TLR4 mRNA Levels Research Articles

High mobility group box-1 regulates expression of EGFR, VEGF, StAR and TIMP1/2 in bovine granulosa cells through a mechanism involving TLR2/NF-κB

Innate immunity is involved in ovarian activity through aseptic inflammation and tissue repair. High-mobility group box-1 (HMGB1) is related to placental inflammation and a driver of inflammation throughout pregnancy. The aim of this study was to investigate the interaction of HMGB1 with TLR2 in bovine ovarian granulosa cells, and the effects of HMGB1 on bovine ovarian granulosa cells in vitro. The viability of granulosa cells were not affected by HMGB1 with the concentration less than 5 μg/mL. The mRNA levels of TLR2, epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), the steroidogenic acute regulatory protein (StAR), tissue inhibitors of matrix (TIMP1/2) of ovarian granulosa cells were upregulated by HMGB1(P < 0.05). The protein levels of TLR2, TLR1 and phosphorylation-NF-κB (p-NF-κB) p65 in ovarian granulosa cells increased in 5 μg/mL HMGB1 group (P < 0.05), and TLR2 decreased in siRNA-2 group (P < 0.05). IL-6 of ovarian granulosa cells was increased by 1 μg/mL and 5 μg/mL HMGB1 (P < 0.05). These results implicate that HMGB1 has interaction with TLR2, TLR1 and p-NF-κB p65 in ovarian granulosa cells, which lead to nuclear translocation of NF-κB p65 and the secretion of interleukin-6 (IL-6). HMGB1 regulates the expression of EGFR, VEGF, StAR, TIMP1/2 and the secretion of IL-6 in ovarian granulosa cells of dairy cows through activating the TLR2/NF-κB signaling pathway, which may be interfere with ovarian physiological activity.

Cl-amidine attenuates lipopolysaccharide-induced inflammation in human gingival fibroblasts via the JNK/MAPK, NF-κB, and Nrf2 signalling pathways.

Cl-amidine has been reported to have anti-inflammatory properties in a variety of diseases. However, the role of Cl-amidine in periodontal disease remains unclear. Here, the purpose of this study was to investigate the effect of Cl-amidine on lipopolysaccharide (LPS)-induced inflammation in human gingival fibroblasts (HGFs). The cytotoxic effect of Cl-amidine was measured with the Cell Counting Kit-8 (CCK-8) assay and Annexin V-FITC/PI staining. The protein levels of IL-6 and IL-8 in culture supernatants were measured with enzyme-linked immunosorbent assay (ELISA). The mRNA levels of inflammatory cytokines, TLR4 and MyD88 were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) analysis. The expression patterns of IL-6, TNF-ɑ, and IL-1β in HGFs were tested with western blot. The levels of NF-κB, MAPK, and Nrf2 pathway-related proteins were detected by western blot. Immunofluorescence (IF) staining was used to examine the nuclear translocation of NF-κB p65. Moreover, a rat gingivitis model was established to further clarify the role of Cl-amidine. Our results showed that Cl-amidine suppressed LPS-induced gingival inflammation both in vitro and in vivo. Mechanistically, Cl-amidine inhibited LPS-induced MyD88 expression, NF-κB activation, and JNK phosphorylation. Additionally, Cl-amidine upregulated Nrf2 and Ho-1 expression both with and without LPS stimulation but did not alter ROS levels or Keap1 expression. Overall, our data suggest that Cl-amidine acts as an inhibitor of LPS-induced gingival inflammation via the JNK/MAPK, NF-κB, and Nrf2 signalling pathways.

Alpha-pyrrolidinopentiothiophenone (α-PVT) activates the TLR–NF-κB–MAPK signaling pathway and proinflammatory cytokine production and induces behavioral sensitization in mice

Synthetic cathinones are chemical derivatives of cathinone, a structural analog to amphetamine. It has been shown that synthetic cathinones have abuse potentials similar to psychomotor stimulants such as amphetamine and induce neuroinflammation. Among the novel synthetic cathinones, α-pyrrolidinopentiothiophenone (α-PVT) has been known to produce rewarding and reinforcing effects in rodent models. However, it has not yet been determined whether α-PVT induces neuroinflammation in vivo. In the present study, mice were exposed to repeated saline or α-PVT (20 mg/kg, intraperitoneally) for 7 days to test changes in locomotor activity and neuroinflammation-related factors in the striatum of mice. Repeated administration of α-PVT significantly induced locomotor sensitization. In addition, repeated α-PVT administration significantly increased the number of microglial cells, accompanied by marked increases in TLR1, TLR4, TLR6, and TLR7 mRNA levels in the striatum of mice. Furthermore, acute or repeated α-PVT administration increased the levels of phosphorylated NF-κB, ERK, p38, and JNK MAPK activation and repeated α-PVT, but not acute, increased the levels of TNF-α and IL-6 mRNA in the striatum of mice. Finally, systemic administration of TAK-242 (5 mg/kg, i.p.) or MPLA (50 μg/kg, i.p.), each an inhibitor or activator of TLR4, did not change α-PVT-induced behavioral sensitization in mice. These results suggest that the activation of TLR4 by repeated α-PVT administration may lead to neuroinflammation via TLR-mediated NF-κB and MAPK signaling pathways and the production of TNF-α and IL-6 in the striatum of mice, at least without the regulation of behavioral sensitization.

Breastfeeding Increases the Expression of TLR4, TNF-α, CCL2, and CCL3 in the Prepuce Tissue of Neonates

Innate immunity plays a significant role in the tissue repair process. It is well documented that breastfeeding may alter immune responses. Thus, this study was designed to evaluate the effects of breastfeeding on the levels of TLR1-4, TNF-α, TGF-β, CCL2, and CCL3 in the prepuce tissue of neonates. This study was conducted on 90 samples of prepuce tissue obtained from neonates (45 neonates who were breastfed with human milk [HM] and 45 neonates who were not breastfed and received non-human milk [NHM]). The tissues were homogenized and mRNA levels of TLR1-4 and protein levels of TNF-α, TGF-β, CCL2, and CCL3 were analyzed using Real-Time PCR and ELISA techniques, respectively. Protein levels of TNF-α, CCL2 and CCL3, and mRNA levels of TLR4 were significantly lower in the NHM neonates than in the HM neonates. There was a significant negative correlation between duration of pregnancy and mRNA levels of TLR1 in the NHM neonates. These results indicate that breastfeeding may be associated with the regulated expression of TLR4 and its related cytokines/chemokines and that it may improve wound healing and aid in the fight against pathogens.

RhCNB Improves Cyclophosphamide-Induced Immunodeficiency in BALB/c Mice.

Background This study aims to explore the immunomodulatory effect of rhCNB on mice with cyclophosphamide (CTX)-induced immunodeficiency through TLR4/MAPK pathway. Methods BALB/c mice were randomly divided into three groups: a negative control group, an immunosuppression model group, and a rhCNB treatment group. Tail vein injection of cyclophosphamide (40 mg/kg) was used to establish a mouse immunosuppression model. Intraperitoneal injection of rhCNB (20 mg/kg) was administered to the treatment group, whereas equal quantities of normal saline were given to the control group and model group. Perform peripheral blood routine of CD4, CD8, and CD19 lymphocyte subsets and peripheral blood Th1/Th2 cell subsets 24 hours after the last administration. RT-PCR was used to detect mRNA levels of TLR4, P38, JNK, T-bet, and GATA3, the spleen immune organ index was measured, and the histopathological status of the spleen and thymus was observed. Results The results showed that compared with the control group, WBC, PLT, LYM, NEU, immune organ index, CD4+/CD8+ and CD19+ subgroup ratio, and peripheral blood Th1/Th2 cell subgroups decreased in the model group. The mRNA levels of TLR4, P38, JNK, T-bet, and GATA3 decreased compared with the model group, while they increased in the treatment group. Conclusions rhCNB has an immunomodulatory effect by regulating the expression of Th1/Th2 cytokine balance through the TLR4/MAPK signaling pathway and promoting the differentiation and proliferation of lymphocytes, thereby improving the immune function.

Dietary Supplementation with Eucommia ulmoides Leaf Extract Improved the Intestinal Antioxidant Capacity, Immune Response, and Disease Resistance against Streptococcus agalactiae in Genetically Improved Farmed Tilapia (GIFT; Oreochromis niloticus).

A 7-week rearing trial was designed to investigate the effects of Eucommia ulmoides leaf extract (ELE) on growth performance, body composition, antioxidant capacity, immune response, and disease susceptibility of diet-fed GIFT. The results showed that dietary ELE did not affect growth performance or whole-body composition (p > 0.05). Compared with the control group, plasma ALB contents increased in the 0.06% dietary ELE group (p < 0.05), and plasma ALT and AST activities decreased in the 0.08% dietary ELE group (p < 0.05). In terms of antioxidants, compared with GIFT fed the control diet, 0.06% dietary ELE upregulated the mRNA expression levels of Nrf2 pathway-related antioxidant genes, including CAT and SOD (p < 0.05), and 0.06% and 0.08% dietary ELE upregulated the mRNA levels of Hsp70 (p < 0.05). In terms of immunity, 0.06% dietary ELE suppressed intestinal TLR2, MyD88, and NF-κB mRNA levels (p < 0.05). Moreover, the mRNA levels of the anti-inflammatory cytokines TGF-β and IL-10 were upregulated by supplementation with 0.04% and 0.06% dietary ELE (p < 0.05). In terms of apoptosis, 0.06% and 0.08% ELE significantly downregulated the expression levels of FADD mRNA (p < 0.05). Finally, the challenge experiment with S. agalactiae showed that 0.06% dietary ELE could inhibit bacterial infection, and significantly improve the survival rate of GIFT (p < 0.05). This study demonstrated that the supplementation of 0.04–0.06% ELE in diet could promote intestinal antioxidant capacity, enhance the immune response and ultimately improve the disease resistance of GIFT against Streptococcus agalactiae.

