCD groups, and were administered the respective treatments via intragastric administration for 8 weeks. The levels of 24-h urinary protein, serum creatinine (CRE), and blood urea nitrogen (BUN) were measured in each group. Pathological alterations were detected. IgA deposition was visualized through the use of immunofluorescence staining. The ultrastructure of the kidney was observed using a transmission electron microscope. The expression levels of interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and transforming growth factor-β1 (TGF-β1) were examined by immunohistochemistry and quantitative polymerase chain reaction. Levels of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and nuclear factor-kappa B (NF-κB) P65, were examined by immunohistochemistry, Western blotting, and quantitative polymerase chain reaction. The 24-h urine protein level in each group increased significantly at week 6, and worsen from then on. But this process can be reversed by treatments of telmisartan, and high-, medium-, and low-dose of MHCD, and these treatments did not affect renal function. Telmisartan, and high-, and medium-dose of MHCD reduced IgA deposition. Renal histopathology demonstrated the protective effect of high-, medium-, and low-dose of MHCD against kidney injury. The expression levels of MCP-1, IL-6, and TGF-β1 in kidney tissues were downregulated by low, medium and high doses of MHCD treatment. Additionally, treatment of low, medium and high doses of MHCD decreased the protein and mRNA levels of TLR4, MyD88, and NF-κB. MHCD exerted nephroprotective effects on IgAN rats, and MHCD regulated the expressions of key targets in TLR4/MyD88/NF-κB signaling pathway, thereby alleviating renal inflammation by inhibiting MCP-1, IL-6 expressions, and ameliorating renal fibrosis by inhibiting TGF-β1 expression.

Correlations of expressions of Toll-like receptor 4 and Janus kinase 3 in peripheral blood mononuclear cells with T helper 17 cell/regulatory T cell imbalance in patients with ankylosing spondylitis

Abstract Background We aimed to analyze the correlations of the expressions of Toll-like receptor 4 (TLR4) and Janus kinase 3 (JAK3) in peripheral blood mononuclear cells (PBMCs) with T helper 17 cell (Th17)/regulatory T cell (Treg) imbalance in patients with ankylosing spondylitis (AS). Methods A total of 101 AS patients were enrolled into an AS group. Based on the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) score, they were subdivided into a stable AS (ASS) group (BASDAI score &lt;4 points, n=42) and an active AS (ASA) group (BASDAI score ≥4 points, n=59). Meanwhile, 50 healthy volunteers who received health examination in the same time period were assigned into a healthy control (HC) group. RT-qPCR was performed to determine the mRNA expressions of TLR4 and JAK3 in PBMCs. Enzyme-linked immunosorbent assay was conducted to measure the levels of Th17/Treg-related cytokines [interleukin (IL)-10, IL-17, IL-23, and transforming growth factor-β (TGF-β)] in the serum. Results The mRNA expressions of TLR4 and JAK3 had positive correlations with IL-17 (r=0.592, 0.542) and IL-23 levels (r=0.529, 0.590) (P 0.05). The levels of TLR4 mRNA, JAK3 mRNA, IL-17, and IL-23 were all correlated with the occurrence of AS (P&lt;0.05). The areas under the receiver operating characteristic curves of TLR4, JAK3, and their combination in PBMCs for predicting the occurrence of AS were 0.912, 0.835, and 0.919, respectively. Conclusions TLR4 combined with JAK3 can effectively predict the occurrence of AS.

Mechanism of aqueous extract of Strychni Semen in improving pain in rats with rheumatoid arthritis based on TLR4/TNF-α/MMP-9 signaling pathway

