Inhibitory Effect of Cinobufotalin on Macrophage Inflammatory Factor Storm and Its Mechanism / 中国实验血液学杂志

OBJECTIVE@#To investigate the inflammatory effects of Cinobufotalin on monocytes in resting state and macrophages in activated state and its molecular mechanism.@*METHODS@#THP-1 cells were stimulated with Phorbol 12-myristate 13-acetate to induce differentiation into macrophages. Lipopolysaccharides was added to activate macrophages in order to establish macrophage activation model. Cinobufotalin was added to the inflammatory cell model for 24 h as a treatment. CCK-8 was used to detect cell proliferation, Annexin V /PI double staining flow cytometry was used to detect cell apoptosis, flow cytometry was used to detect macrophage activation, and cytometric bead array was used to detect cytokines. Transcriptome sequencing was used to explore the gene expression profile regulated by Cinobufotalin. Changes in the significantly regulated molecules were verified by real-time quantitative polymerase chain reaction and Western blot.@*RESULTS@#1∶25 concentration of Cinobufotalin significantly inhibited the proliferation of resting monocytes(P<0.01), and induced apoptosis(P<0.01), especially the activated macrophages(P<0.001, P<0.001). Cinobufotalin significantly inhibited the activation of macrophages, and significantly down-regulated the inflammatory cytokines(IL-6, TNF-α, IL-1β, IL-8) released by activated macrophages(P＜0.001). Its mechanism was achieved by inhibiting TLR4/MYD88/P-IκBa signaling pathway.@*CONCLUSION@#Cinobufotalin can inhibit the inflammatory factors produced by the over-activation of macrophages through TLR4/MYD88/P-IκBa pathway, which is expected to be applied to the treatment and research of diseases related to the over-release of inflammatory factors.

Clinicopathological features and BRAF V600E and MYD88 L265P mutation status of nodal marginal zone lymphoma / 中华病理学杂志

Objective: To investigate the clinicopathological features as well as BRAF V600E and MYD88 L265P mutation status of nodal marginal zone B cell lymphoma (NMZL). Methods: Thirty-two cases of NMZL were diagnosed from September 2009 to February 2021 at the Henan Provincial People's Hospital and Peking University School of Basic Medical Sciences. The clinicopathologic characteristics were obtained and analyzed. BRAF V600E and MYD88 L265P mutation status were identified using PCR and Sanger sequencing, respectively. Results: There were 20 males and 12 females patients with a median age of 69 years (ranging 36-82 years). The most prevalent clinical manifestation was multiple lymph nodes enlargement in head and neck (22/32, 68.8%), followed by inguinal (12/32, 37.5%), axillary (11/32, 34.4%), mediastinum (5/32, 15.6%) and retroperitoneal lymph nodes (4/32, 12.5%). Most of the patients were in Ann Arbor stage Ⅰ/Ⅱ (21 cases). The morphologic features included diffuse (24/32, 75.0%), nodular (5/32, 15.6%), interfollicular (2/32,6.3%) and perifollicular (1/32,3.1%) types. The tumor cells showed monocyte-like, centrocyte-like, small lymphocyte-like and plasma cell-like differentiation. Immunophenotyping revealed diffuse expression of CD20 in all tumor cells, whereas CD43 (11/32, 34.4%), bcl-2 (20/32, 62.5%), MNDA (13/32, 40.6%) and CD5 (2/32, 6.3%) were partially expressed. Ki-67 proliferation index varied from 10% to 40%. BRAF V600E mutation was found in two cases (2/32, 6.3%), but MYD88 L265P mutation was not detected. Eighteen patients survived and three died at the end of follow-up period which ranged 6 to 110 months. Conclusions: The morphologic features of NMZL varies across individuals, it should be differentiated from various B-cell lymphomas; however immunological biomarkers with high specificity for NMZL are still lacking. No MYD88 L265P mutation is found in NMZL. Some cases may harbor BRAF V600E mutation and yet the prevalence remains indeterminate; further researches are warranted.

miR-139-5p protects septic mice with acute lung injury by inhibiting Toll-like receptor 4/Myeloid differentiation factor 88/Nuclear factor-&mac_kgr; B signaling pathway

OBJECTIVES: To investigate the role of miR-139-5p and the TLR4/MyD88/NF-κB signaling pathway in acute lung injury in septic mice. METHOD: A total of 140 healthy male SPF C57BL/6 mice were divided into seven groups, i.e., Normal, