MyD88 mRNA Research Articles

LncRNA ANRIL play an important role in renal injury induced by diabetes

IntroductionThe aim of this study was to discuss the effects and mechanisms of lncRNA in renal injury induced by diabetes by vivo and vitro study.Material and methodsCollecting healthy and DN patients, and measuring lncRNA ANRIL expression by RT-qPCR assay in serum. Measuring the ANRIL expression in normal, 10d and 20d model rats in kidney. Measuring LDH, MDA, SOD, IL-6 and TNF-α concentration by Elisa assay, evaluating the kidney pathology by HE staining, cell apoptosis by TUNEL assay, relative proteins expression by IHC assay and relative mRNA expression by RT-qPCR assay. In the cell experiment, Measuring LDH, MDA, SOD, IL-6 and TNF-α concentration by Elisa assay, measuring the cell proliferation by CCK-8, cell apoptosis by flow cytometry, relative mRNA and proteins expression by RT-qPCR and WB assay. The NF-κB(p65) nuclear volume of difference groups were evaluated by cell immunofluorescence.ResultsCompared with healthy people, lncRNA ANRIL was significantly increased in DN patients. Compared with sham group, the ANRIL expression, LDH, MDA, IL-6 and TNF-α concentration was significantly increased, SOD concentrations and cell apoptosis rate were significantly depressed with day increasing. TLR4, MyD88 and NF-κB(p65) mRNA and proteins expressions were significantly up-regulation.ConclusionslncRNA ANRIL knockdown had effects to improve renal injury induced by diabetes via regulation TLR4/NF-κB(p65).

Clostridium butyricum Supernatant Regulates the Expression of RORγt in HCT-116 Cells by Inhibiting the TLR2/MyD88/NF-κB Signaling Pathway.

In this study, we treated HCT-116 cells with Clostridium butyricum (C. butyricum) supernatant and observed its effects on the TLR2/MyD88/NF-κB signaling pathway and RORγt, to further explore the possible immune regulatory mechanism of C. butyricum. Our results showed that C. butyricum supernatant downregulated the mRNA and protein levels of TLR2, MyD88, NF-κBp65, and RORγt in HCT-116 cells and the protein levels of phospho-NF-κBp65. Partial blockage of TLR2 by CD282 weakened the inhibitory effects of C. butyricum supernatant on the above pathway components. Those component levels were still inhibited by C. butyricum supernatant after Pam3CSK4 activation of TLR2. In summary, C. butyricum supernatant can inhibit the TLR2/MyD88/NF-κB signaling pathway and the expression of RORγt in HCT-116 cells. These effects are at least partly achieved through inhibition of TLR2.

Survival and gene expression responses in immune challenged larval lake sturgeon

Larval lake sturgeon, Acipenser fulvescens, reared in hatcheries for stock enhancement of wild populations may be susceptible to early opportunistic bacterial infection. Thus, we examined survival and whole-body mRNA expression of both stress- and immune-related genes (MyD88, IL-1β, StAR, GR1, and HSP70) in 30 days post fertilization larval lake sturgeon following immune challenge with lipopolysaccharides (LPS). Larval sturgeon were exposed to 0, 25, 50, 100, 150, and 200 μg ml−1 LPS and sampled after 30 min, 4 h, and 48 h. Mortality was zero in 0 and 25 μg ml−1 LPS; 37.5% in 50 μg ml−1 LPS and 100% in the higher concentrations. Expression of MyD88 and StAR mRNA were positively correlated and increased with time in the 50 μg ml−1 LPS treatment. There was an influence of both treatment and time on IL-1β mRNA, with expression 10-fold higher than controls after 4 h. Expression of HSP70 mRNA was suppressed within 30 min of 50 μg ml−1 LPS exposure and remained so throughout the time course. Correlated mRNA expression of GR1 with MyD88, StAR and IL-1β suggests a potential relationship between the innate immune and glucocorticoid responses of larval lake sturgeon during this early developmental stage. Data presented suggest that larval lake sturgeon largely responded with predicted changes in gene expression of immune related and stress response genes following LPS challenge. This study provides a foundation for future research examining the effects of hatchery and naturally occurring stressors on the immune responses of larval lake sturgeon.

Astragaloside IV Improves High-Fat Diet-Induced Hepatic Steatosis in Nonalcoholic Fatty Liver Disease Rats by Regulating Inflammatory Factors Level via TLR4/NF-κB Signaling Pathway.

