Myeloid Differentiation Factor 88 Protein Expression Research Articles

Effects of acupuncture on fecal short-chain fatty acids and TLR4/MyD88/NF-κB signaling pathway in spontaneously hypertensive rats

To observe the effects of acupuncture on blood pressure, fecal short-chain fatty acids (SCFAs) and toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor-κB (NF-κB) signaling pathway in spontaneously hypertensive rats (SHR), and to explore the mechanism of acupuncture for anti-hypertension. Twenty-four male SHR of SPF grade were randomly divided into a model group, a western medication group, an acupuncture group and a sham acupuncture group, with 6 rats in each group, and 6 male Wistar-Kyoto rats were selected as the blank group additionally. Hydrochlorothiazide solution was given by gavage in the western medication group; acupuncture was applied at bilateral "Renying" (ST 9) and "Zusanli" (ST 36) in the acupuncture group, 20 min a time; acupuncture was applied at the non-meridian and non-acupoint points close to bilateral "Renying" (ST 9) and "Zusanli" (ST 36) in the sham acupuncture group, 20 min a time. The intervention was adopted once a day for 4 weeks continuously in each group. The systolic blood pressure (SBP) of the caudal artery was measured before intervention and after 1, 2, 3 and 4 weeks of intervention. After intervention, the morphology of colonic tissue was observed by HE staining; the fecal level of SCFAs was detected by gas chromatography; the serum levels of interleukin (IL)-6, IL-1βand tumor necrosis factor-α (TNF-α) were detected by ELISA; the protein expression of TLR4, MyD88 and NF-κB p65 in the mesenteric artery was detected by Western blot. Compared with the blank group, in the model group, the SBP was increased (P<0.05), significant pathological changes could be found in the colonic tissue, the fecal SCFAs level was decreased (P<0.05), the serum levels of IL-6, IL-1β and TNF-α were increased (P<0.05), the protein expression of TLR4, MyD88 and NF-κB p65 in the mesenteric artery was increased (P<0.05). Compared with the model group, the SBP after 2, 3 and 4 weeks of intervention was decreased (P<0.05), the serum levels of IL-6, IL-1β and TNF-α were decreased (P<0.05) in the acupuncture group and the western medication group; the mucosal epithelium of colonic tissue was intact, the number of intestinal glands was abundant, the fecal SCFAs level was increased (P<0.05), and the protein expression of TLR4, MyD88 and NF-κB p65 in the mesenteric artery was decreased (P<0.05) in the acupuncture group. Compared with the sham acupuncture group, the SBP after 2, 3 and 4 weeks of intervention was decreased (P<0.05), the fecal SCFAs level was increased (P<0.05), the serum levels of IL-6, IL-1β and TNF-α were decreased (P<0.05), the protein expression of TLR4, MyD88 and NF-κB p65 in the mesenteric artery was decreased (P<0.05) in the acupuncture group. Acupuncture at bilateral "Renying" (ST 9) and "Zusanli" (ST 36) can effectively play an anti-hypertensive role in SHR. Its mechanism may be related to regulating fecal SCFAs level and inhibiting the TLR4/MyD88/NF-κB signaling pathway.

Acupuncture at "Die E acupoint" alleviates inflammatory reaction via inhibiting TLR4/MyD88/NF-κB signaling in rats with allergic rhinitis.

To observe effects of acupuncture at "Die E acupoint" on the protein expression levels of Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), nuclear transcription factor κB (NF-κB), transcription factor T-bet (T-bet), and GATA-binding protein-3 (GATA-3) in the nasal mucosa and the serum contents of related inflammatory cytokines in rats with allergic rhinitis, so as to explore the mechanism of acupuncture in treating allergic rhinitis. Twenty-four healthy SD rats were randomly divided into blank, model, acupuncture, and sham acupuncture groups, with 6 rats in each group. The rat model of allergic rhinitis was established by using ovalbumin induction. The rats in the acupuncture group received bilateral acupuncture at the "Die E acupoint" with a depth of 15-20 mm, while the rats in the sham acupuncture group received only sham acupuncture (light and shallow acupunture of the skin at the "Die E acupoint" ). Both interventions were performed once daily for a total of 6 days. Behavioral scores of rats in each group were recorded. Pathological changes of nasal mucosa were observed by H.E. staining. Serum contents of IgE, ovalbumin-specific IgE (OVA-sIgE), interferon(IFN)-γ, interleukin(IL)-4, IL-10 and IL-17 were measured by ELISA and the protein expression levels of T-bet, GATA-3, TLR4, MyD88 and NF-κB p65 in the nasal mucosa were detected by Western blot. After modeling, compared with the blank group, rats in the model group showed increased behavioral scores, serum IgE, OVA-sIgE, IL-4, and IL-17 contents, and nasal mucosal GATA-3, TLR4, MyD88, and NF-κB p65 protein expression levels (P<0.05), whereas the contents of serum IFN-γ, IL-10 and the protein expression level of T-bet in the nasal mucosa were decreased (P<0.05). Comparison between the EA and model groups showed that acupuncture intervention can decrease the behavioral scores of rats with allergic rhinitis, the contents of serum IgE, OVA-sIgE, IL-4, IL-17, and the protein expression levels of GATA-3, TLR4, MyD88, and NF-κB p65 in the nasal mucosa (P<0.05), and up-regulate the contents of serum IFN-γ, IL-10, and the nasal mucosal T-bet protein expression level. Sham acupuncture did not have a significant modulating effect on the above indicators. Inflammatory infiltration of nasal mucosa was seen in the model group and sham acupuncture, and the inflammatory reaction was milder in the acupuncture group. Acupuncture at "Die E acupoint" can alleviate the symptoms of allergic rhinitis and suppress the inflammation of nasal mucosa in rats, which may be related to inhibiting the TLR4/MyD88/NF-κB signaling and balancing the levels of cytokines of Th1/Th2 and Treg/Th17, and T-bet/GATA-3.

