The activation of TLR4-MyD88 signaling promotes hepatic dysfunction and fibrotic changes in SD rats resulting from prolonged exposure to sodium arsenite.

Arsenic, a poisonous metalloid element, is linked to liver diseases, but the exactmechanisms for this process are not yet to be completely elucidated. Toll like receptor 4 (TLR4), acting as a pathogenic pattern recognition receptor, plays a pivotal role in various inflammatory diseases via the myeloid differentiation factor 88 (MyD88) pathway. This study aims to investigate the involvement of the TLR4-MyD88 signaling pathway in liver injury induced by prolonged exposure to sodium arsenite (NaAsO2) in Sprague-Dawley rats. Our research findings demonstratethe activation of TLR4-MyD88 signaling pathway in long-term NaAsO2-exposed rat liver tissues, leading to a significant release of inflammatory factors, which suggests its potential involvement in the pathogenesis of NaAsO2-induced liver injury. We further administered lipopolysaccharide (LPS), a natural ligand of TLR4, and TAK-242, a specific inhibitor of TLR4, to rats in order to validate the specific involvement of the TLR4-MyD88 signaling pathway in NaAsO2-induced liver injury. The results showed that, 1 mg/kg.bw LPS treatment significantly activated TLR4-MyD88 signalling pathway and its mediated pro-inflammatory factors, leading to up-regulation of activation indicators in hepatic stellate cells (HSCs) as well as increased secretion levels of extracellular matrix (ECM) in the liver, and ultimately induced liver fibrosis and dysfunction in rats. Relevantly, subsequent administration of 0.5 mg/kg.bw TAK-242 significantly attenuated the expression levels of TLR4 and its associated proteins, mitigated collagen deposition, and partially improved liver fibrosis and dysfunction caused by NaAsO2 in rats. Our study fully confirms the pivotal role of the TLR4-MyD88 signaling in promoting liver injury induced by NaAsO2, thereby providing a novel molecular target for preventing and treating patients with arsenic poisoning-related liver injury.

The Role of the TLR4-MyD88 Signaling Pathway in the Immune Response of the Selected Scallop Strain "Hongmo No. 1" to Heat Stress.

The innate immunity of marine bivalves is challenged upon exposure to heat stress, especially with increases in the frequency and intensity of heat waves. TLR4 serves a classical pattern recognition receptor in recognizing pathogenic microorganisms and activating immune responses. In this study, three genes, HMTLR4, HMMyD88 and HMTRAF6, were characterized as homologs of genes in the TLR4-MyD88 signaling pathway in the selected scallop strain "Hongmo No. 1". According to RT-PCR, acute heat stress (32 °C) inhibited genes in the TLR4-MyD88 signaling pathway, and LPS stimulation-induced activation of TLR4-MyD88 signal transduction was also negatively affected at 32 °C. ELISA showed LPS-induced tumor necrosis factor alpha (TNF-α) or lysozyme (LZM) activity, but this was independent of temperature. RNA interference (RNAi) confirmed that HMTLR4 silencing suppressed the expression of its downstream gene, whether at 24 °C or at 32 °C. The level of TNF-α and the activity of LZM also decreased after injection with dsRNA, indicating a negative effect on the innate immunity of scallops. Additionally, acute heat stress affected the suppression of downstream gene expression when compared with that at 24 °C, which led us to the hypothesis that heat stress directly influences the downstream targets of HMTLR4. These results enrich the knowledge of scallop immunity under heat stress and can be beneficial for the genetic improvement of new scallop strains with higher thermotolerance.

Dihydrotestosterone mediates the inflammation effect under lipopolysaccharides in bovine endometrial epithelial cells via AR blockading TLR4/MyD88 signaling pathway.

Dihydrotestosterone (DHT) is a potent nonaromatizable 5α-reduced androgen with both positive and negative effect on inflammation process. However, it remains unknown whether DHT can regulate Lipopolysaccharides (LPS)-induced inflammation in bovine endometrial epithelial cells (bEECs). Here, we demonstrated that the DHT biosynthesis ability and androgen receptors (AR) expression is defective in bovine endometrial with endometritis, which aggravates endometrial inflammation. In vitro study, we established a LPS-induced inflammation model in bEECs, and found that DHT inhibited the TLR4 and MyD88 protein as well as TNF-α, IL-1ß, and IL-6 mRNA of bEECs in a dose-dependent manner. Moreover, the anti-inflammation effect of DHT was blocked by AR inhibitor flutamide. Together, we demonstrated that supplementing DHT can alleviate the inflammation response of bEECs caused by LPS, which is associated with AR regulating the inhibition of TLR4/MyD88 signaling pathway.