Objective: Astragaloside IV (AS-IV) is the primary bioactive component purified from Astragalus membranaceus which is one of the traditional Chinese medicines. Research studies found that AS-IV has significant pharmacological effects on focal cerebral ischemia/reperfusion, cardiovascular disease, pulmonary disease, liver cirrhosis, and diabetic nephropathy, but little is known about the effects of AS-IV on nonalcoholic fatty liver disease (NAFLD). In this study, we investigated whether AS-IV has beneficial effects on NAFLD in rats and its potential mechanisms. Methods: Male SD rats were fed with high-fat diet (HFD) for 12 weeks to establish NAFLD rat model, and then, the rats were divided into five groups. The control group rats were fed with normal diet for 12 weeks and then were given normal saline (1.0 ml kg−1 day−1) by intragastric administration for 4 weeks. The model group rats were fed with HFD for 12 weeks and then were given normal saline (1.0 ml kg−1 day−1) by intragastric administration for 4 weeks. The AS-IV-L, AS-IV-M, and AS-IV-H groups were treated with 20, 40, and 80 mg kg−1 day−1 of AS-IV by intragastric administration for 4 weeks and given HFD diet. Then, we detected serum transaminase (ALT, AST), blood lipid (TG, TC), inflammatory cytokines (IL-6, IL-8 and TNF-α), liver histology(NAFLD activity score), TLR4/MyD88 signaling pathway in liver tissue. Results: We found AS-IV significantly reduced serum levels of AST, ALT, TG, TNF-α, IL-6, and IL-8 in NAFLD rats and downregulate the expression of TLR4 mRNA, MyD88 mRNA, NF-κB mRNA, and proteins in liver tissue. Moreover, AS-IV could significantly reduce the NAFLD activity score of NAFLD rat liver. Conclusion: In this study, we demonstrated that AS-IV have a protective effect on NAFLD by inhibiting TNF-α, IL-6 and IL-8 levels and down-regulating TLR4, MyD88 and NF-κB expression in rat liver tissues.

Effects of different temperatures and protein levels on growth performance, physiological response and expression of immune-related genes of juvenile oriental river prawn (Macrobrachium nipponense)

To investigate effects of different temperatures and protein levels on growth performance, physiological response and expression of immune-related genes of M. nipponense. A 3 × 3 factorial design feeding trail was conducted with three levels of water temperature (20 °C, 25 °C and 30 °C) and three levels of dietary protein (P34, P38 and P42) for 8 weeks. The results indicated that weight gain rate (WGR) was significantly affected by water temperature and their interaction. WGR at 25 °C was significantly higher than that at 20 °C and 30 °C. WGR of prawns fed with 38% protein diets was higher than that of the other two treatments at 20 °C. Alanine aminotransferase (ALT) and complement component 4 (C4) were significantly influenced by water temperature and their interaction. Superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA) and inducible nitric oxide synthase (iNOS) were significantly affected by their interaction. SOD, CAT, MDA and NO were increased with increasing dietary protein level at 20 °C. P42 diets positively increased the levels of SOD, CAT, NO, iNOS and reduced the level of MDA at 25 °C. However, P34 diets positively increased the levels of SOD, CAT, NO and iNOS at 30 °C. The interaction significantly affected Toll, Myd88, Dorsal, IMD, Relish, HSP60 and HSP70 mRNA levels. P38 diets significantly up-regulated the Toll, Myd88, Dorsal, IMD and Relish mRNA levels at 20 °C. Also, P42 diets positively increased their mRNA levels at 25 °C, while negatively down-regulated at 30 °C. Our study indicated that based on WGR, the optimal dietary protein requirement of shrimp is 38%, 42% and 34% at 20 °C, 25 °C and 30 °C, respectively.

Is there an Inflammation Role for MYD88 in Rheumatoid Arthritis?

Rheumatoid arthritis (RA) is an autoimmune and inflammatory disease with strong genetic influence, especially upon immune response components. Several cytokines from the toll-like receptors activation pathway display recognized role for RA establishment. However, few studies have verified the role of key mediators such as MYD88 gene and its genetic variants. In the present study, we aim to evaluate the rs6853 functional single-nucleotide variation (SNV) role in RA etiopathogenesis, clinical severity status, and its impact in MYD88 mRNA levels and IL-lβ protein levels. For the association study, a total of 423 RA patients and 346 health individuals, enrolled as control, from Northeast and Southeast Brazil were genotyped using specific Taqman probe. For the gene expression assays, we performed a MYD88 rs6853 genotype-guided monocyte cell culture divided into non-stimulated and lypopolysaccharides (LPS)-stimulated cells from healthy individuals. MYD88 gene expression was measured using primer specifics while IL-1β levels were evaluated by ELISA. We observed that A allele and AA genotype were associated to an increased risk to RA development (OR = 1.60; 95% CI 1.24-2.08; p = 0.0004/OR = 2.83; 95% CI 1.25-6.41; p = 0.0152). The AA genotype exhibited lower MYD88 mRNA levels than GG genotype in non-stimulated monocyte cell culture (FC-3.83; p = 0.003). Additionally, we verified an increase of IL-1β levels when AA genotype non-stimulated monocytes were compared to AA genotype LPS-stimulates (p = 0.021). In summary, MYD88 rs6853 polymorphism associated to RA development in our Brazilian cohort and showed influence upon MYD88 mRNA levels' expression and IL-lβ production.