Ghrelin Alleviates Inflammation, Insulin Resistance, and Reproductive Abnormalities in Mice with Polycystic Ovary Syndrome via the TLR4-NF-κB Signaling Pathway.

Polycystic ovary syndrome (PCOS) commonly impacts fertile females with potentially severe effects on fertility and metabolism. Blood ghrelin levels are lower in PCOS patients, and exogenous supplements have been proposed for their potential to trigger anti-inflammatory effects at the cellular level. This study aimed to investigate whether pretreatment with ghrelin reduced inflammation, insulin resistance, and reproductive abnormalities in PCOS and the underlying mechanism of this disorder. Ghrelin supplementation was first tested in an inflammation model using human ovarian granulosa cells (KGN cells) that were built by treated with Lipolyaccharide. KGN cells were pretreated with ghrelin and exposed to lipopolysaccharide (LPS). Inflammatory gene expression and cytokine production were analyzed by Enzyme-linked immunosorbent assay (ELISA). Based on these results, the PCOS mice model was built with Dehydroepiandrosterone (DHEA) and a high-fat diet. The mRNA and protein expressions of inflammatory factors including Toll-like receptor 4 (TLR4), nuclear factor kappa-B-p65 (NF-κB-p65), Phospho-NF-κB-p65 (p-NF-κB-p65) and myeloid differentiation factor 88 (MYD88) related to the TLR4/NF-κB signaling pathway were evaluated in KGN cells and mouse ovarian tissues using Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) and western blot, respectively. Lipid metabolism was quantified via an automated biochemical analyzer. The mRNA and protein expressions of interleukin-6 (IL-6), interleukin-1 beta (IL-1β), and tumor necrosis factor alpha (TNF-α) in ghrelin pretreated KGN cells were lower than the LPS group (p < 0.05). Protein expression was reduced for TLR4, NF-κB-p65, and MYD88 within KGN cells of ghrelin groups compared to the LPS group (p < 0.05). Ghrelin treatment restored the estrous cycle and slowed weight gain and abdominal fat weight of PCOS mice (p < 0.05). Ghrelin treatment decreased the serum concentrations of testosterone, luteinizing hormone, insulin, IL-6, IL-1β, and TNF-α compared to the PCOS group (p < 0.05). Estradiol concentrations of mice treated with ghrelin were higher than the PCOS group (p < 0.05). The concentrations of low and high-density lipoprotein, triglyceride, and cholesterol in mice treated with ghrelin were lower than in the PCOS mice (p < 0.05). Inflammatory gene expression for IL-6, IL-1β, TNF-α, TLR4, NF-κB-p65, and MYD88 decreased in the ovarian tissues of ghrelin-treated mice compared to the PCOS group (p < 0.05), along with reduced protein expression of TLR4, p-NF-κB-p65, and MYD88 (p < 0.05). In the present study, ghrelin treatment effectively reduced inflammation in vitro, and attenuated insulin resistance and reproductive abnormalities in PCOS mice through the TLR4/NF-κB signaling pathway, highlighting potential therapeutic avenues for future PCOS treatments and research directions.

Moxibustion improves experimental colitis in rats with Crohn’s disease by regulating bile acid enterohepatic circulation and intestinal farnesoid X receptor

ObjectiveThis study was conducted to explore the mechanism of intestinal inflammation and barrier repair in Crohn’s disease (CD) regulated by moxibustion through bile acid (BA) enterohepatic circulation and intestinal farnesoid X receptor (FXR). MethodsSprague-Dawley rats were randomly divided into control group, CD model group, mild moxibustion group and herb-partitioned moxibustion group. CD model rats induced by 2,4,6-trinitrobenzene sulfonic acid were treated with mild moxibustion or herb-partitioned moxibustion at Tianshu (ST25) and Qihai (CV6). The changes in CD symptoms were rated according to the disease activity index score, the serum and colon tissues of rats were collected, and the pathological changes in colon tissues were observed via histopathology. Western blot, immunohistochemistry (IHC) and immunofluorescence were used to evaluate the improvement of moxibustion on intestinal inflammation and mucosal barrier in CD by the BA-FXR pathway. ResultsMild moxibustion and herb-partitioned moxibustion improved the symptoms of CD, inhibited inflammation and repaired mucosal damage to the colon in CD rats. Meanwhile, moxibustion could improve the abnormal expression of BA in the colon, liver and serum, downregulate the expression of interferon-γ and upregulate the expression of FXR mRNA, and inhibit Toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88) mRNA. The IHC results showed that moxibustion could upregulate the expression of FXR and mucin2 and inhibit TLR4 expression. Western blot showed that moxibustion inhibited the protein expression of TLR4 and MyD88 and upregulated the expression of FXR. Immunofluorescence image analysis showed that moxibustion increased the colocalization sites and intensity of FXR with TLR4 or nuclear factor-κB p65. In particular, herb-partitioned moxibustion has more advantages in improving BA and upregulating FXR and TLR4 in the colon. ConclusionMild moxibustion and herb-partitioned moxibustion can improve CD by regulating the enterohepatic circulation stability of BA, activating colonic FXR, regulating the TLR4/MyD88 pathway, inhibiting intestinal inflammation and repairing the intestinal mucosal barrier. Herb-partitioned moxibustion seems to have more advantages in regulating BA enterohepatic circulation and FXR activation.Please cite this article as: Shen JC, Qi Q, Han D, Lu Y, Huang R, Zhu Y, Zhang LS, Qin XD, Zhang F, Wu HG, Liu HR. Moxibustion improves experimental colitis in rats with Crohn’s disease by regulating bile acid enterohepatic circulation and intestinal farnesoid X receptor. J Integr Med. 2023; 21(2): 194–204.

Brain-gut-liver axis: Chronic psychological stress promotes liver injury and fibrosis via gut in rats.

The effect of chronic psychological stress on hepatitis and liver fibrosis is concerned. However, its mechanism remains unclear. We investigated the effect and mechanism of chronic psychological stress in promoting liver injury and fibrosis through gut. Sixty male SD rats were randomly assigned to 6 groups. Rat models of chronic psychological stress (4 weeks) and liver fibrosis (8 weeks) were established. The diversity of gut microbiota in intestinal feces, permeability of intestinal mucosa, pathologies of intestinal and liver tissues, collagen fibers, protein expressions of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), nuclear factor kappa β (NF-κβ), tumor necrosis factor α (TNF-α) and interleukin 1 (IL-1) in liver tissue, liver function and coagulation function in blood and lipopolysaccharide (LPS) in portal vein blood were detected and analyzed. The diversities and abundances of gut microbiota were significant differences in rats among each group. The pathological lesions of intestinal and liver tissues, decreased expression of occludin protein in intestinal mucosa, deposition of collagen fibers and increased protein expression of TLR4, MyD88, NF-κβ, TNF-α and IL-1 in liver tissue, increased LPS level in portal vein blood, and abnormalities of liver function and coagulation function, were observed in rats exposed to chronic psychological stress or liver fibrosis. There were significant differences with normal rats. When the dual intervention factors of chronic psychological stress and liver fibrosis were superimposed, the above indicators were further aggravated. Chronic psychological stress promotes liver injury and fibrosis, depending on changes in the diversity of gut microbiota and increased intestinal permeability caused by psychological stress, LPS that enters liver and acts on TLR4, and active LPS-TLR4 pathway depend on MyD88. It demonstrates the possibility of existence of brain-gut-liver axis.

Genistein attenuates LPS-induced inflammatory injury of rat dorsal root ganglion neuron via down-regulating HDAC6.

Neuropathic pain (NP) is a chronic pain caused by somatosensory neuropathy or disease, and genistein (Gen) might be a potential drug for the treatment of NP. Therefore, this study aims to investigate the effect of Gen on lipopolysaccharide (LPS)-induced inflammatory injury of dorsal root ganglion neuron (DRGn) in rats and the possible molecular mechanism. The DRGn of 1-day-old juvenile rats were taken for isolation and culture. The DRGn in logarithmic growth phase were divided into a control group, a LPS group, a tubastatin hydrochloride (TSA)+LPS group, a Gen1+LPS group, a Gen2+LPS group, a Gen2+LPS+TSA group, a Gen2+pcDNA-histone deacetylase 6 (HDAC6)+LPS group, and a Gen2+pcDNA3.1+LPS group. The LPS group was treated with 1 μg/mL LPS for 24 h; the TSA+LPS group, the Gen1+LPS group, the Gen2+LPS group were treated with 5 μmol/L TSA, 5 μmol/L Gen, 10 μmol/L Gen respectively for 0.5 h, and then added 1 μg/mL LPS for 24 h; the Gen2+TSA+LPS group was treated with 10 μmol/L Gen and 5 μmol/L TSA for 0.5 h and then added 1 μg/mL LPS for 24 h; the Gen2+pcDNA-HDAC6+LPS group and the Gen2+pcDNA3.1+LPS group received 100 nmol/L pcDNA-HDAC6 and pcDNA3.1 plasmids respectively, and 24 h after transfection, 10 μmol/L Gen was pretreated for 0.5 h, and then added 1 μg/mL LPS for 24 h. Real-time RT-PCR was used to detect the HDAC6 mRNA expression in DRGn; CCK-8 method was used to detect cell viability of DRGn; flow cytometry was used to detect cell apoptosis of DRGn; ELISA was used to detect the levels of IL-1β, IL-6, and TNF-α in DRGn culture supernatant; Western blotting was used to detect the protein expression of HDAC6, Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and NF-κB p65 in DRGn. Compared with the control group, the expression levels of HDAC6 mRNA and protein, the expression levels of TLR4 and MyD88 protein in DRGn of LPS group rats were significantly up-regulated, the ratio of p-NF-κB p65/NF-κB p65 was significantly increased, and the activity of DRGn was significantly decreased, the apoptosis rate was significantly increased, and the levels of IL-1β, IL-6 and TNF-α in the DRGn culture supernatant were significantly increased (all P<0.05). Compared with the LPS group, the expression levels of HDAC6 mRNA and protein, TLR4 and MyD88 protein expression levels in DRGn of the TSA+LPS group, the Gen1+LPS group, the Gen2+LPS group and the Gen2+TSA+LPS group were significantly down-regulated, the ratio of p-NF-κB p65/NF-κB p65 was significantly decreased, the activity of DRGn was significantly increased, the apoptosis rate was significantly decreased, and the levels of IL-1β, IL-6 and TNF-α in the DRGn culture supernatant were significantly decreased (all P<0.05), and the above changes were most obvious in the Gen2+TSA+LPS group. Compared with the Gen2+LPS group, the expression levels of HDAC6 mRNA and protein, TLR4 and MyD88 protein expression levels in DRGn of the Gen2+pcDNA-HDAC6+LPS group were significantly up-regulated, the ratio of p-NF-κB p65/NF-κB p65 was significantly increased, the activity of DRGn was significantly decreased, and the apoptosis rate was significantly increased, and the levels of IL-1β, IL-6 and TNF-α in the DRGn culture supernatant were significantly increased (all P<0.05). Gen can alleviate LPS-induced DRGn inflammatory injury in rats, which might be related to down-regulating the expression of HDAC6 and further inhibiting the activation of TLR4/MyD88/NF-κB signaling pathway.