NLRP3 inflammasome inhibition and M1-to-M2 microglial polarization shifting via scoparone-inhibited TLR4 axis in ovariectomy/D-galactose Alzheimer's disease rat model.

Neuroinflammation mediated by microglia activation is a critical contributor to Alzheimer's disease (AD) pathogenesis. Dysregulated microglia polarization in terms of M1 overactivation with M2 inhibition is involved in AD pathological damage. Scoparone (SCO), a coumarin derivative, displays several beneficial pharmacological effects including anti-inflammatory and anti-apoptotic properties, however, its neurological effect in AD is still elusive. This study investigated the neuroprotective potential of SCO in AD animal model focusing on determining its effect on M1/M2 microglia polarization and exploring the plausible mechanism involved via investigating its modulatory role on TLR4/MyD88/NF-κB and NLRP3 inflammasome. Sixty female Wistar rats were randomly allocated into four groups. Two groups were sham-operated and treated or untreated with SCO, and the other two groups were subjected to bilateral ovariectomy (OVX) and received D-galactose (D-Gal; 150 mg/kg/day, i.p) alone or with SCO (12.5 mg/kg/day, i.p) for 6 weeks. SCO improved memory functions of OVX/D-Gal rats in the Morris water maze and novel object recognition tests. It also reduced the hippocampal burden of amyloid-ß42 and p-Tau, additionally, the hippocampal histopathological architecture was prominently preserved. SCO inhibited the gene expression of TLR4, MyD88, TRAF-6, and TAK-1, additionally, p-JNK and NF-κBp65 levels were significantly curbed. This was associated with repression of NLRP3 inflammasome along with M1-to-M2 microglia polarization shifting as exemplified by mitigating pro-inflammatory M1 marker (CD86) and elevating M2 neuroprotective marker (CD163). Therefore, SCO could promote microglia transition towards M2 through switching off TLR4/MyD88/TRAF-6/TAK-1/NF-κB axis and inhibiting NLRP3 pathway, with consequent mitigation of neuroinflammation and neurodegeneration in OVX/D-Gal AD model.

The anti-inflammatory effect of lutein in broilers is mediated by regulating Toll-like receptor 4/myeloid-differentiation-factor 88 signaling pathway.

The anti-inflammatory role of lutein has been widely recognized, however, the underlying mechanism is still not fully elucidated. Hence, the effects of lutein on the intestinal health and growth performance of broilers and the action of mechanism were investigated. 288 male yellow-feathered broilers (1-day old) were randomly allocated to 3 treatment groups with 8 replicates of 12 birds each, and the control group was fed a broken rice-soybean basal diet, while the test groups were fed a basal diet added with 20 mg/kg and 40 mg/kg of lutein (LU20, LU40), respectively. The feeding trial lasted for 21 d. The results showed that 40 mg/kg lutein supplementation tended to increase ADFI (P = 0.10) and ADG (P = 0.08) of broilers. Moreover, the addition of lutein caused a decreasing trend of gene expression and concentration of proinflammatory cytokines IL-1ß (P = 0.08, P = 0.10, respectively) and IL-6 (P = 0.06, P = 0.06, respectively) and also tended to decrease the gene expression of TLR4 (P = 0.09) and MyD88 (P = 0.07) while increasing gene expression and concentration of anti-inflammatory cytokines IL-4 and IL-10 (P < 0.05) in the jejunum mucosa of broilers. Additionally, lutein supplementation increased the jejunal villi height of broilers (P < 0.05) and reduced villi damage. The experiment in vitro showed that lutein treatment reduced the gene expression of IL-1ß, IL-6, and IFN-γ in chicken intestinal epithelial cells (P < 0.05). However, this effect was diminished after knock-down of TLR4 or MyD88 genes using RNAi technology. In conclusion, lutein can inhibit the expression and secretion of proinflammatory cytokines in the jejunum mucosa and promote intestinal development of broilers, and the anti-inflammatory effect may be achieved by regulating TLR4/MyD88 signaling pathway.