Evodiamine protects against airway remodelling and inflammation in asthmatic rats by modulating the HMGB1/NF-κB/TLR-4 signalling pathway

Context Evodiamine, which is isolated from Evodia rutaecarpa (Rutaceae), possess strong anti-inflammatory, immunomodulatory, and antibacterial properties. Objective The protective effects of evodiamine in asthma were evaluated. Materials and methods Thirty-two Sprague-Dawley (SD) rats were used, asthma was induced by injecting intraperitoneally with a mixture of Al(OH)3 (100 mg) and ovalbumin (OA; 1 mg/kg), further exposing them to a 2% OA aerosol for 1 week. All animals were divided into four groups: control, asthma, and evodiamine 40 and 80 mg/kg p.o. treated group. Serum levels of inflammatory cytokines, interferon gamma (IFN-γ), and immunoglobulin E (IgE) and infiltrations of inflammatory cells in the bronchoalveolar lavage fluid (BALF) of the animals were determined. The thickness of the smooth muscle layer and airway wall in the intact small bronchioles of asthmatic rats was examined as well. Results Cytokine levels in the serum and BALF were lower in the evodiamine-treated group than in the asthma group. Evodiamine treatment reduced IgE and IFN-γ levels as well as the inflammatory cell infiltrate in the lung tissue of asthmatic rats. The thickness of the smooth muscle layer and airway wall of intact small bronchioles was less in the evodiamine-treated group than in the asthma group. Lower levels of TLR-4, MyD88, NF-κB, and HMGB1 mRNA in lung tissue were measured in the evodiamine-treated group than in the asthma group. Discussion and conclusion The effect of evodiamine treatment protects the asthma, as evodiamine reduces airway inflammation and remodelling in the lung tissue by downregulating the HMGB1/NF-κB/TLR-4 pathway in asthma.

Therapeutic Role of miR-30a in Lipoteichoic Acid-Induced Endometritis via Targeting the MyD88/Nox2/ROS Signaling.

Staphylococcus aureus (S. aureus), a notorious pathogenic bacterium prevalent in the environment, causes a wide range of inflammatory diseases such as endometritis. Endometritis is an inflammatory disease in humans and mammals, which prolongs uterine involution and causes great economic losses. MiR-30a plays an importan trole in the process of inflammation; however, the regulatory role of miR-30a in endometritis is still unknown. Here, we first noticed that there was an increased level of miR-30a in uterine samples of cows with endometritis. And then, bovine endometrial epithelial (BEND) cells stimulated with the virulence factor lipoteichoic acid (LTA) from S. aureus were used as an in vitro endometritis model to explore the potential role of miR-30a in the pathogenesis of endometritis. Our data showed that the induction of the miR-30a expression is dependent on NF-κB activation, and its overexpression significantly decreased the levels of IL-1β and IL-6. Furthermore, we observed that the overexpression of miR-30a inhibited its translation by binding to 3′−UTR of MyD88 mRNA, thus preventing the activation of Nox2 and NF-κB and ROS accumulation. Meanwhile, in vivo studies further revealed that upregulation of miR-30a using chemically synthesized agomirs alleviates the inflammatory conditions in an experimental mouse model of endometritis, as indicated by inhibition of ROS and NF-κB. Taken together, these findings highlight that miR-30a can attenuate LTA-elicited oxidative stress and inflammatory responses through the MyD88/Nox2/ROS/NF-κB pathway and may aid the future development of novel therapies for inflammatory diseases caused by S. aureus, including endometritis.

Early pregnancy affects the expression of toll-like receptor pathway in ovine thymus

Toll-like receptors (TLRs) participates in regulation of the maternal immune tolerance during pregnancy, and the thymus is critical for the adaptive immune system. This study hypothesized whether early pregnancy affected the expression of toll-like receptor pathway in the thymus of ewes. In this study, expression of TLRs, tumor necrosis factor receptor associated factor 6 (TRAF6), interleukin 1 receptor associated kinase 1 (IRAK1) and myeloid differentiation primary response gene 88 (MyD88) was detected in maternal thymus during early pregnancy in sheep. Ovine thymuses were collected on day 16 of the estrous cycle, and days 13, 16 and 25 of pregnancy, and expression of TLR members was analyzed by real-time quantitative PCR, western blot and immunohistochemistry analysis. The results revealed that there were decreases in the expression of the mRNA and proteins of TLR2, IRAK1, TRAF6 and MyD88, but increase in TLR5 mRNA and protein. Furthermore, expression of TLR3 and TLR4 proteins peaked at days 13 and 16 of gestation, and MyD88 protein was located in the epithelial reticular cells and thymic corpuscles. In summary, TLR signaling is implicated in regulation of maternal thymic immune, which may be via downregulation of TLR2, IRAK1, TRAF6 and MyD88 during early pregnancy in sheep.

Carvacrol inhibits the excessive immune response induced by influenza virus A via suppressing viral replication and TLR/RLR pattern recognition

Ethnopharmacological relevanceCarvacrol, a monoterpene phenol from Mosla chinensis Maxim, which is a commonly Chinese herbal medicine. The most important pharmacology of it is dispelling exogenous evils by increasing perspiration. And it is the gentleman medicine in the Chinese herbal compound prescription of Xin-Jia-Xiang-Ru-Yin, mainly for the treatment of summer colds with dampness including influenza virus A infection. Aim of the studyOur preliminary study verified that the Xin-Jia-Xiang-Ru-Yin could inhibit acute lung injury of mice with influenza virus A infection. And there have been some reports implicating the high antimicrobial activity of carvacrol for a wide range of product preservation, but little research including the effects of it on viral infection. The aim of this study was to reveal the antiviral effects of carvacrol, the main constituent in Mosla chinensis Maxim. Materials and methodsInitially, C57BL/6 mice were grouped and intranasally administered FM1 virus to construct viral infection models. After treatment with ribavirin and carvacrol for 5 days, all mice were euthanized, and specimens were immediately obtained. Histology, flow cytometry and Meso Scale Discovery (MSD) analysis were used to analyze pathological changes in lung tissue, the expression levels of cytokines and the differentiation and proportion of CD4+ T cells subsets, while Western blot and qRT-PCR were used to detect the expression of related proteins and mRNA. ResultsCarvacrol attenuated lung tissue damage, the proportions of Th1, Th2, Th17 and Treg in CD4+ T cells and the relative proportions of Th1/Th2 and Th17/Treg cells. Carvacrol inhibited the expression of inflammation-associated cytokines including IFN-γ, IL-2, IL-4, IL-5, IL-12 and TNF-ɑ, IL-1, IL-10, IL-6. Decreased levels of TLR7, MyD88, IRAK4, TRAK6, NF-κB, RIG-I, IPS-I and IRF mRNA in carvacrol-treated mice were observed comparing to the mice in VC group. Further, the total expression of RIG-I, MyD88 and NF-κB proteins had increased significantly in the VC group but reduced obviously in the group treated with ribavirin or carvacrol. ConclusionsThese results indicate that carvacrol is a potential alternative treatment for the excessive immune response induced by influenza virus A infection, the cold-fighting effect of Mosla chinensis Maxim may depend on the anti-virus of carvacrol.

Ability of Post-treatment Glycyrrhizic Acid to Mitigate Cerebral Ischemia/Reperfusion Injury in Diabetic Mice.

BackgroundDiabetes aggravates cerebral ischemia/reperfusion (I/R) injury by increasing inflammatory reactions, but its specific mechanism is currently unclear.Material/MethodsDiabetes was induced in mice with a high-fat diet combined with streptozotocin. These mice were subjected to transient middle cerebral artery occlusion (tMCAO) for 60 min, followed by reperfusion for 24–72 h and post-treatment glycyrrhizic acid (GA). Control and diabetic mice were randomly allocated to 8 groups of 18 mice each. Blood glucose, brain infarction, brain edema, and neurological function were monitored. Necrosis was determined by Nissl staining, loss of neurons by immunofluorescent (IF) staining for NeuN, and activation of inflammatory microglia by IF staining for Iba-1. Levels of HMGB1, TLR4, Myd88, and NF-κB mRNA and protein in ischemic brain were determined by qRT-PCR and western blotting, respectively, and serum concentrations of IL-1β, IL-6, and TNF-α by ELISA.ResultsInfarction volume, brain edema, and neurological function after tMCAO were significantly aggravated in diabetes, but ameliorated by post-treatment GA. GA also reduced neuronal loss and microglial activation. Cerebral Myd88 level showed a positive correlation with neurological scores. GA suppressed the expression of Myd88 and a proinflammatory pathway that included Myd88, HMGB1, TLR4, and NF-κB, as well as reducing serum concentrations of IL-1β, IL-6, and TNF-α.ConclusionsPost-treatment inhibited inflammatory responses and provided therapeutic benefits in diabetic mice with cerebral I/R injury, suggesting that GA may be a candidate drug to suppress cerebral I/R in diabetic patients.