Fuzi-Lizhong Decoction Alleviates Nonalcoholic Fatty Liver Disease by Blocking TLR4/MyD88/TRAF6 Signaling.

Background Fuzi-Lizhong decoction (FLD) derives from the ancient Chinese Pharmacopoeia and has been clinically used for years. The present study aimed to investigate the activities and underlying mechanisms of FLD against nonalcoholic fatty liver disease (NAFLD). Methods In vivo studies were conducted by inducing NAFLD in rats with a high-fat diet, and in vitro studies were performed on HL-7702 cells treated with oleic and linoleic acids. Total cholesterol (TC), triglyceride (TG), and blood glucose (Glu) levels were detected using an automatic biochemical analyzer. The expression of IL-2, IL-6, and TNF-α in sera and cell culture supernatants was measured by ELISA. The mRNA and protein levels of TLR4, MyD88, and TRAF6 were measured in liver tissue and HL-7702 cells using reverse transcription-quantitative polymerase chain reaction and western blot. Results FLD significantly reduced the TC, TG, Glu, FFA, IL-2, IL-6, and TNF-α levels in NAFLD rats and HL-7702 cells. Analysis of liver lipid content by Oil Red O staining revealed a significant increase in hepatic lipid accumulation in rats with NAFLD, but this lipid accumulation was reversed by FLD treatment. In addition, the mRNA expression levels of TLR4, MyD88, TRAF6, and NF-κB p65 as well as the protein levels of TLR4, MyD88, TRAF6, and NF-κB p65 were decreased after FLD treatment. FLD significantly reduced inflammation and improved collagen accumulation in vivo and in vitro by inhibiting the activation of the TLR4/MyD88/TRAF6 signaling pathway. Conclusions FLD exerted potent protective effects against NAFLD via TLR4/MyD88/TRAF6 signaling. These findings provide novel insights into the mechanisms whereby this compound acts as an anti-inflammatory agent and highlight the potential application of FLD in the treatment of acute liver failure (ALF).

Efficacy of Sishen Wan on dinitrobenzene sulfonic acid-induced ulcerative colitis and its effect on toll-like receptor 2/interleukin-1 receptor-associated kinase-4/nuclear factor-κB signal pathway.

To investigate the therapeutic effect of Sishen Wan (, SSW) on ulcerative colitis (UC) induced by dinitrobenzene sulfonic acid and its effect on toll-like receptor 2/interleukin-1 receptor-associated kinase-4/nuclear factor-κB (TLR2/IRAK4/NF-κB) sig-naling pathway in colonic tissue. In this study, 120 Sprague-Dawley rats were randomly divided into blank and model groups. The experimental UC model in rats was established by subcutaneous injection of hydrocortisone + senna gavage for 21 d + dinitrobenzene sulfonic acid (DNBS)/ ethanol solution enema. The successful model rats were randomly divided into the model group; mesalazine (0.36 g/kg) group; and high-, medium-, and low- dose SSW (24, 12, and 6 g/kg) groups. The model and blank groups were gavaged with equal volumes of distilled water once a day for 21 d. The general condition of the rats was observed, and the body mass, fecal properties, and occult blood were recorded for calculating the disease activity index (DAI) score. The colonic tissue of the rats was collected, and its general morphology and pathological form were noted for obtaining the colonic mucosal injury index (CMDI) score. Hematoxylin-eosin staining was used to view the pathological changes of the colon tissue in each group, apoptosis of the cells was detected using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining, and quantitative real-time polymerase chain reaction was used to measure the expressions of TLR2, myeloid differentiation primary response gene 88 (MyD88), IRAK4, and NF-κB p65 mRNA in the colon tissue. The expressions of TLR2, MyD88, IRAK4, and NF-κB p65 protein were detected using western blotting and immunohistochemistry assay, and the levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the colon tissue were determined using enzyme linked immunosorbent assay. Compared with the blank group, the general condition of the model group was relatively poor. The DAI and CMDI scores of the model group increased significantly (< 0.01), the glands and intestinal mucosa disappeared partially, and several inflammatory cells infiltrated and gathered in the mucosal layer and base layer of the rats in the model group. Furthermore, the cell apoptosis and expression levels of TLR2, MyD88, IRAK4, and NF-κB p65 mRNA and protein in the colon tissue of rats in the model group increased significantly (< 0.01). The levels of IL-1β and TNF-α increased significantly in the colon tissue of rats in the model group (< 0.01). After treatment with SSW, compared with the model group, the general condition of the UC rats improved. Moreover, the DAI and CMDI scores of the UC rats decreased significantly (< 0.05), and the pathological changes in the colon tissue of the UC rats tended to be normal. The cell apoptosis and expression levels of TLR2, MyD88, IRAK4, and NF-κB p65 mRNA and protein in the colon tissue of the UC rats decreased gradually ( < 0.01), and the levels of IL-1β and TNF-α decreased significantly (< 0.01). SSW can improve the general condition and alleviate the intestinal mucosal injury of UC model rats. Additionally, SSW can inhibit the TLR2/IRAK4/ NF-κB signaling pathway, but further studies are required to confirm it.