Protective effects and mechanism of Dachengqi decoction on intestinal septic mice / 中国药房

OBJECTIVE To study the protective effects of Dachengqi decoction （DCQD） on intestinal septic mice， and to explore the possible mechanisms from the Toll-like receptor 4（TLR4）/myeloid differentiation factor 88（MyD88） signaling pathway. METHODS The SPF male C57BL/6J mice were randomly divided into Sham group， Sham+DCQD-H group， model （CLP） group， DCQD-L group， DCQD-H group and Positive group. The model of intestinal sepsis was established by cecal ligation and puncture in CLP group， DCQD-L group， DCQD-H group and Positive group. Three days before the operation and seven days after the operation， DCQD-L group and DCQD-H group were given DCQD intragastrically at 4， 8 g/kg （calculated by crude drug）， respectively. Positive group was given ulinastatin intraperitoneally 2 h before operation and 7 d after the operation （at 50 000 U/kg）. In Sham group and Sham+DCQD-H group， only cecum of mice was exposed without ligation and puncture. Sham+DCQD- H group was given DCQD intragastrically （8 g/kg，calculated by crude drug） 3 days before the operation and 7 days after the operation. Both the Sham group and CLP group were given normal saline 0.2 mL intragstrically and intraperitoneally each day， for 10 consecutive days. After the operation， the severity of sepsis was assessed， and the 7 d survival rate of mice was assessed. One hour after the last medication， the levels of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） in serum and ileum of mice were determined； the pathological and morphological changes of mice’s liver， lung， kidney and ileum were observed； mRNA expressions of the TLR4 and MyD88 in ileum were tested. RESULTS Compared with CLP group， sepsis score， the levels of TNF-α and IL-6 in serum and ileum （except for IL-6 in ileum of DCQD-L group）， damage score of the liver， lung， kidney and ileum， mRNA expressions of TLR4 and MyD88 in ileum were all decreased significantly in DCQD-L group and DCQD-H group （P＜0.05 or P＜0.01）， while 7 d survival rate （except for DCQD-L group） was increased significantly （P＜0.05）. The damage to liver tissue in mice was significantly improved， and inflammation infiltration and apoptosis were reduced； lung tissue damage had been alleviated， with varying degrees of improvement in alveolar atrophy， bleeding and edema； the renal tissue damage was improved and weakened dilation of renal tubular lumen was weakened； the damage and edema of ileal tissue were significantly improved. CONCLUSIONS DCQD may exert a protective role on intestinal septic model mice. The mechanism may be related to the inhibition of systemic inflammation， the reduction of multiple organ damage， and down-regulation of TLR4/MyD88 signaling pathway.

Polyphenol-Rich Liupao Tea Extract Prevents High-Fat Diet-Induced MAFLD by Modulating the Gut Microbiota.

The modulation of gut microbiota dysbiosis might regulate the progression of metabolic-associated fatty liver disease (MAFLD). Here, we found that polyphenol-rich Liupao tea extract (PLE) prevents high-fat diet (HFD)-induced MAFLD in ApoE-/- male mice accompanied by protection of the intestinal barrier and downregulation of lipopolysaccharide (LPS)-related Toll-like receptor 4 (TLR4)-myeloid differentiation primary response 88 (MyD88) signaling in the liver. Fecal microbiome transplantation (FMT) from PLE-and-HFD-treated mice delayed MAFLD development significantly compared with FMT from HFD-treated mice. In this case, 16S rRNA gene sequencing revealed that Rikenellaceae and Odoribacter were significantly enriched and that Helicobacter was significantly decreased in not only the HFD+PLE group but also the HFD+PLE-FMT group. Furthermore, the level of 3-sulfodeoxycholic acid was significantly decreased in the HFD+PLE-FMT group compared with the HFD-FMT group. In conclusion, our data demonstrate that PLE could modulate the MAFLD phenotype in mice and that this effect is partly mediated through modulation of the gut microbiota.

Synthesis and anti-inflammatory activity of paeonol derivatives with etherized aryl urea by regulating TLR4/MyD88 signaling pathway in RAW264.7 cell.

Thirty-three novel paeonol etherized aryl urea derivatives (PEUs) were synthesized via a bromination-Williamson Ether Synthesis-deprotection-nucleophilic addition reaction sequence. The structures of PEUs were characterized by LC-MS, HRMS, 1H NMR and 13C NMR spectra. The levels of nitric oxide (NO), tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1ß) in lipopolysaccharide (LPS)-induced RAW264.7 macrophages were initially employed to evaluate the anti-inflammatory effects of all compounds. Remarkably, b16 exhibited a good anti-inflammatory activity at 2.5 µm which is the same as the potency of paeonol at 20 µm. The results of mechanism research displayed that the anti-inflammatory effect of b16 was ascribed to the inhibition of the TLR4/MyD88 signaling pathway and inflammatory factors. Additionally, b16 distinctly reduced the generation of free radicals in macrophages and strikingly increased the mitochondrial membrane potential. According to the structure-activity relationships (SAR) of PEUs, the incorporation of halogens on the benzene ring and the hydrogen of phenol hydroxyl substituted by aryl urea, were beneficial to enhance the anti-inflammatory activities. Molecular docking results illustrated that the binding ability of b16 to TLR4 was stronger than that of paeonol. In summary, the novel aryl urea-derivied paeonol b16 could be a new promising candidate for the treatment of inflammation-related diseases.