Treatment Effect of Mometasone Furoate Cream on Lichen Sclerosus et Atrophicus of External Genitalia in Boys and Its Correlation with Toll-Like Receptor 4 and Myeloid Differentiation Factor 88.

Objective The objective is to explore the treatment effect of mometasone furoate cream on lichen sclerosus et atrophicus (LSeA) of external genitalia in boys and its correlation with Toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88). Methods A total of 100 boys treated in our hospital from January 2021 to January 2023 due to clinical manifestations in the external genitalia were selected as the study objects. All boys received redundant circumcision treatment, their protein expression of TLR4 and MyD88 in foreskin tissues was measured by BCA protein assay and western blot, and mometasone furoate cream was applied to those who were pathologically diagnosed with LSeA, so as to compare the levels of serum inflammatory factors and urodynamic indicators in the child patients before and after treatment. Results The total clinical efficacy rate of LSeA child patients was up to 90.79%; after treatment, the maximum and mean urinary flow rates of child patients were significantly higher than before treatment (P < 0.001); compared with the non-LSeA group, the protein expression of TLR4, MyD88, and NF-κB was increased in the LSeA group (P < 0.001), and the mRNA expression of TLR4, MyD88, and NF-κB was significantly increased in the LSeA group (P < 0.001); the results of ROC curves showed that TLR4 had the highest AUC value, and during treatment, the incidence rate of adverse reactions in child patients was 6.58%. Conclusion LSeA will increase the inflammatory reactions in child patients, and its pathogenesis may be related to the upregulation of TLR4, MyD88, NF-κB expression and thus activation of TLR4/MyD88/NF-κB signaling pathways. Applying mometasone furoate cream to LSeA patients after redundant circumcision can effectively reduce the inflammatory reactions in the body and improve their urodynamic indicators, with exact efficacy. Further research will be conducive to establishing a better treatment scheme for such child patients.

Antiatherosclerotic effects of corilagin via suppression of the LOX-1/MyD88/NF-κB signaling pathway in vivo and in vitro.

Corilagin, a natural polyphenol compound isolated from Phyllanthus urinaria L., exerts various pharmacological effects, such as antihyperglycemic, antitumor, and antioxidative stress properties, but the mechanisms underlying the antiatherosclerotic effects of corilagin have not been entirely elucidated. In the present study, we investigated the antiatherosclerotic effects of corilagin using a high-fat diet (HFD)-induced atherosclerotic rabbit model and ox-LDL-induced vascular smooth muscle cells (VSMCs) and explored the underlying molecular mechanisms. The serum lipid levels were measured through an enzymatic colorimetric assay. A histological analysis of rabbit aortas was performed after hematoxylin-eosin and oil red O staining. The proliferation of ox-LDL-induced VSMCs was detected using MTT assays, and the migration of cells was determined by wound scratch assays. In addition, the mRNA and protein expression levels of lectin-like ox-LDL receptor-1 (LOX-1), myeloid differentiation factor 88 (MyD88), nuclear factor-kappa B (NF-κB), monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor α (TNF-α) were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting assays. Our results indicate that corilagin significantly reduced the serum levels of TC, TG and LDL-C, increased the HDL-C levels, decreased the intimal thickening in the thoracic aorta, and reduced the formation of foam cells in an HFD-induced rabbit atherosclerosis model. Moreover, corilagin suppressed the proliferation and migration of ox-LDL-induced VSMCs and reduced LOX-1, MyD88, NF-κB, MCP-1, and TNF-α mRNA and protein expression in vivo and in vitro. These data demonstrate that corilagin exerts antiatherosclerotic effects in vivo and in vitro and that the mechanisms may be closely associated with downregulation of the LOX-1/MyD88/NF-κB pathway.

Time-serial expression of toll-like receptor 4 signaling during polymicrobial sepsis in rats.