A behavioral and molecular study; ameliorated anxiety-like behavior and cognitive dysfunction in a rat model of chronic unpredictable stress treated with oregano extract.

ObjectiveChronic stress is considered a severe risk factor leading to various disorders, including anxiety and cognitive decline. The present study aimed to investigate the effects of Origanum vulgare (oregano) extract on improving anxiety‐like behavior and learning and memory defection caused by chronic unpredictable stress (CUS).MethodA 10‐day CUS protocol was executed on male rats, and on day 10, their anxiety, learning, and memory status were evaluated. After that, in addition to the CUS, the rats were treated with the oregano extract for 2 weeks. Then, the expression of BDNF, TrkB, and TLR2/4 genes in the hippocampus and prefrontal cortex of the rats was evaluated. Also, the liver‐ and kidney‐related serum parameters, including triglycerides, total cholesterol, HDL, LDL, creatinine, urea, serum glucose, alanine aminotransferase, and aspartate aminotransferase were assessed. Further, the extract's lethal effect and its impact on animals’ body weight were investigated.ResultsBehavioral tests confirmed the anxiety‐like behavior and learning–memory function impairment caused by CUS. In contrast, the administration of the extract could significantly alleviate the mental deficiencies and diminished anxiety‐like behaviors. Molecular assessments showed that CUS could markedly decrease the BDNF and TrkB genes’ expression levels while increasing that of TLR2 and TLR4. In contrast, in extract‐treated animals, mRNA levels of BDNF and TrkB considerably increased, yet TLR2 and TLR4 mRNA levels reduced. Additionally, consumption of the extract caused weight gain, while having no lethality and detrimental effect on the liver and kidneys functions.ConclusionsThese findings indicate the anxiolytic properties of the extract and its improving effect on cognitive dysfunction.

Baicalin Inhibits Inflammation in Rats with Chronic Obstructive Pulmonary Disease by the TLR2/MYD88/NF-κBp65 Signaling Pathway.

Objective Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease with a relatively high morbidity and death rate. This study aimed to investigate the inhibitory effect of baicalin (BA) on inflammation in COPD rats and its possible mechanism. Methods The experimental COPD of SD rats were induced by LPS, smoking, and cold stimulation, and they were randomly divided into the control group, COPD group, COPD + LB group, COPD + MB group, and COPD + HB group. The test of pulmonary function and the HE staining were carried out in COPD rats. The levels of TNF-α, IL-1β, IL-6, IL-10, and IL-8, as well as GSH, SOD, and MDA in serum, were detected by ELISA. The levels of TLR2, MYD88, TNF-α, and IL-1β mRNA in BALF were detected by qPCR. The expression of TLR2/MYD88/NF-κBp65 pathway-related proteins was also detected by the Western blot and immunohistochemistry assays. Results Compared to the COPD model group, BA treatment significantly improved the pulmonary function and pathologic changes, reduced the levels of TNF-α, IL-1β, IL-6, IL-10, IL-8, and MDA, and increased the levels of IL-10, SOD, and GSH in COPD rats. In addition, BA could also decrease the protein levels of MYD88, p–NF–κBp65/NF-κBp65, TLR2, and TLR4 but increase the protein level of p-IκBa/IκB in lung tissue of COPD rats. Conclusion BA ameliorated inflammatory response and oxidative stress in COPD rats by regulating the TLR2/MYD88/NF-κBp65 signaling pathway.

Hexavalent Chromium Exposure Induces Intestinal Barrier Damage via Activation of the NF-κB Signaling Pathway and NLRP3 Inflammasome in Ducks.

Hexavalent chromium [Cr(VI)] is a dangerous heavy metal which can impair the gastrointestinal system in various species; however, the processes behind Cr(VI)-induced intestinal barrier damage are unknown. Forty-eight healthy 1-day-old ducks were stochastically assigned to four groups and fed a basal ration containing various Cr(VI) dosages for 49 days. Results of the study suggested that Cr(VI) exposure could significantly increase the content of Cr(VI) in the jejunum, increase the level of diamine oxidase (DAO) in serum, affect the production performance, cause histological abnormalities (shortening of the intestinal villi, deepening of the crypt depth, reduction and fragmentation of microvilli) and significantly reduced the mRNA levels of intestinal barrier-related genes (ZO-1, occludin, claudin-1, and MUC2) and protein levels of ZO-1, occludin, cand laudin-1, resulting in intestinal barrier damage. Furthermore, Cr(VI) intake could increase the contents of hydrogen peroxide (H2O2) and malondialdehyde (MDA), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-18 (IL-18) but decrease the activities of total superoxide dismutase (T-SOD), catalase (CAT), and glutathione reductase (GR), as well as up-regulate the mRNA levels of TLR4, MyD88, NF-κB, TNFα, IL-6, NLRP3, caspase-1, ASC, IL-1β, and IL-18 and protein levels of TLR4, MyD88, NF-κB, NLRP3, caspase-1, ASC, IL-1β, and IL-18 in the jejunum. In conclusion, Cr(VI) could cause intestinal oxidative damage and inflammation in duck jejunum by activating the NF-κB signaling pathway and the NLRP3 inflammasome.

Correlations between Kidney and Heart Function Bioindicators and the Expressions of Toll-Like, ACE2, and NRP-1 Receptors in COVID-19.

Background: COVID-19 impacts the cardiovascular system resulting in myocardial damage, and also affects the kidneys leading to renal dysfunction. This effect is mostly through the binding with angiotensin-converting enzyme 2 (ACE2) and Neuropilin-1 (NRP-l) receptors. Toll-Like Receptors (TLRs) typically combine with microbial pathogens and provoke an inflammatory response. Aim: This work aims to compare the changes in kidney and heart function bioindicators and expressions of TLRs (TLR2 and TLR2) as well as ACE2 and NRP-l receptors in moderate and severe COVID-19 patients. The correlations between kidney and heart function bioindicators and expressions of these receptors are also studied. Patients and Methods: In this study, 50 healthy control and 100 COVID-19 patients (55 males and 45 females) were enrolled. According to WHO guidelines, these participants were divided into severe (50 cases) and moderate (50 cases). Serum creatinine, blood urea, CK-MB, LDH, and Troponin I were estimated. We measured the gene expression for Toll-Like Receptors (TLR2 and TLR4), ACE2, and NRP-1 in the blood samples using quantitative real-time PCR (qRT-PCR). Results: In comparison with the healthy group, all patients exhibited a significant elevation in serum creatinine, urea, cardiac enzymes (CK-MB and LDH), and CRP. Serum Troponin I level was significantly increased in severe COVID-19 patients. Furthermore, all studied patients revealed a significant elevation in the expression levels of TLR2, TLR4, ACE2, and NRP-1 mRNA. In all patients, CK-MB, ACE2, and NRP-1 mRNA expression levels were positively correlated with both TLR2 and TLR4 expression levels. Moreover, serum creatinine and urea levels were positively correlated with both TLR2 and TLR 4 expression levels in the severe group only. In the moderate group, serum CK-MB activity and Troponin I level had a significant positive correlation with both NRP-1 and ACE2 expression levels, while serum urea level and LDH activity had a significant positive correlation with NRP-1 only. In severe patients, the increases in serum creatinine, urea, CK-MB, and LDH were significantly associated with the elevations in both ACE2 and NRP-1 expression levels, whereas serum Troponin I level had a positive direct relationship with NRP-1 only. Conclusions: Our study concluded that expression levels for TLR2, TLR4, ACE2, and NRP-1 mRNA in both severe and moderate patients were positively correlated with renal biomarkers and cardiac enzymes. Innate immune markers can be important because they correlate with the severity of illness in COVID-19.