Ketamine alleviates HMGB1-induced acute lung injury through TLR4 signaling pathway.

Acute lung injury (ALI) is a common critical respiratory disease that seriously threatens human health. Ketamine has good anti-inflammatory and immune-regulating properties that can delay the lung injury process. High mobility group box protein 1 (HMGB1) plays an important role in the occurrence, development and treatment of ALI. Toll-like receptor 4 (TLR4) is the receptor for HMGB1. The aim of this study was to determine the role of the HMGB1 TLR4 signaling pathway in the treatment of ALI using ketamine. A total of 30 healthy, male, 8-week-old Sprague-Dawley rats were randomly, equally divided into a control group, an lipopolysaccharide (LPS) group and a ketamine group. In order to establish a rat ALI model, 15 mg/kg of LPS was injected into the femoral veins. Ketamine was intravenously injected (10 mg/kg) into the experimental group rats. The rats were euthanized 24 h after modeling and lung tissue samples were collected. Western blot was used to test TLR4, MyD88, TRAF-6, LOX-1, and HMGB1 protein expression in the lung tissue. Real-time polymerase chain reaction (RT-PCR) was performed to detect TLR4, MyD88, TRAF-6, LOX-1, and HMGB1 mRNA levels. Compared with the controls, the LPS group had significantly higher TLR4, MyD88, TRAF-6, LOX-1, and HMGB1 mRNA and protein levels (p < 0.05). These levels were significantly lower after ketamine intervention in comparison with the LPS group (p < 0.05). A positive correlation was found between TLR4 and HMGB1 expression in the LPS and ketamine groups (r = 0.952, p < 0.001; r = 0.941, p < 0.001). Ketamine attenuates HMGB1-induced ALI, possibly by regulating the TLR4 signaling pathway.

Cyclic Peptide Extracts Derived From Pseudostellaria heterophylla Ameliorates COPD via Regulation of the TLR4/MyD88 Pathway Proteins.

We have explored the method of extraction and purification of cyclic-peptide extract (CPE) from Pseudostellaria heterophylla (Miq.) Pax. (Taizishen, TZS), characterized the structure about cyclic-peptide compounds and investigated the biological activity of CPE attenuating chronic obstructive pulmonary disease (COPD) in rats. The CPE from TZS was obtained by ethyl acetate, petroleum ether, hot water extraction, and alcohol-precipitation. Cyclic-peptide structures were distinguished using ultra-high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS). Rats were induced by solid combustibles smoke (SCS) for the COPD model, and the anti-COPD activity of CPE was detected using lung airway resistance and dynamic lung compliance, as well as pulmonary tissue hematoxylin and eosin (HE) staining. The relevant inflammatory cytokines were assayed by enzyme-linked immunosorbent assay (ELISA). CPE obtained from TZS contained 12 cyclic-peptide constituents; the purity was up to 92.94%. CPE (200, 400, or 500 mg/kg/day) was given to SCS-induced COPD model rats orally for 15 days. The results showed that in rats given CPE (400 mg/kg/day) there was a sharp fall in lung airway resistance but a rise in dynamic lung compliance. The image analysis of lung tissue sections suggested that CPE could decrease the degree of alveolar destruction (p <0.05), alleviate lung inflammation, increase alveolar space, and improve the infiltration of inflammatory cells. CPE was found to reduce the levels of TNF-α, but increase IL-10, adjusting multiple cytokines in rat serum; the TLR4 mRNA, MyD88 mRNA and AP-1 mRNA levels, the expressing levels of p-JNK, p-p38 and p-TAK1 protein were significantly down regulated in rat alveolar macrophages. CPE intervention could improve the pulmonary ventilation function on COPD rats, which may be related to its effect in inhibiting the abnormal activation of the TLR4-MyD88-JNK/p38 pathway. This is the first report that the CPE of TZS lessens the severity of COPD episodes. The new preparation process of CPEs implements the anticipated goal, which is to refine CPE and actualize quality control.

Traditional Chinese Medicine Li-Zhong-Tang accelerates the healing of indomethacin-induced gastric ulcers in rats by affecting TLR-2/MyD88 signaling pathway