Sepsis caused by aggressive infection is a severe clinical problem with an increasing incidence worldwide. Toll-like receptors and their common adapter myeloid differentiation factor 88 (MyD88) can activate immune responses by recognizing a foreign microbe’s product. This study aimed to identify the different time expression of TLR four signaling pathway in an experimental rodent model of polymicrobial sepsis. A randomized animal study was investigated in rats with septic peritonitis induced by cecal ligation and puncture (CLP). The expressions of MyD88-dependent pathway biomarkers, including MyD88, nuclear factor-κB (NF-κB), and serum tumor necrosis factor-α (TNF-α), were analyzed and compared to the sham controls at the different time points after CLP surgery. CLP-induced sepsis increased liver MyD88 mRNA expression and protein expression compared to the control groups at 2 h after surgery. The MyD88 mRNA and protein expressions in rats with CLP-induced sepsis marked increased at 4 and 6 h, and their NF-κB activities and serum TNF-α levels also increased at 4 h after CLP surgery (both p < .05). The different serial expression of MyD88-ependent pathway during sepsis may be used as biomarkers during sepsis. These results may provide further helpful information for using pro-inflammatory biomarkers of innate immunity such as MyD88 and TNF-α in clinical sepsis or related abdominal surgical emergency in the future.

Gyejibokryeong-Hwan improves recovery of injured muscles in mouse model of muscle contusion.

To investigate the effects of Gyejibokryeong-Hwan (Guizhifuling-wan, GBH) on muscle injury in a mouse model of muscle contusion. C57/BL6 mouse biceps femoris muscles were injured using the drop-mass method and injured animals were treated orally with GBH (50, 100, or 500 mg/kg) once a day for 7 d. Open field and treadmill running tests were performed to assess functional recovery from muscle injury. The production of pro-inflammatory cytokines was examined by enzyme-linked immunosorbent assay and Western blotting analysis. Expression of the muscle regeneration biomarkers, myoblast determination (MyoD), myogenic factor 5 (Myf5), and smooth muscle actin (α-SMA), in the biceps femoris muscle was investigated at the protein and mRNA level by Western blotting and real time-PCR, respectively. Histological analysis was performed using hematoxylin and eosin staining. Finally, myosin heavy chain production was investigated in differentiated C2C12 myoblasts in the presence of GBH. GBH treatment markedly improved locomotion and running behavior. GBH significantly inhibited the secretion of monocyte chemoattractant protein-1 into the bloodstream in muscle-contused animals. The levels of MyoD, Myf5, and α-SMA protein and mRNA were significantly up-regulated by GBH in injured muscle tissue. Histological studies suggested that GBH facilitated recovery from muscle damage. However, GBH did not induce the production of myosin heavy chain in vitro. Overall, the present study suggested that GBH improves the recovery of the injured muscles in the mouse model of muscle contusion.

Circulating Exosomes From Patients With Graves' Disease Induce an Inflammatory Immune Response.

Exosomes are extracellular vesicles that can participate in autoimmune diseases. The purpose of this study was to explore whether circulating exosomes are involved in Graves' disease (GD) pathogenesis. In this study, serum exosomes were extracted from 26 healthy controls (HC-EXO), 26 GD patients (GD-EXO), and 7 Graves' ophthalmopathy patients (GO-EXO). For each group, the total protein content was detected, and thyrotropin receptor, insulin-like growth factor 1 receptor (IGF-1R), heat shock protein 60 (HSP60), and cluster of differentiation (CD) 63 expression were analyzed by Western blotting (WB). Healthy volunteer-derived peripheral blood mononuclear cells (PBMCs) and HC-EXO or GD-EXO were cocultured for 24 h, and immunofluorescence was used to observe the locations of the exosomes and toll-like receptor (TLR) 2/3. CD11c+TLR2+ and CD11c+TLR3+ cell percentages were determined by flow cytometry. Myeloid differentiation factor 88 (MyD88), toll/interleukin (IL)-1 receptor domain-containing adaptor inducing interferon-β (TRIF) and p-P65 expression were analyzed by WB. IL-6 and IL-1β supernatant levels were detected using enzyme-linked immunosorbent assay. The results showed that the total protein concentration was similar among GD-EXO, GO-EXO, and HC-EXO. IGF-1R and HSP60 expression was significantly higher in GD-EXO and GO-EXO than in HC-EXO. After coculturing PBMCs with GD-EXO or HC-EXO for 24 h, GD-EXO could bind to TLR2/3. GD-EXO significantly increased CD11c+TLR2+ and CD11c+TLR3+ cell percentages; MyD88, TRIF, and p-P65 protein expression; and IL-6 and IL-1β levels. In conclusion, we first demonstrated that GD-EXO and GO-EXO highly expressed IGF-1R and HSP60. GD-EXO may induce an inflammatory response through the TLR/NF-κB signaling pathway and be involved in the pathogenesis of GD.

Prognostic value of MYD88 L265P mutation in diffuse large B cell lymphoma via droplet digital PCR.