Curcumae longae Rhizoma (Jianghuang) extract reverses the 5-Fluoruracil resistance in colorectal cancer cells via TLR4/PI3K/Akt/mTOR pathway

ObjectivesIntensive efforts have been made in the area of identifying drug resistance modulators from traditional Chinese medicine. Various natural plant extracts have been reported for their reversal effect of drug resistance in cancers. This study was to assess the reversal potential of Curcumae longae Rhizoma extract (CLRE) in 5-Fluorouracil (5-Fu) resistance to colon cancer and explore its underly mechanism. MethodsIncreased concentrations of 5-Fu were used to culture SW480. A series of concentrations of CLRE were used to treat the 5-Fu resistant SW480 cells. WST-8 assay was used to detect the cell viability. Cell apoptosis was assessed by SuperView™ 488 Caspase-3 Assay Kit. The quantification of mentioned factors was archived by RT-qPCR. Network pharmacology analysis was used to explore the target of CLRE. Results5-Fu resistant cell line (SW480/5-FuR) was established. The IC50 value of CLRE against SW480/5-FuR was 181.0 ± 14.12 µg/ml. CLRE can resensitize the SW480/5-FuR to 5-Fu by inhibiting cell growth. The combination treatment (CLRE+5-Fu) induced cell apoptosis via inhibition of bcl-2 and activation of caspase-3 and bax. Three active ingredients from CLRE were identified. TLR4 was targeted by these three ingredients and linked these ingredients to PI3K/Akt/mTOR pathway. The levels of TLR4, PI3K, AKT1, and mTORC1 mRNA were decreased when 5-Fu was combined with CLRE. ConclusionsCLRE could reverse 5-Fu resistance in colon cancer by inactivating TLR4/PI3K/AKT/mTORC1 pathway. This finding might provide a molecular basis and a valuable direction for further clinical applications and research for treating 5-Fu resistant colon cancer.

Role of TLR9 methylation on its active transcription in apical inflammation.

To explore the methylation pattern, its role in transcriptional regulation and potential modifiers of methylation of the TLR9 gene in chronic periapical inflammation. In this cross-sectional study, apical lesions of endodontic origin (ALEO, n=61) and healthy periodontal ligaments (HPL, n=15) were included. Products from bisulfited and PCR-amplified DNA were analysed for their methylation profiles in the promoter region and at each CpG island. Additionally, TLR9 mRNA levels were quantified by qPCR and bivariate and multiple modelling were performed to better understand the influence of methylation on gene transcription. TLR9 mRNA levels were upregulated in ALEO compared to HPL (p<.001). TLR9 promoter CpG sites and CpG +2086 in the intragenic island 1 were demethylated in ALEO compared to HPL (p<.05). Multivariate analysis, adjusted by smoking and gender, revealed that demethylation of TLR9 promoter sites enhanced transcriptional activity, specifically demethylated CpGs at positions -736 and -683, (p=.02), which are close to CRE binding. Although ALEO reduced the global methylation of the gene promoter and intragenic-island 2 (p<.05) by -42.5 and -9.5 percentage points, respectively, age reduced the global methylation of intragenic-island 3 within the exon 2. Demethylations of TLR9 promoter CpG sites, along with the intragenic DNA methylation status, were involved in higher transcription in ALEO. Hence, chronic periapical inflammation and ageing modify the methylation status both in the gene promoter and in intragenic CpG islands.

Effect of oral administration of Limosilactobacillus reuteri on intestinal barrier function and mucosal immunity of suckling piglets

The aim of present study was to evaluate the effect of Limosilactobacillus reuteri (L. reuteri) on the intestinal barrier function and mucosal immunity of piglets at different suckling phase. In this study, fifty-four neonatal piglets (Rongchang × Yorkshire) were orally administered with 1.0 × 1010 CFU L. reuteri I5007 (dissolved in 2 ml 0.1% peptone) or 2 ml 0.1% peptone by gavage every day. The experiment had lasted for 20 days from the birth. Six piglets from each group were slaughtered at 10- and 20-day-old, respectively. Intestinal samples were collected for the evaluation of target genes by real-time PCR and western blot. The results showed that L. reuteri I5007 significantly increased the average daily gain, decreased the diarrhoea incidence, and improved the intestinal morphology of piglets (p < .05). Oral administration of L. reuteri I5007 also elevated the mRNA and protein level of jejunal and ileal Zonula Occludens-1, Claudin-1, and Occludin to enhance the intestinal mucosal barrier function. Additionally, L. reuteri I5007 treatment modulated toll-like receptors (TLRs) and porcine host defense peptides (HDPs) expression, such as increased the mRNA levels of ileal TLR9 and PBD2 at 10-day-old, jejunal TLR9 and ileal PG1-5 at 20-day-old, the protein level of ileal TLR2 at 20-day-old, and influenced the protein and mRNA level of ileal TLR4. These findings indicated that oral administration of L. reuteri I5007 could improve the intestinal mucosal barrier and enhance the intestinal innate immunity of piglets during suckling period. Highlights L. reuteri increased the average daily gain and decreased the diarrhoea incidence of piglets. L. reuteri increased the expression of Zonula Occludens-1, Claudin-1, and Occludin in jejunal and ileal mucosal. L. reuteri influenced the expression of toll-like receptors and porcine host defense peptides.