Anti-Influenza Effect and Mechanisms of Lentinan in an ICR Mouse Model.

Influenza virus is a serious threat to global human health and public health security. There is an urgent need to develop new anti-influenza drugs. Lentinan (LNT) has attracted increasing attention in recent years. As potential protective agent, LNT has been shown to have anti-tumor, anti-inflammatory, and antiviral properties. However, there has been no further research into the anti-influenza action of lentinan in vivo, and the mechanism is still not fully understood. In this study, the anti-influenza effect and mechanism of Lentinan were studied in the Institute of Cancer Research (ICR) mouse model. The results showed that Lentinan had a high degree of protection in mice against infection with influenza A virus, delayed the emergence of clinical manifestations, improved the survival rate of mice, significantly prolonged the middle survival days, attenuated the weight loss, and reduced the lung coefficient of mice. It alleviated the pathological damage of mice infected with the influenza virus and improved blood indices. Lentinan treatment considerably inhibited inflammatory cytokine (TNF-α, IL-1ß, IL-4, IL-5, IL-6) levels in the serum and lung and improved IFN-γ cytokine levels, which reduced cytokine storms caused by influenza virus infection. The underlying mechanisms of action involved Lentinan inhibiting the inflammatory response by regulating the TLR4/MyD88 signaling pathway. This study provides a foundation for the clinical application of Lentinan, and provides new insight into the development of novel immunomodulators.

Geniposidic acid protects lipopolysaccharide-induced acute lung injury via the TLR4/MyD88 signaling pathway in vitro and in vivo.

BACKGROUND: Acute lung injury (ALI) is a common respiratory disease and is a serious threat to human health due to the lack of effective treatment. Geniposidic acid (GPA) is an iridoid glucoside extracted from Gardeniae jasminoides Ellis and can treat inflammation-related diseases. This study aimed to investigate the regulatory functions of GPA on lipopolysaccharide (LPS)-induced ALI and its potential mechanism, providing effective strategies for the clinical treatment of ALI. METHODS: ALI models were constructed by LPS in Sprague-Dawley rats and pulmonary epithelial cells. The function of GPA was investigated by hematoxylin-eosin staining, lung function assessment, Western blot, Masson staining, and Sirius Red staining, quantitative real-time PCR, enzyme-linked immunosorbent assay, cell counting kit-8 assay, apoptosis analysis, and immunofluorescence assays. RESULTS: Functionally, GPA increased survival, relieved pulmonary epithelial function in response to LPS, repressed pulmonary fibrosis and inflammation caused by ALI in vivo; GPA also repressed pulmonary epithelial cell injury and inflammation induced by LPS in vitro. Mechanistically, GPA decreased the protein levels of TLR4 and MyD88 and accelerated the nuclear export of p65, suggesting that GPA repressed the activation of p65. CONCLUSION: GPA protected LPS-induced ALI through the TLR4/MyD88 signaling pathway.

[Effect of transcutaneous auricular vagus nerve stimulation on the expression of TLR4、MyD88、IL-18 protein in the prefrontal cortex of depression rats].

OBJECTIVE: To observe the effect of transcutaneous auricular vagus nerve stimulation (taVNS) on the depressive behavior in depression rats, so as to explore its mechanism underlying improvement of depression. METHODS: A total of 24 male Sprague Dawley rats were randomly divided into normal, depression model, taVNS, tnVNS (transcutaneous auricular none-vagus nerve stimulaton) groups (n=6 in each group). The depression model was established by chronic unpredictable mild stimulation combined with solitary raising for 35 consecutive days. After 14 days modeling, transcutaneous electrostimulation (2 mA, 2 Hz/15 Hz) was applied to auricular concha or auricular margin, respectively. Each intervention was conducted for 30 minutes, once daily for 21 days. The depression-like behavior was evaluated by forced swimming immobility time and body weight. The expression levels of Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88) and interleukin-18 (IL-18) protein in the prefrontal cortex were detected by Western blot. RESULTS: Following modeling, the increase amount of body weight was decreased, the forced swimming immobility time and expression TLR4, MyD88 and IL-18 protein in the prefrontal cortex were increased in the model group than those in the normal group (P<0.01). Following the treatment and compared with the model group, the increase amount of body weight in the taVNS group was obviously increased (P<0.05), the swimming immobility time and the expression of TLR4, MyD88, IL-18 protein in the taVNS and tnVNS groups were significantly decreased (P<0.01). CONCLUSION: TaVNS is able to improve depression in depression rats, which is probably related to its effect in inhibiting inflammatory response of TLR4/MyD88 signaling pathway in prefrontal cortex.