To assess the prevalence and prognostic value of myeloid differentiation factor 88 (MYD88) expression and mutational status in diffuse large B cell lymphoma (DLBCL), a total cohort of 100 patients with DLBCL were studied using immunohistochemistry (IHC) and droplet digital polymerase chain reaction (DDPCR), and the association between MYD88 expression and clinicopathological parameters was analyzed. Overall, the positive expression rate of MYD88 protein was 38% and the gene mutation rate was 29%. The positive expression and mutation rates were the highest in the primary central nervous system lymphomas (58.33 and 66.67%, respectively). The coincidence rate of the results of MYD88 expression between IHC and DDPCR results was 73% (73/100). Univariate survival analysis showed that age (≥60 years old), high neutrophil/lymphocyte count ratio, low lymphocyte count, c-Myc ≥40%, positive MYD88 protein expression, and gene mutation were associated with poorer prognosis rates. Multivariate survival analysis revealed that MYD88 expression was an independent prognostic factor affecting overall survival. In conclusion, the results of this study demonstrated that MYD88 mutation was a valuable index to evaluate the prognosis of DLBCL. DDPCR can be used as a method for detecting MYD88 mutations, although it was not completely consistent with the results of IHC.

Gegen Qinlian Decoction Downregulates the TLR7 Signalling Pathway to Control Influenza A Virus Infection

Ethnopharmacological relevanceIn recent years, Gegen Qinlian decoction (GQD) has been applied to treat influenza virus infection, and its clinical effectiveness has been shown. However, the potential mechanism by which GQD acts on influenza A virus (IAV) has not been fully elucidated. Traditional Chinese medicine (TCM) formulas are well known to have multiple targets and effects. Our previous experiments examined the mechanism by which TCM can be used to treat influenza from the perspective of the influenza immune mechanism. Aim of the studyTo explore the possible mechanism by which GQD affects mice infected with the FM1 strain of influenza virus. Materials and MethodsForty-eight C57BL/6 mice were divided randomly into four groups: a normal control (NG) group, an IAV infection (VG) group, an IAV + oseltamivir (30.44 mg/kg) treatment (VO) group, and an IAV + GQD (9.74 g/kg) treatment (VQ) group. We also grouped forty-eight Toll-like receptor 7 knockout (TLR7−/−) mice in the same manner. The pulmonary mRNA expression of TLR7, myeloid differentiation factor 88 (MyD88), and nuclear factor (NF)-κB p65 was measured by RT-qPCR, and the pulmonary protein expression of TLR7, MyD88, and NF-κB p65 was measured by western blot. The proportions of T helper (Th) 1, Th2, Th17 and regulatory T (Treg) cells were measured by flow cytometry. ResultsIAV infection led to low body weights and high viral load. Compared with those in the NG group, the mRNA expression levels of TLR7, MyD88, and NF-κB p65 in the VG group were upregulated (P < 0.05). However, the mRNA and protein expression levels of TLR7, MyD88, and NF-κB p65 were lower in the VO and VQ groups than in the VG group (P < 0.05). IAV infection led to increased proportions of Th1/Th2 and Th17/Treg cells in the VG group. In the VO and VQ groups, both Th2 and Th1 cell numbers were increased, resulting in a lower Th1/Th2 proportion than that in the VG group. ConclusionsGQD downregulated the expression of some key TLR signalling pathway factors. GQD also affected the differentiation of CD4+ T cells, thereby exerting a protective systemic effect on influenza virus infection. In conclusion, GQD activated a balanced inflammatory response in the host to limit immune pathological injury and improve clinical and survival outcomes.

Reduced meal frequency alleviates high-fat diet-induced lipid accumulation and inflammation in adipose tissue of pigs under the circumstance of fixed feed allowance.

The present study was conducted to determine whether reduced meal frequency (MF) could restore high-fat diet (HFD)-modified phenotypes and microbiota under the condition of fixed feed allowance. A total of 32 barrows with initial weight of 61.6 ± 0.8kg were assigned to two diets [control diet (CON) versus HFD] and two meal frequencies [12 equal meals/day (M12) versus 2 equal meals/day (M2)], the trial lasted 8 weeks. The lipid metabolism and inflammatory response in adipose tissue as well as the profiles of intestinal microbiota and bacterial-derived metabolites were determined. M2 versus M12 feeding regimen decreased perirenal fat weight and serum triglyceride and liposaccharide (LPS) concentrations in HFD-fed pigs (P < 0.05). Reduced MF down-regulated mRNA expression of lipoprotein lipase, CD36 molecule, interleukin 1 beta, tumor necrosis factor alpha, toll-like receptor 4, myeloid differentiation factor 88 (MYD88), and nuclear factor kappa beta 1 as well as protein expression of MYD88 in perirenal fat of HFD-fed pigs (P < 0.05). M2 feeding regimen increased abundance of Prevotella and decreased abundance of Bacteroides in colonic content of HFD-fed pigs (P < 0.05). No difference in short-chain fatty acids (SCFAs) profile in colonic content was observed among four groups (P > 0.05). Our results suggested that M2 versus M12 feeding regimen ameliorated HFD-induced fat deposition and inflammatory response by decreasing fatty acid uptake and deactivating LPS/TLR4 signaling pathway in adipose tissue and restoring microbiota composition in distal intestine, without affecting SCFAs profile in distal luminal content.