The Effect of Natural-Based Formulation (NBF) on the Response of RAW264.7 Macrophages to LPS as an In Vitro Model of Inflammation.

Macrophages are some of the most important immune cells in the organism and are responsible for creating an inflammatory immune response in order to inhibit the passage of microscopic foreign bodies into the blood stream. Sometimes, their activation can be responsible for chronic inflammatory diseases such as asthma, tuberculosis, hepatitis, sinusitis, inflammatory bowel disease, and viral infections. Prolonged inflammation can damage the organs or may lead to death in serious conditions. In the present study, RAW264.7 macrophages were exposed to lipopolysaccharide (LPS; 20 ng/mL) and simultaneously treated with 20 µg/mL of natural-based formulation (NBF), mushroom–cannabidiol extract). Pro-inflammatory cytokines, chemokines, and other inflammatory markers were analyzed. The elevations in the presence of interleukin-6 (IL-6), cycloxygenase-2 (COX-2), C-C motif ligand-5 (CCL5), and nitrite response, following exposure to LPS, were completely inhibited by NBF administration. IL-1β and tumor necrosis factor alpha (TNF-α) release were inhibited by 3.9-fold and 1.5-fold, respectively. No toxic effect of NBF, as assessed by lactate dehydrogenase (LDH) release, was observed. Treatment of the cells with NBF significantly increased the mRNA levels of TLR2, and TLR4, but not NF-κB. Thus, it appears that the NBF possesses anti-inflammatory and immunomodulatory effects which can attenuate the release of pro-inflammatory markers. NBF may be a candidate for the treatment of acute and chronic inflammatory diseases and deserves further investigation.

Identification of Potential Biomarkers of Type 2 Diabetes Mellitus-Related Immune Infiltration Using Weighted Gene Coexpression Network Analysis.

Background Type 2 diabetes mellitus (T2DM) is characterized by chronic low-grade inflammation, showing an increasing trend. The infiltration of immune cells into adipose tissue has been shown to be an important pathogenic cause of T2DM. The purpose of this study is to use the relevant database to identify some abnormally expressed or dysfunctional genes related to diabetes from the perspective of immune infiltration. Methods Weighted gene coexpression network analysis (WGCNA) was employed to systematically identify the coexpressed gene modules and hub genes associated with T2DM development based on a microarray dataset (GSE23561) from the Gene Expression Omnibus (GEO) database. The key genes in modules highly related to clinical features were calculated and screened by using R software, and their participation in T2DM was determined by gene enrichment analysis. The mRNA levels of CSF1R, H2AFV, LCK, and TLR9 in pre-T2DM mice and normal wild-type mice were detected by WGCNA screening and real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Results We constructed 14 coexpressed gene modules, and the brown module was shown to be significantly related to T2DM. Through verification of the protein-protein interaction (PPI) network, four upregulated hub genes, CSF1R, H2AFV, LCK, and TLR9, were screened from the brown module and successfully distinguishedT2DM patients from healthy people. These hub genes may be used as biomarkers and important indicators for patient diagnosis. Enrichment analysis showed that these hub genes were highly associated with IL-6-related inflammatory metabolism, immune regulation, and immune cell infiltration. Finally, we verified the hub genes CSF1R, LCK, and TLR9 in a T2DM animal model and found that their mRNA levels were significantly higher in animals with T2DM than in control group mice (NC). Conclusions In summary, our results suggest that these hub genes (CSF1R, LCK, and TLR9) can serve as biomarkers and immunotherapeutic targets for T2DM.

Identification of the Association Between Toll-Like Receptors and T-Cell Activation in Takayasu's Arteritis.