This study aims to explore the mechanism of aqueous extract of Strychni Semen(SA) in relieving pain in the rat model of rheumatoid arthritis(RA) via Toll-like receptor 4(TLR4)/tumor necrosis factor-α(TNF-α)/matrix metalloproteinase-9(MMP-9) signaling pathway. Firstly, the main chemical components of Strychni Semen were searched against TCMSP, TCMID, ETCM, and related literature, and the main targets of the chemical components were retrieved from TargetNet and SwissTargetPrediction. The main targets of RA and pain were searched against GeneCards, Online Mendelian Inheritance in Man(OMIM), and Therapeutic Target Database(TTD). Venny 2.1.0 was used to obtain the common targets shared by Strychni Semen, RA, and pain, and STRING and Cytoscape 3.6.1 were used to build the protein-protein interaction network. Then, molecular docking was carried out in AutoDock Vina. Finally, the rat model of type Ⅱ collagen-induced arthritis(CIA) was established. The up-down method and acetone method were employed to examine the mechanical pain threshold and cold pain threshold of rats, and the pain-relieving effect of SA on CIA rats was evaluated comprehensively. Hematoxylin-eosin(HE) staining was employed to evaluate the histopathological changes of joints in CIA rats. The expression levels of key target proteins was determined by immunohistochemistry and Western blot, and the mRNA levels of key targets were determined by real-time fluorescence quantitative polymerase chain reaction(real-time PCR). The results of network prediction showed that Strychni Semen may act on the TLR4/TNF-α/MMP-9 signaling pathway to exert the pain-relieving effect. The results of molecular docking showed that brucine, the main active component of SA, had strong binding ability to TLR4, TNF-α, and MMP-9. The results of animal experiments showed that SA improved the mechanical and cold pain sensitivity(P&lt;0.05, P&lt;0.01) and reduced the joint histopathological score of CIA rats(P&lt;0.01). In addition, medium and high doses of SA down-regulated the protein and mRNA levels of TNF-α, TLR4, and MMP-9(P&lt;0.05,P&lt;0.01). In conclusion, SA alleviated the mechanical pain sensitivity, cold pain sensitivity, and joint histopathological changes in CIA rats by inhibiting the over activation of TLR4/TNF-α/MMP-9 signaling pathway.

Three Artemisia pollens trigger the onset of allergic rhinitis via TLR4/MyD88 signaling pathway.

The prevalence of allergic rhinitis is high, making it a relatively common chronic condition. Countless patients suffer from seasonal Allergic rhinitis (AR). The objective of this investigation is to examine the potential involvement of common pollen allergens in seasonal allergic rhinitis, and study the proposed mechanism of Toll-like receptor 4 (TLR4)/Myeloid differentiation primary response gene 88 (MyD88) signaling pathway in the induction of AR. A mouse AR model (sensitized group) was constructed with pollen extracts and ovalbumin(OVA) of Artemisia annua (An), Artemisia argyi (Ar) and Artemisia Sieversiana (Si), and thereafter, AR symptom score was performed. After successful modeling, mouse serum and nasal mucosa tissues were extracted for subsequent experiments. The expression levels of immunoglobulin E (IgE), Interleukin (IL)-4, IL-5, IL-13 and Tumor Necrosis Factor-α (TNF-α) in serum were detected using Enzyme-linked immunosorbent assay (ELISA); Hematoxylin-eosin (H&E) staining methods were used to observe the pathological changes of the nasal mucosal tissue; Utilizing immunohistochemistry (IHC) staining, the expression levels of TLR4, MyD88 and Nuclear factor kappa B (NF-κB) p65 in mouse nasal mucosa were quantified; The mRNA and protein expression levels of TLR4, MyD88 and NF-κB p65 in nasal mucosa of sensitized mice were detected with Quantitative reverse transcription PCR (qRT-PCR) and Western Blot. Finally, the in vitro culture of Human nasal mucosal epithelial cells (HNEpC) cells was conducted, and cells were treated with 200µg/ml Artemisia annua pollen extractand OVA for 24h. Western Blot assay was used to detect the expression level of TLR4, MyD88 and NF-κB p65 proteins before and after HNEpC cells were treated with MyD88 inhibitor ST-2825. On the second day after AR stimulation, the mice showed obvious AR symptoms. H&E results showed that compared to the control group, the nasal mucosal tissue in the sensitized group was significantly more inflamed. Furthermore, ELISA assay showed increased expression levels of IgE, IL-4, IL-5, IL-13 and TNF-α in serum of mice induced by OVA and Artemisia annua pollen, Artemisia argyi pollen and Artemisia Sieversiana pollen than those of the control group. However, the expression level of IL-2 was lower than that of the control group (P < 0.05). Using Immunohistochemistry staining visually observed the expression levels of TLR4, MyD88 and NF-κB p65 in mouse nasal mucosa tissues and quantitatively analyzed. The expression levels of TLR4, MyD88 and NF-κB p65 in the sensitized group were higher than those in the control group, and the differences were statistically significant (P < 0.05). The results from qRT-PCR and Western Blot showed that the mRNA and protein expression levels of TLR4, MyD88 and NF-κB p65 in nasal mucosa of the sensitized group were significantly higher than those in the control group (P < 0.05). Finally, HNEpC cells were cultured in vitro and analyzed using Western Blot. The expression levels of TLR4, MyD88 and NF-κB p65 in OVA and An groups were significantly increased (P < 0.05). After ST-2825 treatment, TLR4 protein expression was significantly increased (P < 0.05) and MyD88 and NF-κB p65 protein expression were significantly decreased (P < 0.05). To sum up, the occurrence and development of AR induced by OVA and pollen of Artemisia annua, Artemisia argyi and Artemisia Sieversiana were related to TLR4/MyD88 signal pathway.