Rehabilitation training inhibits neuronal apoptosis by down-regulation of TLR4/MyD88 signaling pathway in mice with cerebral ischemic stroke.

OBJECTIVE: To investigate the role of rehabilitation training and TLR4/MyD88 signaling pathway on neuronal apoptosis in mice with cerebral ischemic stroke. METHODS: Mice were randomized into six groups, which were normal group (healthy mice, n=20), control group (sham surgery, n=20), model group (middle cerebral artery occlusion (MCAO) model, n=20), training (MCAO model, continuous rehabilitation training for 4 weeks, n=20), TAK-242 group (MCAO model, TL R4 inhibitor TAK-242, n=20), and TAK-242 + Training group (MCAO model, TLR4 inhibitor TAK-242 + rehabilitation training, n=20). RESULTS: Neurobehavioral assessment was performed, and cerebral infarction area of mice was detected by triphenyl tetrazolium chloride staining. Compared with the normal group, no significant differences in all indicators were found in the control group (all P>0.05), while the other groups had higher neurological function scores, cerebral infarction area, neuronal apoptosis rate, increased expressions of TLR4, MyD88, Bax, NF-κB, TNF-α, Caspase-3, IL-1ßA and decreased mRNA and protein expressions of Bcl-2 (all P<0.05). CONCLUSION: Rehabilitation training can effectively reduce the apoptosis of hippocampal neurons in mice with ischemic stroke by inhibiting the TLR4/MyD88 signaling pathway.

Olanzapine-Induced Activation of Hypothalamic Astrocytes and Toll-Like Receptor-4 Signaling via Endoplasmic Reticulum Stress Were Related to Olanzapine-Induced Weight Gain.

The antipsychotic drug olanzapine is associated with serious obesity side effects. Hypothalamic astrocytes and associated toll-like receptor-4 (TLR4) signaling play an essential role in obesity pathogenesis. This study investigated the effect of olanzapine on astrocytes and TLR4 signaling both in vitro and in the rat hypothalamus and their potential role in olanzapine-induced weight gain. We found that olanzapine treatment for 24 h dose-dependently increased cell viability, increased the protein expression of astrocyte markers including glial fibrillary acidic protein (GFAP) and S100 calcium binding protein B (S100B), and activated TLR4 signaling in vitro. In rats, 8- and 36-day olanzapine treatment caused weight gain accompanied by increased GFAP and S100B protein expression and activated TLR4 signaling in the hypothalamus. These effects still existed in pair-fed rats, suggesting that these effects were not secondary effects of olanzapine-induced hyperphagia. Moreover, treatment with an endoplasmic reticulum (ER) stress inhibitor, 4-phenylbutyrate, inhibited olanzapine-induced weight gain and ameliorated olanzapine-induced changes in hypothalamic GFAP, S100B, and TLR4 signaling. The expression of GFAP, S100B, and TLR4 correlated with food intake and weight gain. These findings suggested that olanzapine-induced increase in hypothalamic astrocytes and activation of TLR4 signaling were related to ER stress, and these effects may be related to olanzapine-induced obesity.

Anti-Inflammatory Activity of Adenosine 5'-Trisphosphate in Lipopolysaccharide-Stimulated Human Umbilical Vein Endothelial Cells Through Negative Regulation of Toll-Like Receptor MyD88 Signaling.