To explore the relationships between Toll-like receptors (TLRs) and the activation and differentiation of T-cells in Takayasu’s arteritis (TAK), using real-time fluorescence quantitative polymerase chain reaction, mRNA abundance of 29 target genes in peripheral blood mononuclear cells (PBMCs) were detected from 27 TAK patients and 10 healthy controls. Compared with the healthy control group, the untreated TAK group and the treated TAK group had an increased mRNA level of TLR2 and TLR4. A sample-to-sample matrix revealed that 80% of healthy controls could be separated from the TAK patients. Correlation analysis showed that the inactive-treated TAK group exhibited a unique pattern of inverse correlations between the TLRs gene clusters (including TLR1/2/4/6/8, BCL6, TIGIT, NR4A1, etc) and the gene cluster associated with T-cell activation and differentiation (including TCR, CD28, T-bet, GATA3, FOXP3, CCL5, etc). The dynamic gene co-expression network indicated the TAK groups had more active communication between TLRs and T-cell activation than healthy controls. BCL6, CCL5, FOXP3, GATA3, CD28, T-bet, TIGIT, IκBα, and NR4A1 were likely to have a close functional relation with TLRs at the inactive stage. The co-expression of TLR4 and TLR6 could serve as a biomarker of disease activity in treated TAK (the area under curve/sensitivity/specificity, 0.919/100%/90.9%). The largest gene co-expression cluster of the inactive-treated TAK group was associated with TLR signaling pathways, while the largest gene co-expression cluster of the active-treated TAK group was associated with the activation and differentiation of T-cells. The miRNA sequencing of the plasma exosomes combining miRDB, DIANA-TarBase, and miRTarBase databases suggested that the miR-548 family miR-584, miR-3613, and miR-335 might play an important role in the cross-talk between TLRs and T-cells at the inactive stage. This study found a novel relation between TLRs and T-cell in the pathogenesis of autoimmune diseases, proposed a new concept of TLR-co-expression signature which might distinguish different disease activity of TAK, and highlighted the miRNA of exosomes in TLR signaling pathway in TAK.

Ginsenosides affect the system of Toll-like receptors in the brain of rats under conditions of long-term alcohol withdrawal

Long-term alcohol consumption causes the development of neuroinflammation in various brain structures. One of the mechanisms involved in this process is the increased activity of TLR-signaling intracellular pathways. Studies confirm the ability of ginseng extract or its individual ginsenosides to reduce the increased activity of TLR-signaling pathways. The aim of our study was to study the effect of the amount of ginsenosides obtained from the extract of the Panax japonicus cell line on the state of the TLR-signaling system in the nucleus accumbens and hippocampus of the rat brain in a model of long-term alcohol consumption during alcohol withdrawal. The results of the study showed that ginsenosides were able to make changes in the TLR signaling system, which has been altered by long-term alcohol consumption. A significant effect of ginsenosides on the level of TLR3 and TLR4 mRNA in the nucleus accumbens was found, while in the hippocampus, ginsenosides significantly affected the level of TLR7 mRNA. The effect of ginsenosides on the level of mRNA of transcription factors and cytokines involved in TLR-signaling was evaluated. Thus, results of our study confirm that ginsenosides are able to influence the state of TLR-signaling pathways, but this effect is multidirectional in relation to different brain structures. In the future, it seems interesting to evaluate the role of individual ginsenosides in relation to genes of TLR-signaling, as well as the effect of ginsenosides on other brain structures.

Effects of Dietary Resveratrol Supplementation on Growth Performance and Anti-Inflammatory Ability in Ducks (Anas platyrhynchos) through the Nrf2/HO-1 and TLR4/NF-κB Signaling Pathways.

Simple SummaryThis study clarified for the first time that the Nrf2 and NF-κB signaling pathways in ducks are related to the antioxidant and anti-inflammatory effects. For the first time, we found that dietary resveratrol (RES) effectively alleviated the inflammatory response caused by lipopolysaccharide (LPS) by reducing oxidative stress and endoplasmic reticulum stress, alternating the blood biochemical index, and restoring the destruction of hepatocyte morphology. The results of this study provide strong evidence that dietary RES improved the anti-inflammatory ability and the growth performance of ducks.The aim of this study was to explore the effect of dietary resveratrol on the growth performance and anti-inflammatory mechanism in ducks. A total of 280 one-day-old specific pathogen-free male ducklings (Anas platyrhynchos) with an average body weight of 35 ± 1 g were randomly divided into two dietary treatment groups with different supplementation levels of resveratrol for growth performance experiments: R0 and R400 (0 and, 400 mg kg−1 resveratrol, respectively). At the age of 28 days, 16 ducks were selected from each treatment group and divided into four subgroups for a 2 × 2 factorial pathological experiment: R0; R400; R0 + LPS; R400 + LPS, (0 mg kg−1 resveratrol, 400 mg kg−1 resveratrol, 0 mg kg−1 resveratrol, 400 mg kg−1 resveratrol + 5 mg lipopolysaccharide/kg body weight). The results showed that resveratrol significantly improved final body weight and average daily gain (p < 0.01) and alleviated the lipopolysaccharide-induced inflammatory response with a reduction in IL-1β and IL-6 in the plasma and the liver (p < 0.05). Resveratrol improved mRNA levels of Nrf2 and HO-1 and decreased the mRNA levels of TLR4 and NF-κB in duck liver (p < 0.05). Dietary resveratrol can improve growth performance and reduce inflammation through the Nrf2/HO-1 and TLR4/NF-κB signaling pathways in duck.