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&lt;0.01), lowered IL-10 level(P&lt;0.01), up-regulated protein levels of TLR4, MyD88, p-p65/NF-κB p65, and p-p38 MAPK/p38 MAPK(P&lt;0.01) and mRNA levels of TLR4 and MyD88(P&lt;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&lt;0.01), and elevated the IL-10 level(P&lt;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&lt;0.01) and the mRNA levels of TLR4 and MyD88 in the lung tissue(P&lt;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-1β-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-1β(IL-1β) 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-1β, 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-1β-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.

Mogroside Alleviates Diabetes Mellitus and Modulates Intestinal Microflora in Type 2 Diabetic Mice.

Mogroside, the main component of Siraitia grosvenorii (Swingle) C. Jeffrey (Cucurbitaceae) is a natural product with hypoglycemic and intestinal microbiota regulating properties. However, whether the alteration of intestinal microbiota is associated with the antidiabetic effect of mogroside remains poorly understood. This study investigated the mechanism underlying the hypoglycemic effect of mogroside in regulating intestinal flora and attenuating metabolic endotoxemia. Kunming mice with type 2 diabetes mellitus (T2DM) induced by high-fat diet and intraperitoneal injection of streptozotocin were randomly divided into model, pioglitazone (2.57 mg/kg) and mogroside (200, 100, and 50 mg/kg) groups. After 28 d of administration, molecular changes related to glucose metabolism and metabolic endotoxemia in mice were evaluated. The levels of insulin receptor substrate-1 (IRS-1), cluster of differentiation 14 (CD14) and toll-like receptor 4 (TLR4) mRNAs were measured, and the composition of intestinal microflora was determined by 16s ribosomal DNA (rDNA) sequencing. The results showed that mogroside treatment significantly improved hepatic glucose metabolism in T2DM mice. More importantly, mogroside treatment considerably reduced plasma endotoxin (inhibition rate 65.93%, high-dose group) and inflammatory factor levels, with a concomitant decrease in CD14 and TLR4 mRNA levels. Moreover, mogroside treatment reduced the relative abundance of Firmicutes and Proteobacteria (the inhibition rate of Proteobacteria was 85.17% in the low-dose group) and increased the relative abundance of Bacteroidetes (growth rate up to 40.57%, high-dose group) in the intestines of diabetic mice. This study reveals that mogroside can relieve T2DM, regulating intestinal flora and improving intestinal mucosal barrier, indicating that mogroside can be a potential therapeutic agent or intestinal microbiota regulator in the treatment of T2DM.

Anti-inflammatory effects of Mentha pulegium L. extract on human peripheral blood mononuclear cells are mediated by TLR-4 and NF-κB suppression