Activation of TLR4-MyD88-NF-κB signaling by lipopolysaccharide (LPS) evokes a proinflammatory immune response, and plays a pivotal role in initiation and progression of atherosclerosis (AS). ATP (adenosine 5'-trisphosphate), a powerful extracellular signal transduction molecule, functions to regulate immune inflammatory responses depending on the type of P2 receptors and cell lines. In this study, we first performed RT-PCR to detect the mRNA expression of monocyte chemoattractant protein-1 (MCP-1), IL-8, and IL-1ß induced by different concentrations of LPS in human umbilical vein endothelial cells (HUVECs). Protein level of TLR4 signaling including TLR4, myeloid differentiation factor (MyD88), and CD14 induced by LPS (1 µg/mL) at different times (0, 10, 30, 60, 120 min) was analyzed by Western blot. Then, RT-PCR was performed to detect the effect of different concentrations of ATP on mRNA expression of IL-1ß and MCP-1 induced by LPS (1 µg/mL) and the TLR4 signaling pathway. Western blot was performed to detect the effect of low concentrations of ATP on phosphorylation of p65 induced by 1 µg/mL LPS. Finally, we used P2Y receptor blocker Suramin to verify whether the role of ATP on LPS-induced inflammatory cytokine expression was through P2Y receptors. The results showed that LPS upregulated the expression of MCP-1, IL-8, and IL-1ß in a dose-dependent manner accompanied by the activation of TLR4-MyD88 signaling in HUVECs. Only low concentration ATP (1, 10 µM) inhibited LPS-induced mRNA expression of IL-1ß and MCP-1. ATP at low concentrations also downregulated the mRNA expression of TLR4, CD14, and MyD88 and inhibited LPS-induced phosphorylation of p65. Furthermore, Suramin, a nonspecific P2Y receptor antagonist, did not attenuate the inhibition of ATP on LPS-induced IL-1ß and MCP-1 expression. Taking this together, low concentration ATP inhibited LPS-induced inflammation in HUVECs by negatively regulating TLR4-MyD88 signaling, and P2Y receptors were not involved in this process, which might provide new ideas for prevention and treatment of inflammatory diseases such as AS.

Anti-inflammatory and Regulatory Effects of Huanglian Jiedu Decoction on Lipid Homeostasis and the TLR4/MyD88 Signaling Pathway in LPS-Induced Zebrafish.

Huanglian Jiedu decoction (HLJDD) has been used in the clinical treatment of inflammatory conditions. To clarify the mechanism of its comprehensive anti-inflammatory activities, the correlation between lipid homeostasis and the TLR4/MyD88 signaling pathway in zebrafish was established in the present study. In the lipopolysaccharide (LPS)-induced inflammation in zebrafish model, RT-PCR assays of five inflammatory cytokines and six targeted proteins were measured. Lipidomics analysis was conducted to identify potential lipid markers. HLJDD displayed strong efficacies, with a 61% anti-inflammatory rate at a concentration of 50 µg/mL. The activation of TLR4/MyD88 played an essential role in the inflammatory process. All protein indexes in the HLJDD group exhibited a tendency to reverse back to normal levels. Moreover, 79 potential pathological lipid biomarkers were identified. Compared with the model group, 61 therapeutic lipid biomarkers were detected in HLJDD group. Most perturbations of lipids were ameliorated by HLJDD, mainly through the glycerophospholipid metabolic pathway. In the visual network study, the corresponding lipoproteins such as PLA2, SGMS, and SMDP were observed as important intermediates between lipid homeostasis and the TLR4/MyD88 signaling pathway.

Effect of TLR4/MyD88 signaling pathway on sepsis-associated acute respiratory distress syndrome in rats, via regulation of macrophage activation and inflammatory response.

The present study aimed to investigate the effects of the Toll-like receptor (TLR)4/myeloid differentiation primary response (MyD)88 signaling pathway on sepsis-associated acute respiratory distress syndrome (ARDS) in rats, and the involvement of macrophage activation and the inflammatory response. A total of 36 specific pathogen-free male Sprague-Dawley rats were selected to establish the rat model of sepsis-associated ARDS using cecal ligation and puncture (CLP). Rats were assigned into the Ab (anti-TLR4 monoclonal antibody)-CLP, CLP and Sham groups. Arterial partial pressure of oxygen (PaO2) was detected using blood gas analysis. Bronchoalveolar lavage fluid (BALF) and alveolar macrophages were collected. The pathological structure of lung tissue was observed following hematoxylin-eosin staining. The ultrastructural alterations of alveolar epithelial cells were observed under transmission electron microscope. The ratios of wet/dry weight of lung tissue and total protein content in BALF were measured. The concentration of tumor necrosis factor (TNF)-α and interleukin (IL)-1ß in BALF and peripheral blood was determined by enzyme-linked immunosorbent assay. The TLR4, TLR9, MyD88 and nuclear factor (NF)-κΒ mRNA and protein expression levels in alveolar macrophages were measured by reverse transcription-quantitative polymerase chain reaction and western blotting. Compared with the Sham group, the rats in the CLP group demonstrated significantly increased respiratory frequency, lung permeability, lung edema, inflammatory infiltration, TNF-α and IL-1ß expression levels in BALF and peripheral blood and TLR4, TLR9, MyD88 and NF-κΒ expression levels in macrophages, however decreased arterial PaO2. Following pretreatment with anti-TLR4 monoclonal antibody, rats exhibited decreased lung injury, inflammatory infiltration, lung edema, TNF-α and IL-1ß expressions in BALF and peripheral blood, and TLR4, TLR9, MyD88 and NF-κΒ expression levels in macrophages, with increased arterial PaO2. These results suggested that the inhibition of TLR4/MyD88 signaling pathway may relieve sepsis-associated ARDS in rats through regulating macrophage activation and the inflammatory response.