Ethnopharmacological relevanceLi-Zhong-Tang (LZT) is a well-known Chinese herbal formulation first described in one of traditional Chinese medicine (TCM) scriptures, Treatise on Febrile Diseases. LZT has been commonly prescribed for the treatment of various gastrointestinal diseases for over 1800 years, and has demonstrated pronounced therapeutic effects on patients with gastric ulcers. Aim of the studyThe present study aimed to scientifically evaluate protective effects of LZT on indomethacin (IND)-induced gastric injury in rats and to elucidate whether LZT exerts its gastro-protective effects via enhancing mucosal immunity by regulating TLR-2/MyD88 signaling pathway. Material and methodsGastric ulcers were induced in male Sprague-Dawley (SD) rats with a single oral dose of 150 mg/kg IND. Ulcer index (UI) and curative index (CI) were evaluated. Histopathological examinations were performed and microscopic score (MS) was macroscopically calculated. The volume of gastric juice, free acidity, total acidity, and gastric pH was measured. The gastroprotective and inflammatory biomarkers including levels of nitric oxide (NO), tumor necrosis factor-α (TNF-α), prostaglandin E2 (PGE2), and malondialdehyde (MDA) were determined. Expression levels of TLR-2 and MyD88 mRNA were assessed by qRT-PCR. The expression, distribution, and co-localization of TLR-2 and MyD88 protein were determined by Western blot, immunohistochemistry, and immunofluorescence, respectively. ResultsInduction of gastric ulcers in rats resulted in very significantly increased UI and elevated volume and acidity of gastric juice, which were markedly attenuated by LZT treatment. Microscopic examinations of the IND-induced gastric ulcers revealed severe gastric hemorrhagic necrosis, submucosal edema, and destruction of epithelial cells, which were significantly attenuated in LZT-treated rats. Moreover, treatment with LZT remarkably increased gastric mucosal levels of PGE2 and NO, and lowered highly elevated levels of TNF-α and MDA in gastric ulcerative rats. Mechanistically, LZT inhibited mRNA and protein expression of TLR-2 and MyD88 and enhanced immune function in gastric mucosa. Immunohistochemical analyses and immunofluorescent detection further confirmed a markedly decreased co-localization of TLR-2 and MyD88 protein in the gastric mucosa of LZT-treated rats as compared to that of gastric ulcerative rats. ConclusionsThese findings indicate that LZT alleviates serious gastric mucosal ulcerations induced by IND. Protective effects of LZT on gastric ulcers are believed to be associated with the intensification of the anti-oxidative defense system, mitigation of proinflammatory cytokines, stimulation of the production of cytoprotective mediators, and improvement of the mucosal immunity through TLR-2/MyD88 signaling pathway.

Molecular cloning and expression analysis of myd88 from oriental weatherfish (Misgurnus anguillicaudatus) in response to bacterial challenge

Myeloid differentiation factor 88 (Myd88) plays an important role in both innate and adaptive immune response. In this study, the full-length complementary DNA (cDNA) of myd88 from Misgurnus anguillicaudatus was characterized. The myd88 cDNA is 1333 bp in length and contains an 855 bp open reading frame encoding a predicted protein of 284 amino acids. The predicted protein possesses typical Myd88 domain structural features including a death domain in the N-terminus, and box 1, 2, and 3 motifs of the Toll/IL-1 receptor domain in the C-terminus. Quantitative real-time PCR (qRT-PCR) revealed that myd88 messenger RNA (mRNA) was ubiquitously expressed in all examined tissues, especially highly in brain, kidney, blood, intestines and liver. qRT-PCR and western blotting were used to determine the mRNA and protein levels of Myd88 after Aeromonas veronii challenge, respectively. The Myd88 was remarkably upregulated in response to infection of A. veronii. These results suggested that Myd88 may play a vital role during the immune response of M. anguillicaudatus against bacterial infection.

Hermetia illucens larvae as a Fishmeal replacement alters intestinal specific bacterial populations and immune homeostasis in weanling piglets

Hermetia illucens larvae meal (HILM) are rich in proteins and chitin, and represent an innovative feed ingredient for animals. However, little is known about the intestinal bacteria and immune homeostasis response of HILM as a fishmeal replacement on weanling piglets. Thus, this study aimed to investigate the changes in specific ileal and cecal bacterial populations and their metabolic profiles, and ileal immune indexes in weanling piglets fed with a diet containing HILM. A total of 128 weanling piglets were fed either a basal diet or 1 of 3 diets with 1%, 2%, and 4% HILM (HI0, HI1, HI2, and HI4, respectively). Each group consisted of 8 pens (replicates), with 4 pigs per pen. After 28 d of feeding, 8 barrows per treatment were euthanized, the ileal and cecal digesta, and ileal mucosa were collected for analyzing bacterial population and metabolic profiles, and immune indexes, respectively. Results showed that HILM increased (P < 0.05, maximum in HI2) the number of Lactobacillus and Bifidobacterium in the ileum and cecum, but quadratically decreased (P < 0.05, minimum in HI2) the number of Escherichia coli. In the cecum, the number of Firmicutes, Ruminococcus, Clostridium cluster IV, and Prevotella showed a quadratic response to increasing (P < 0.05, maximum in HI2) HILM levels. Lactate and butyrate concentrations in the ileum and cecum were quadratically increased (P < 0.05, maximum in HI2) with increasing HILM levels. In the cecum, the amines, phenol, and indole compounds concentrations were quadratically decreased (P < 0.05, minimum in HI2) with increasing HILM levels, while total short-chain fatty acids and acetate concentrations were quadratically increased (P < 0.05, maximum in HI2). In the ileum, the TLR4, NF-κB, MyD88, and TNF-α mRNA expressions were quadratically decreased (P < 0.05, minimum in HI2) with increasing HILM levels, while the mRNA expression of IL-10, barrier function (MUC1, ZO-1, Occludin, and Claudin-2), and development-related genes (IGF-1, GLP-2, and EGF) was quadratically increased (P < 0.05, maximum in HI2). Furthermore, the changes in the mucosal gene expression were associated with changes in the bacterial populations and their metabolites. Collectively, these results showed that a diet supplemented with 2% HILM affected specific bacterial populations and metabolic profiles, and maintained ileal immune status. These findings provide new insights into the use of insect meal as a suitable alternative protein source for swine feeding.