There is great interest in evaluating the anti-inflammatory properties of new herbal products. Thus, the effects of Mentha pulegium L. extract on gene and protein expressions of pro-inflammatory mediators and transcription factors were determined.The hydro-ethanolic extract of Mentha pulegium L. was obtained and optimal non-cytotoxic concentrations of the extract were determined by MTT assay. Then, three different concentrations of Mentha pulegium L. (10, 30, and 90 μg/mL) were used to pre-treat the lipopolysaccharide (LPS)-stimulated and non-stimulated peripheral blood mononuclear cells (PBMCs) of 10 healthy individuals. Finally, the tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, Toll-like receptor-4 (TLR-4), nuclear factor-kappa B (NF-κB) p65, activator protein-1 (AP-1), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) gene expressions and TNF-α, IL-1β, IL-6, TLR-4, prostaglandin E2 (PGE2), and COX-2 protein levels were measured.MTT results showed that there is no significant difference in cell viability among 10, 20, 40, and 80 μg/mL concentrations of Mentha pulegium L. extract at 24, 48, and 72 h (P > 0.05). The IC50 values were 236.1, 147.0, and 118.0 μg/mL after 24, 48, and 72 h respectively. TNF-α, IL-1β, IL-6, TLR-4, iNOS, and NF-κB p65 mRNA levels in the pre-treated LPS-stimulated PBMCs were concentration-dependently reduced (P < 0.01 for TNF-α, TLR-4, and NF-κB p65; P < 0.05 for IL-1β, IL-6, and iNOS). Also, the protein levels of pro-inflammatory mediators decreased and these differences were significant for TNF-α, IL-1β, and TLR-4 (P < 0.001, P < 0.01, and P < 0.001, respectively).Mentha pulegium L. extract decreased the expression and biosynthesis of pro-inflammatory mediators. These effects are mainly mediated by TLR-4 and NF-κB suppression. Thus, Mentha pulegium L. could be useful in treating or ameliorating chronic inflammatory diseases.

Toll-Like Receptor mRNA Levels in Schizophrenia: Association With Complement Factors and Cingulate Gyrus Cortical Thinning.

Previous studies revealed innate immune system activation in people with schizophrenia (SZ), potentially mediated by endogenous pathogen recognition receptors, notably Toll-like receptors (TLR). TLRs are activated by pathogenic molecules like bacterial lipopolysaccharides (TLR1 and TLR4), viral RNA (TLR3), or both (TLR8). Furthermore, the complement system, another key component of innate immunity, has previously been linked to SZ. Peripheral mRNA levels of TLR1, TLR3, TLR4, and TLR8 were compared between SZ and healthy controls (HC). We investigated their relationship with immune activation through complement expression and cortical thickness of the cingulate gyrus, a region susceptible to immunological hits. TLR mRNA levels and peripheral complement receptor mRNA were extracted from 86 SZ and 77 HC white blood cells; structural MRI scans were conducted on a subset. We found significantly higher TLR4 and TLR8 mRNA levels and lower TLR3 mRNA levels in SZ compared to HC. TLRs and complemental factors were significantly associated in SZ and HC, with the strongest deviations of TLR mRNA levels in the SZ subgroup having elevated complement expression. Cortical thickness of the cingulate gyrus was inversely associated with TLR8 mRNA levels in SZ, and with TLR4 and TLR8 levels in HC. The study underscores the role of innate immune activation in schizophrenia, indicating a coordinated immune response of TLRs and the complement system. Our results suggest there could be more bacterial influence (based on TLR 4 levels) as opposed to viral influence (based on TLR3 levels) in schizophrenia. Specific TLRs were associated with brain cortical thickness reductions of limbic brain structures.

Resveratrol alleviates heat-stress-induced impairment of the jejunal mucosa through TLR4/MAPK signaling pathway in black-boned chicken

Heat stress in chickens caused by high temperatures in summer is a serious issue faced by the poultry industry globally, which reduces product quality. The aim of this study is to investigate the role of resveratrol in alleviating heat stress injury and inflammatory response of jejunal mucosa in black-boned chickens through TLR4/MAPK signaling pathway. In total, 240 black-boned chickens (28-day old) were randomly divided into 4 treatment groups as follows. The normal temperature (NT) and normal temperature with resveratrol (NT+Res) groups received a basal diet without and with 400 mg/kg resveratrol, respectively, and treated at 24℃ ± 2℃, 24 h/d. The high temperature (HT) and high temperature with resveratrol (HT+Res) groups received basal diet without and with 400 mg/kg resveratrol, respectively, and treated at 37℃ ± 2℃ for 8 h/d and 24°C ± 2°C for the rest of the time for 12 d. The results revealed the heat-stress responses impaired the villous structure of the jejunum, causing a rough and uneven surface of the jejunal villus, and local intestinal villi were even more prone to rupture. However, resveratrol significantly improved the morphology and structure of jejunal mucosa under heat stress. Heat stress increased the mRNA levels of toll-like receptor 4 (TLR4), c-Jun, c-fos, caspase-3, and p38 (P < 0.05), reduced mRNA level of Bcl-2, and reduced the expression of tight junction proteins Occludin, ZO-1, and Claudin1 (P < 0.05) in the jejunal mucosa. However, resveratrol inhibited the TLR4/ mitogen-activated protein kinase (MAPK) signaling pathway via downregulating TLR4, c-Jun, p38, and caspase-3 (P < 0.05); upregulating Bcl-2 (P < 0.05); decreasing the protein levels of MKK3, p53, and myeloid differentiation factor 88 (MYD88); and increasing the protein levels of Occludin, ZO-1, and Claudin1. In addition, it reduced the levels of JNK and p38 proteins (P < 0.05) and inflammatory factors like tumor necrosis factor-α (TNF-α) in the jejunal mucosa of black-boned chickens under heat stress. In conclusion, resveratrol may play a regulatory role in heat-stress-induced damage and inflammatory response in the intestinal mucosa of black-boned chickens under heat stress.

Elevated expression of Toll-like receptor 4 and cytokines in both serum and myometrium at term may serve as promising biomarkers for uterine activation preceding labor.

Increased inflammation and cytokine levels are considered risk factors and promoters of preterm birth (PTB). However, the regulatory mechanism of pregnancy-related inflammation remains unclear. Toll-like receptor 4 (TLR4) plays a critical role in inflammatory responses in various diseases. Therefore, our study aimed to investigate whether TLR4 is involved in the inflammatory responses during uterine activation for labor, with the goal of identifying potential biomarkers for uterine activation at term. We used flow cytometry to detect TLR4 expression on CD14+ maternal blood monocytes in the first, second, and third trimesters. ELISA was employed to measure TLR4 and cytokines levels in the maternal serum of term non-labor (TNL), term labor (TL) women and LPS induced preterm labor and PBS injected controls. TLR4siRNA was transfected into the human myometrial smooth muscle cells (HMSMCs), which were subsequently treated with IL-1β. The mRNA and protein levels of TLR4, uterine contraction-related protein connexin 43 (CX43), oxytocin receptor (OTR), MAPK/NF-κB signaling pathway, and cytokines were analyzed using qRT-PCR, western blotting, and immunohistochemistry. The study revealed TLR4 expression on CD14+ maternal blood monocytes was higher in the third trimester group compared to the first and second trimester groups (p<0.001). Maternal serum concentrations of TLR4 and cytokines were significantly higher in the TL group than the TNL group (p<0.001). TLR4, OTR, CX43, activated MAPK/NF-κB expression, and cytokines levels were upregulated in TL group, and similarly significantly higher in the LPS-induced preterm group than in the control group. Using the HMSMCs we demonstrated that TLR4siRNA transfection suppressed contractility. Interfering with TLR4 expression reduced the expression of OTR, CX43, cytokines, and MAPK/NF-κB activation. There was a significant positive relationship between TLR4 expression and the inflammatory status in the myometrium. ROC analysis indicated that TLR4 and cytokines may serve as potential biomarkers for predicting uterine activation for labor. Our data suggest that TLR4 and cytokines can act as stimulators of uterine activation for labor at term. Furthermore, the MAPK/NF-κB pathway appears to be one of the potential signaling pathways mediating TLR4's regulation of parturition initiation.

Aucubin combined with ADSCs-exos protects TBHP-induced nucleus pulposus cells via TLR4/NF-κB pathway

This paper aims to investigate the effects and mechanisms of adipose-derived stem cells-exosomes(ADSCs-exos) toge-ther with aucubin in protecting human-derived nucleus pulposus cells(NPCs) from inflammatory injury, senescence, and apoptosis. The tert-butyl hydroperoxide(TBHP)-induced NPCs were assigned into normal, model, aucubin, ADSCs-exos, and aucubin+ADSCs-exos groups. The cell viability was examined by cell counting kit-8(CCK-8), cell proliferation by EdU staining, cell senescence by senescence-associated-β-galactosidase(SA-β-Gal), and cell cycle and apoptosis by flow cytometry. Enzyme-linked immunosorbent assay was employed to examine the expression of interleukin-1β(IL-1β), IL-10, and tumor necrosis factor-α(TNF-α). Real-time fluorescence quantitative PCR and Western blot were employed to determine the mRNA and protein levels of aggregated proteoglycan(aggrecan), type Ⅱ collagen alpha 1(COL2A1), Toll-like receptor 4(TLR4), and nuclear factor-kappa B(NF-κB). The results showed that compared with the model group, the aucubin or ADSCs-exos group showed enhanced viability and proliferation of NPCs, decreased proportion of G_0/G_1 phase cells, increased proportion of S phase cells, reduced apoptosis and proportion of cells in senescence, lowered IL-1β and TNF-α levels, elevated IL-10 level, down-regulated mRNA and protein levels of TLR4 and NF-κB, and up-regulated mRNA and protein levels of aggrecan and COL2A1. Compared with the aucubin or ADSCs-exos group, the aucubin+ADSCs-exos combination further increased the viability and proliferation of NPCs, decreased the proportion of G_0/G_1 phase cells, increased the proportion of S phase cells, reduced the apoptosis and proportion of cells in senescence, lowered the IL-1β and TNF-α levels, elevated the IL-10 level, down-regulated the mRNA and protein levels of TLR4 and NF-κB, and up-regulated the mRNA and protein levels of aggrecan and COL2A1. In summary, both aucubin and ADSCs-exos could exert protective effects by inhibiting inflammatory responses, reducing apoptosis and senescence of NPCs, improving cell viability and proliferation as well as extracellular matrix synthesis, which may be associated with the inhibition of TLR4/NF-κB signaling pathway activation. The combination of both plays a synergistic role in the protective effects.

Effects of Buyang Huanwu Decoction and Astragali Radix-Angelicae Sinensis Radix combination on inflammatory responses in atherosclerotic mice

The study aims to observe the effects and explore the mechanisms of Buyang Huanwu Decoction and Astragali Radix-Angelicae Sinensis Radix combination in the treatment of the inflammatory response of mice with atherosclerosis(AS) via the Toll-like receptor 4(TLR4)/myeloid differentiation primary response protein 88(MyD88)/nuclear factor-κB(NF-κB) signaling pathway. Male ApoE~(-/-) mice were randomly assigned into a model group, a Buyang Huanwu Decoction group, an Astragali Radix-Angelicae Sinensis Radix combination group, and an atorvastatin group, and male C57BL/6J mice of the same weeks old were used as the control group. Other groups except the control group were given high-fat diets for 12 weeks to establish the AS model, and drugs were administrated by gavage. Aortic intimal hyperplasia thickness, blood lipid level, plasma inflammatory cytokine levels, M1/M2 macrophage markers, and expression levels of proteins in TLR4/MyD88/NF-κB pathway in the vessel wall were measured to evaluate the effects of drugs on AS lesions and inflammatory responses. The results showed that the AS model was successfully established with the ApoE~(-/-) mice fed with high-fat diets. Compared with the control group, the model group showed elevated plasma total cholesterol(TC), triglyceride(TG), and low-density lipoprotein cholesterol(LDL-c) levels(P&lt;0.05), thickened intima(P&lt;0.01), and increased plasma tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6) levels(P&lt;0.01). Moreover, the model group showed increased expression of vascular cell adhesion molecule-1(VCAM-1) and inducible nitric oxide synthase(iNOS)(P&lt;0.01), inhibited expression of endothelial nitric oxide synthase(eNOS) and cluster of differentiation 206(CD206)(P&lt;0.01), and up-regulated mRNA and protein levels of TLR4, MyD88, NF-κB inhibitor alpha(IκBα), and NF-κB in the vessel wall(P&lt;0.05). Compared with the model group, Buyang Huanwu Decoction and Astragali Radix-Angelicae Sinensis Radix combination lowered the plasma TC and LDL-c levels(P&lt;0.01), alleviated the intimal hyperplasia(P&lt;0.01), and reduced the plasma TNF-α and IL-6 levels(P&lt;0.05). Moreover, the two interventions promoted the expression of eNOS and CD206(P&lt;0.05), inhibited the expression of VCAM-1 and iNOS(P&lt;0.01), and down-regulated the mRNA and protein levels of TLR4, MyD88, IκBα, and NF-κB(P&lt;0.05) in the vessel wall. This study indicated that Buyang Huanwu Decoction and Astragali Radix-Angelicae Sinensis Radix combination could delay the progression of AS, inhibit the polarization of vascular wall macrophages toward M1 type, and attenuate vascular inflammatory response by inhibiting the activation of TLR4/MyD88/NF-κB signaling pathway in the vascular wall. Astragali Radix and Angelicae Sinensis Radix were the main pharmacological substances in Buyang Huanwu Decoction for alleviating the AS vascular inflammatory response.