Ramulus Cinnamomi extract attenuates neuroinflammatory responses via downregulating TLR4/MyD88 signaling pathway in BV2 cells.

Ramulus Cinnamomi (RC), a traditional Chinese herb, has been used to attenuate inflammatory responses. The purpose of this study was to investigate the effect of RC extract on lipopolysaccharide (LPS)-induced neuroinflammation in BV2 microglial cells and the underlying mechanisms involved. BV2 cells were incubated with normal medium (control group), LPS, LPS plus 30 µg/mL RC extract, or LPS plus 100 µg/mL RC extract. The BV2 cell morphology was observed under an optical microscope and cell viability was detected by MTT assay. Nitric oxide level in BV2 cells was detected using Griess regents, and the levels of interleukin-6, interleukin-1ß, and tumor necrosis factor α in BV2 cells were determined by ELISA. The expression levels of cyclooxygenase-2, Toll-like receptor 4 and myeloid differentiation factor 88 proteins were detected by western blot assay. Compared with the LPS group, both 30 and 100 µg/mL RC extract had no significant effect on the viability of BV2 cells. The levels of nitric oxide, interleukin-6, interleukin-1ß and tumor necrosis factor α in BV2 cells were all significantly increased after LPS induction, and the levels were significantly reversed after treatment with 30 and 100 µg/mL RC extract. Furthermore, RC extract significantly inhibited the protein expression levels of cyclooxygenase-2, Toll-like receptor 4 and myeloid differentiation factor 88 in LPS-induced BV2 cells. Our findings suggest that RC extract alleviates neuroinflammation by downregulating the TLR4/MyD88 signaling pathway.

Inhibition of miR-873 provides therapeutic benefit in lipopolysaccharide-induced Parkinson disease animal model / 中国药理学与毒理学杂志

OBJECTIVE Neuroinflammation plays a critical role in neurodegenerative disorders, although the inflammation may not the initiating factor. Parkinson disease (PD) is characterized patho?logically by the accumulation of alpha synuclein (α-syn) and the loss of the dopamine (DA) neurons in the substantia nigra (SN), which has been reported to be induced by the stereotaxic injection of lipopolysaccharide (LPS) to the SN region in rodents. This study is to investigate the therapeutic benefit of the inhibition of miR-873 in PD. METHODS Rats received the right-unilaterally injection with concentrated LV-sponge or LV-EGFP 3 d before LPS treatment, 7 or 14 d after LPS treatment. The animals were tested for rotational behavior with the dopaminergic agonist apomorphine dissolved in sterile saline at 21 d after LPS injection. The regulation of miR-873 on the genes related with cholesterol transport and inflammation was assayed in SH-SY5Y cells and U251 cells. RESULTS TLR4-MyD88 signaling pathway was involved the regulation of miR-873 by LPS. The luciferase assay showed that HMGCR, ABCA1 and A20 were down- stream genes of miR- 873. The transfection of miR- 873 decreased the cholesterol levels in cell membrane, but increased in lysosome in SH-SY5Y cells. Compared with the control SH-SY5Y cells, cholesterol levels were higher in lysosome with α-synuclein overexpression or LPS treatment. The transfection of miR-873 increased the α-syn levels in lysosome in cells with α-synuclein overexpression. The loss of dopaminergic neuorns induced by LPS was significantly respectively decreased by 22.8%, 35.6% and 57% after the inhibition of miR-873 at 3 d before LPS treatment, 7 or 14 d after LPS treatment. Compared with LPS-treated group, the number of the rotation of rats was decreased by 60.4%, 33.5% and 13.2% after the inhibition of miR-873 at 3 d before LPS treatment, 7 or 14 d after LPS treatment. The inhibition of miR-873 significantly decreased accumulation of α-syn. The mRNA levels of HMGCR, ABCA1 and A20 in SN were decreased by LPS treatment, which was attenuated by the injection of LV- sponge. CONCLUSION The selective regulation of miR- 873 can protect the dopaminergic neurons from the LPS-induced damage. The inhibition of miR-873 can attenuate the relocation of cholesterol in lysosome and the accumulation of α-syn in neurons induced by LPS via the regulation of HMGCR, ABCA1 and A20.

In vitro and in vivo protective effect of arginine against lipopolysaccharide induced inflammatory response in the intestine of juvenile Jian carp (Cyprinus carpio var. Jian).

The present study was designed to assess the possible protective effects of arginine (Arg) against lipopolysaccharide (LPS) induced inflammatory response in juvenile Jian carp (Cyprinus carpio var. Jian) in vivo and in enterocytes in vitro. Firstly, inflammatory response was established by exposing enterocytes to different concentrations of LPS for 24 h. Secondly, the protective effects of Arg against subsequent LPS exposure were studied in enterocytes. Finally, we investigated whether dietary Arg supplementation could attenuate immune challenge induced by LPS in vivo. The result indicated that 10 mg/L LPS could induced inflammatory response in enterocytes. Cells exposed to LPS (10-30 mg/L) alone for 24 h resulted in a significant increase in lactate dehydrogenase release (LDH) (P < 0.05). The cell viability, protein content, alkaline phosphatase activity were decreased by LPS (P < 0.05). Moreover, LPS exposure significantly increased TNF-α, IL-1ß, and IL-6 mRNA expression in vitro (P < 0.05). However, pre-treatment with Arg remarkably prevented the increase of TNF-α, IL-1ß, and IL-6 by inhibiting the excessive activation of TLR4-Myd88 signaling pathway through down-regulating TLR4, Myd88, NFκB p65, and MAPK p38 mRNA expression (P < 0.05). Interestingly, the experiment in vivo showed that Arg pre-supplementation could attenuate immune challenge induced by LPS via TLR4-Myd88 signaling pathway, and thus protect fish against LPS-induced inflammatory response. In conclusion, all of these results indicated pre-supplementation with Arg decreased LPS induced immune damage and regulated TLR4-Myd88 signaling pathway in juvenile Jian carp in vivo and in enterocytes in vitro.

Effect of isoflurane preconditioning on TLR4-MyD88 signaling pathway in ischemic penumbra following focal cerebral ischemia-reperfusion in rats / 中华麻醉学杂志

Objective To investigate the effect of isoflurane preconditioning on Toll-like receptor 4 (TLR4)-myeloid differentiation factor 88 (MyD88) signaling pathway in ischemic penumbra following focal cerebral ischemia-reperfusion (I/R) in rats.Methods Fifty-four healthy male SD rats,aged 3 months,weighing 250-280 g,were randomly divided into 3 groups (n =18 each):sham operation group (group S),I/R group and isoflurane preconditioning group (group IP).Focal cerebral I/R was induced by middle cerebral artery occlusion.In groups I/R and IP,a nylon thread with rounded tip was inserted into the right internal jugular vein and threaded cranially until resistance was met.The middle cerebral artery was occluded for 2 h,followed by 24 h reperfusion.In group IP,the animals inhaled 2.0％ isoflurane for 2 h,and middle cerebral artery occlusion was performed at 24 h after the end of preconditioning.Neurological deficit was scored at 24 h of reperfusion and then the rats were sacrificed.Five rats in each group were chosen and the brains removed for measurement of the cerebral infarct volume.The right cerebral ischemic penumbra was removed for detection of the expression of HSP60,TLR4,MyD88 protein and mRNA by Western blot analysis and real time-PCR.Apoptosis was detected in the ischemic penumbra in the left 3 rats in each group using TUNEL.Apoptosis index (AI) was calculated.Results Neurological deficit scores and AI were significantly increased,the cerebral infarct volume was significantly enlarged,and the expression of HSP60,TLR4,MyD88 protein and mRNA was up-regulated in groups I/R and IP as compared with group S ( P ＜ 0.05).Isoflurane preconditioning significantly reduced the cerebral infarct volume and decreased neurological deficit scores and AI,and down-regulated the expression of HSP60,TLR4,MyD88 protein and mRNA (P ＜ 0.05).Conclusion The mechanisn by which isoflurane preconditioning protects ischenic penumbra following focal cerebral I/R may be related to inhibition of TLR4-MyD88 signaling pathway.