Effects of aminolevulinic acid-based photodynamic therapy on the Toll-like receptor 2 signaling pathway in the murine macrophage line RAW264.7

Objective To evaluate the effect of aminolevulinic acid-based photodynamic therapy (ALA-PDT) on the expression of Toll-like receptor 2 (TLR2) and downstream signaling pathway molecules, and secretion of cytokines in murine RAW264.7 cells. Methods The RAW264.7 murine macrophages were induced by inactivated Propionibacterium acnes suspension for the establishment of a cell model of inflammation. The cultured RAW264.7 cells were divided into 5 groups: blank control group receiving normal culture followed by the treatment with phosphate buffer saline (PBS) , model group treated with inactivated Propionibacterium acnes suspension followed by the treatment with PBS, and three ALA groups treated with inactivated Propionibacterium acnes suspension followed by the treatment with 0.03, 0.06 and 0.12 mmol/L ALA, respectively, and infrared radiation at a dose of 16 J/cm2. Enzyme-linked immunosorbent assay (ELISA) was performed to detect levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the culture supernatant of RAW264.7 cells, and Western blot analysis to determine the protein expression of TLR2 and myeloid differentiation factor 88 (MyD88) , as well as p38, c-Jun N-terminal kinase (JNK) , extracellular signal-regulated kinase (ERK) , inhibitor of κB kinase α (IκBα) and their phosphorylated forms (p-p38, p-JNK, p-ERK and p-IκBα) . Results Compared with the blank control group, the model group showed significantly higher levels of TNF-α ([0.34 ± 0.02]ng/L, P 0.05) , but a significantly lower level of IL-6 ([0.07 ± 0.01]ng/L, F= 114.813, P < 0.01) . The protein expression of TLR2, MyD88, p-p38, p-IκBα, p-JNK and p-ERK was all significantly higher in the model group (0.90 ± 0.14, 1.11 ± 0.13, 0.84 ± 0.04, 1.45 ± 0.20, 2.56 ± 0.06, 3.70 ± 0.40) than in the blank control group (all P < 0.01) , and gradually decreased along with the increase of ALA concentration in a dose-dependent manner. Conclusion Photodynamic therapy can suppress the expression of TLR2 in RAW264.7 murine macrophages, and decrease the secretion of cytokines likely by the TLR2 signaling pathway. Key words: Propionibacterium acnes; Acne vulgaris; Aminolevulinic acid; Photochemotherapy; Toll-like receptor 2; Signal transduction

Anti-inflammatory Effects of PMX205 in Mouse Macrophage Periodontitis Model.

C5a receptor antagonist PMX205 is a synthetic hexapeptide capable of blocking C5a-C5a receptor (C5aR) axis by simulating C5a active C-terminal amino acid residues. This hexapeptide presents good anti-inflammatory effects in a series of inflammation models. The anti-inflammatory effect of PMX205 on periodontitis is yet to be fully fathomed. To examine the anti-inflammatory effects of PMX205 on RAW264.7 mouse macrophages exposed to gingipain extracts and Porphyromonas gingivalis (P. gingivalis). MTT assay was carried out so as to specify the cytotoxicity of PMX205. RAW264.7 cells were co-cultured in vitro with gingipain extracts or P. gingivalis to simulate the periodontitis inflammatory milieu. Real-time quantitative PCR, ELISA and Griess assay were performed in order to detect tumor necrosis factor-α (TNF-α), IL-6, IL-23, nitric oxide (NO), IL-10, transforming growth factor-β1 (TGF-β1), andarginase-1 (Arg-1). Furthermore, phagocytosis assay was done to evaluate the phagocytic capacity of RAW 264.7 cells. Finally, western blot analysis was conducted to evaluate myeloid differentiation factor 88 (MyD88). PMX205 increased the expression levels of bacteriostatic substances (NO and IL-23) and anti-inflammatory cytokines (TGF-β1, IL-10 and Arg-1); however, it reduced the expression levels of proinflammatory cytokines TNF-α and IL-6 once RAW 264.7 macrophages were stimulated via gingipain extracts or P. gingivalis. In addition, PMX205 promoted the macrophage phagocytosis and down-regulated protein expression of MyD88. PMX205 has recognizable anti-inflammatory effects in RAW 264.7 cell inflammation model, a finding which probably opens doors to future investigations on new targets for the prevention and treatment of chronic periodontitis.

Extract from Mulberry (Morus australis) leaf decelerate acetaminophen induced hepatic inflammation involving downregulation of myeloid differentiation factor 88 (MyD88) signals

Acetaminophen (APAP) induced inflammation and oxidative stress can cause cell death to induce liver damage. The antioxidative and anti-inflammatory effect of Mulberry (Morus australis) leaf extract (MLE) was shown in previous studies. In this study, we investigated the modulation of MLE on APAP induced inflammation and oxidative stress in rat liver injury or liver cancer cell (HepG2). Wistar rat was fed orally with MLE (0.5% or 1.0 %) for 1 week, and then, 900 mg/kg of APAP was injected intraperitoneally (i.p.). Pretreatment of MLE decreased obvious foci of inflammatory cell infiltration in liver. It also reduced the expression of inflammatory parameters including cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and nuclear factor kappa B (NF-κB) in liver. Treating with MLE increased the antioxidative enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase. Giving APAP to HepG2 hepatocyte was conducted to elucidate the mechanism of MLE or its functional components. The result showed that APAP upregulated hepatic protein expression of (myeloid differentiation factor 88) MyD88, nuclear factor kappa B (NF-kB), inhibitor of kappa B (IkB), c-Jun N-terminal kinases (JNK), and receptor interacting proteins (RIP1 and RIP3). Pretreatment of MLE, gallic acid (GA), gallocatechin gallate (GCG), or protocatechuic acid (PCA) suppressed the indicated protein expression. These findings confirmed that MLE has the potential to protect liver from APAP-induced inflammation, and the protecting mechanism might involve decreasing oxidative stress and regulating the innate immunity involving MyD88.