Effects of Carvedilol on the Expression of TLR4 and its Downstream Signaling Pathway in the Liver Tissues of Rats with Cholestatic Liver Fibrosis.

This study was designed to investigate the effects of carvedilol on the expression of TLR4 and its downstream signaling pathway in the liver tissues of rats with cholestatic liver fibrosis and provide experimental evidence for clinical treatment of liver fibrosis with carvedilol. A total of fifty male Sprague Dawley rats were randomly divided into five groups (10 rats per group): sham operation (SHAM) control group, bile duct ligation (BDL) model group, low-dose carvedilol treatment group (0.1mg·kg-1·d-1), medium-dose carvedilol treatment group (1mg·kg-1·d-1), and high-dose carvedilol treatment group (10mg·kg-1·d-1). Rat hepatic fibrosis model was established by applying BDL. Forty-eight hours after the operation, carvedilol was administered twice a day. The blood and liver were simultaneously collected under the aseptic condition for further detection in two weeks after the operation. The alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBil) and albumin (Alb) in serum were measured. HE and Masson staining were used to determine hepatic fibrosis degree. Hydroxyproline assay was employed to detect liver collagen synthesis. Western Blot was used to measure the expression of TLR4, NF-κB p65 and β-arrestin2 protein. Quantitative analysis of TLR4, MyD88, TNF-α and IL-6 mRNA was performed by Realtime-PCR. Compared with the SHAM group, the BDL group showed obvious liver injury, increased levels of inflammatory factors, and continued progression of liver fibrosis. The above changes in the BDL group were alleviated in the carvedilol treatment groups. The improvement effects augmented as dosages increased. In addition, compared with the BDL group, the reduction of the expressions of TLR4, MyD88 and NF-κB p65 in liver tissues and the increase of the expression of β -arrestin2 in the high-dose carvedilol group were more significant. Carvedilol can reduce the release of inflammatory mediators by downregulating TLR4 expression and inhibiting its downstream signaling pathway, thus playing a potential therapeutic role in cholestatic liver fibrosis.

Ethanol extracts from Ilex pubescens promotes cerebral ischemic tolerance via modulation of TLR4-MyD88/TRIF signaling pathway in rats

Ethnopharmacological relevancePubescent Holly Root is the dry root of Ilex pubescens Hook. et Arn. It is clinically using in the treatment for stroke and coronary artery disease. It remains unclear whether the ethanol extracts of Ilex pubescens(IPEE) treatment can promote cerebral ischemic tolerance (CIT) and exert endogenous neuroprotective effects and thus to alleviate the nerve injury caused by the subsequent persistent cerebral ischemic attacks. Aim of the studyTo investigate the effects of IPEE on CIT and its underlying molecular mechanisms. Materials and methods: Adult male Wistar rats were used in the present study. The bilateral common carotid arteries were blocked for 10 min followed a subsequent reperfusion to create the cerebral ischemic preconditioning (CIP); After 3 days post CIP, rats were subjected to middle cerebral artery occlusion/reperfusion (MCAO/R)-injury. Rats were continuously fed with IPEE for 5 days throughout the experiment period at the dose of 100 mg/kg and 200 mg/kg, respectively. Then, the brain infarct volume, histopathology, neurological deficits, and the gene/protein expression related with the TLR4-MyD88/TRIF signaling pathway were evaluated after 24 h of MCAO/R experiment. ResultsIPEE pretreatment significantly reduced the cerebral infarct volume, the neurological deficit scores, and the plasma level of neuron specific enolase (NSE) at the dose of 100 mg/kg. Meanwhile, IPEE pretreatment significantly decreased the levels of inflammatory cytokines including TNF-α, IL-6, MCP-1, MIP-1α and RANTES, while it increased the levels of anti-inflammatory cytokines, such as IL-10 and TGF-β, when compared with the group with CIP treatment alone. Moreover, the effect of IPEE treatment on CIT was in a dose-dependent manner, showing as a better effect in the group pretreated with IPEE with the dose of 100 mg/kg than that in group pretreated with IPEE with the dose of 200 mg/kg. In addition, IPEE pretreatment significantly inhibited the expressions of MyD88 mRNA and the protein expression of COX-2 and NF-κBp65, while it strengthened the expressions of TRIF mRNA and protein. The effects of IPEE pretreatment on the expression of these genes were better than that in the group treated with CIP alone. ConclusionsThe present study demonstrates that IPEE pretreatment can enhance cerebral ischemic tolerance with a underlying mechanism involved in the toll-like receptor 4 (TLR4) signaling pathway through inhibiting the production of proteins or cytokines in the downstream of MyD88 and activating TRIF dependent anti-inflammatory pathways.