Effects of toll like receptor 4 overexpression on blood parameters, body composition, and gastrointestinal microbial diversity in sheep

Genetic modifications can improve the health status and disease resistance of farm animals. In the previous study, genetically modified (GM) sheep overexpressing toll like receptor 4 (TLR4) were produced. The GM sheep had stronger ability to eliminate invasive microbes compared to the wild-type (WT) sheep. Physiological status and immune homeostasis, blood parameters, body composition, and gastrointestinal microbial diversity were evaluated in this study to elucidate the effects of TLR4 overexpression. In a set of 10 GM rams, the TLR4 mRNA and protein expression levels in the peripheral blood mononuclear cells, muscle, kidney, and spleen were higher than those of the WT (n = 10; P < 0.05). No significant differences in the parameters of red blood cells, white blood cells, platelets and in the composition of circulating T lymphocyte subsets were observed between the GM and WT sheep (P > 0.05). Of the biochemical parameters, only total protein levels differed (were higher) in the GM sheep than in the WT sheep (P < 0.05) while no significant differences were observed for the other indices (P > 0.05). GM and WT sheep showed similar offal weights and muscle fatty acid and amino acid compositions (P > 0.05). Furthermore, TLR4 overexpression did not affect the community structure of the gastrointestinal tract microbiota. Eleven KEGG pathways associated with the TLR4 gene, physiology and biochemistry, growth and metabolism, fatty acids, and amino acids did not demonstrate significant differences between the GM and WT sheep (P > 0.05). In conclusion, TLR4 overexpression had no principal or adverse effects on the physiological development and health of sheep, except for the general mRNA and protein expression levels.

Expression Improvement of Recombinant Plasmids of the Interleukin-7 Gene in Chitosan-Derived Nanoparticles and Their Elevation of Mice Immunity.

To investigate a safe and effective approach for enhancing the in vivo expression of recombinant genes and improving the systemic immunity of animals against infectious diseases, we employed the interleukin-7 (IL-7) gene from Tibetan pigs to construct a recombinant eukaryotic plasmid (VRTPIL-7). We first examined VRTPIL-7's bioactivity on porcine lymphocytes in vitro and then encapsulated it with polyethylenimine (PEI), chitosan copolymer (CS), PEG-modified galactosylated chitosan (CS-PEG-GAL) and methoxy poly (ethylene glycol) (PEG) and PEI-modified CS (CS-PEG-PEI) nanoparticles using the ionotropic gelation technique. Next, we intramuscularly or intraperitoneally injected mice with various nanoparticles containing VRTPIL-7 to evaluate their immunoregulatory effects in vivo. We observed a significant increase in neutralizing antibodies and specific IgG levels in response to the rabies vaccine in the treated mice compared to the controls. Treated mice also exhibited increased leukocytes, CD8+ and CD4+ T lymphocytes, and elevated mRNA levels of toll-like receptors (TLR1/4/6/9), IL-1, IL-2, IL-4, IL-6, IL-7, IL-23, and transforming growth factor-beta (TGF-β). Notably, the recombinant IL-7 gene encapsulated in CS-PEG-PEI induced the highest levels of immunoglobulins, CD4+ and CD8+ T cells, TLRs, and cytokines in the mice's blood, suggesting that chitosan-PEG-PEI may be a promising carrier for in vivo IL-7 gene expression and enhanced innate and adaptive immunity for the prevention of animal diseases.