Effect of sinomenine on the expression of rheumatoid arthritis fibroblast-like synoviocytes MyD88 and TRAF6.

The effect of sinomenine (SIN) on the toll-like receptor (TLR) signal transduction pathway as well as the expression of myeloid differentiation factor 88 (MyD88) and tumor necrosis factor (TNF) receptor-associated factor-6 (TRAF6) was investigated. SIN inhibition of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) proliferation and RA cartilage and subchondral bone destruction was also investigated. RA-FLS were cultured in vitro and the intracellular alkaline phosphatase (ALP) activity was determined in order to obtain the optimal drug concentration. The rate of cell proliferation was determined. Fluorescence quantitative polymerase chain reaction (PCR) was applied to determine the MyD88 and TRAF-6 gene expression and western blot was used to detect the MyD88 and TRAF-6 protein expression. The ALP activity in the SIN groups was lower than that in the control group, among which the 0.5 mM SIN group had the lowest ALP activity (P < 0.01). The rate of RA-FLS proliferation detected by CCK-8 assay in the 0.5-mM SIN group was lower than that in the control group (P < 0.01) and was the highest 4 days after SIN induction. Gene and protein expression of MyD88 and TRAF-6 were downregulated significantly in the 0.5-mM SIN group compared to that in the control group (P < 0.01). SIN effectively inhibited MyD88 and TRAF-6 expression in RA-FLS, which may be one of the important molecular mechanisms involved in RA treatment and prevention of cartilage and subchondral bone destruction.

Role of cardiac- and myeloid-MyD88 signaling in endotoxin shock: a study with tissue-specific deletion models.

Myeloid differentiation factor 88 (MyD88) is an adaptor molecule critical for host innate immunity. Studies have shown that signaling via MyD88 contributes to cytokine storm, cardiac dysfunction, and high mortality during endotoxin shock.However, the specific contribution of MyD88 signaling of immune and cardiac origins to endotoxin shock remains unknown. Tissue-specific MyD88 deletion models: Cre-recombinase transgenic mice with α-myosin heavy chain (α-MHC) or lysozyme M promoters were cross-bred with MyD88-loxP (MyD88fl/fl) mice, respectively, to generate cardiomyocyte- (α-MHCMyD88−/−) or myeloid-specific (Lyz-MyD88−/−) MyD88 deletion models and their respective MyD88fl/fl littermates. Endotoxin shock model: Mice were subjected to 15 mg/kg lipopolysaccharide (intraperitoneal injection). Cardiac function was measured by echocardiography and cytokines by multiplex assay and quantitative reverse transcription-polymerase chain reaction. α-MHC-MyD88−/− mice had 61 and 87% reduction in MyD88 gene and protein expression in cardiomyocytes,respectively, whereas Lyz-MyD88−/− had 73 and 67% decrease, respectively, in macrophages (n=3 per group). After lipopolysaccharide treatment, the two groups of MyD88fl/fl littermates had 46% (n=10) and 60% (n=15) of mortality, respectively.Both α-MHC-MyD88−/− and Lyz-MyD88−/− mice had markedly improved survival. Compared with the MyD88fl/fl littermates, Lyz-MyD88−/− mice had warmer body temperature, attenuated systemic and cardiac inflammatory cytokine production,and significantly improved cardiac function, whereas α-MHC-MyD88−/− mice had decreased myocardial inducible nitricoxide synthase induction and modestly preserved cardiac function. Both cardiomyocyte- and myeloid-MyD88 signaling play a role in cardiac dysfunction and mortality during endotoxin shock. Myeloid-MyD88 signaling plays a predominant role in systemic and cardiac inflammation after endotoxin challenge.

Mechanism of focal cerebral ischemic tolerance in rats with ischemic preconditioning involves MyD88- and TRIF-dependent pathways

The aim of this study was to explore the involvement of Toll-like receptor 4 (TLR4) and the downstream myeloid differentiation factor 88 (MyD88)-dependent and -independent pathways in the mechanisms of cerebral ischemic tolerance. Using an improved middle cerebral artery occlusion method, we constructed a preconditioned ischemic brain model in rats. Sham and ischemia-reperfusion groups were also established. The expression levels of proteins in the MyD88/nuclear factor-κB (NF-κB) pathway (MyD88-dependent) were compared with those in the Toll/interleukin-1 receptor-domain-containing adaptor-inducing interferon-β (TRIF)/interferon regulatory factor-3 (IRF-3) pathway (MyD88-independent) by western blot analysis. NF-κB and IRF-3 protein expression levels within cells were determined by immunofluorescence staining of frozen tissue sections. Western blot analysis showed a downregulation of MyD88 protein expression in the brain tissue of ischemic preconditioned rats; however, NF-κB, TRIF and IRF-3 protein expression levels were upregulated. Immunofluorescence staining showed that NF-κB protein was mainly located in the cytoplasm in ischemic preconditioned rats and IRF-3 was predominantly located in the nucleus. The results indicate that changes in the two TLR4 downstream pathways are the main mechanisms involved in the development of brain ischemic tolerance with ischemic pretreatment.