Regulatory effect of Di'ao Xinxuekang on TLR4/MyD88/NF-κB signaling pathway in atherosclerotic rats

The aim of this paper was to observe the effect of Di'ao Xinxuekang(DXXK) on TLR4/MyD88/NF-κB signaling pathway in atherosclerotic rats, and to explore its anti-atherosclerotic mechanism. Sixty SD rats were randomly divided into normal group, model group, atorvastatin group(4.0 mg·kg~(-1)), and DXXK groups(100, 30, 10 mg·kg~(-1)), with 10 rats in each group. The atherosclerosis model was induced by high fat diet plus vitamin D_2. Experimental drugs were administered intragastrically once daily for 8 weeks starting from the 9 th week. Biochemical analyzers were used to detect levels of triglyceride(TG), total cholesterol(TC), low-density lipoprotein cholesterol(LDL-C) and high-density lipoprotein cholesterol(HDL-C) in blood lipid. The levels of serum tumor necrosis factor(TNF)-α, interleukin(IL)-6 and IL-1β were detected by ELISA. Pathological changes of aortic tissues were observed by using Sudan Ⅳ and HE staining. The mRNA and protein expressions of TLR4, MyD88 and NF-κB p65 in aortic tissues were detected by RT-PCR and Western blot, respectively. As compared with the model group, TC, TG, and LDL-C levels in serum were significantly decreased, HDL-C content was significantly increased, and levels of TNF-α, IL-6, and IL-1β in serum were significantly decreased in atorvastatin group and DXXK high and middle dose groups. Aortic lesions in atorvastatin group and DXXK group were significantly improved, and the mRNA and protein expressions of TLR4, MyD88, NF-κB p65 in the aorta were decreased. DXXK has a preventive and therapeutic effect on atherosclerosis in rats, and its mechanism may be related to inhibiting inflammatory reaction by regulating TLR4/MyD88/NF-κB signal transduction, thereby inhibiting the progression of atherosclerosis.

Anti-inflammation Effects of Sinomenine on Macrophages through Suppressing Activated TLR4/NF-κB Signaling Pathway.

Sinomenine (SN) has been used in the clinical treatment of systemic lupus erythematosus and rheumatoid arthritis for many years. Studies showed that SN held protective effects such as anti-inflammation, scavenging free radicals and suppressing immune response in many autoimmune diseases. The purpose of the present study is to explore the mechanism of anti-inflammation of SN on lipopolysaccharide (LPS)-induced macrophages activation and investigate whether the TLR4/NF-κB signaling pathway participated in. Macrophages isolated from mouse peritoneal cavity were stimulated by 1 µg/mL LPS for 24 h. And then the cells were treated with various concentrations of SN, TLR4 inhibitor respectively for additional 48 h. Drug toxicity was detected by MTT assay and Transwell experiment was used to assess chemotaxis. Furthermore, TLR4 and MyD88 mRNA levels were detected by real-time PCR. Western blotting was used to examine TLR4, MyD88 and phosphorylated IκB protein expression in macrophages. Immunofluorescence assay was applied to observe p65 NF-κB protein expression in macrophage nucleus. We extracted macrophages with high purity and activity from the abdominal cavity of mice. SN remarkably inhibited the chemotaxis and secretion function of LPS-stimulated macrophages. It also down-regulated both the protein levels of inflammatory cytokines (TNF-α, IL-1β and IL-6) and the RNA and protein levels of the key factors (TLR4, MyD88, P-IκB) in TLR4 pathway. The expression of p65 NF-κB protein in nuclei was down-regulated, which was correlated with a similar decrease in P-IκB protein level. In conclusion, SN can inhibit the LPS induced immune responses in macrophages by blocking the activated TLR4/NF-κB signaling pathway. These results may provide a therapeutic approach to regulate inflammatory responses.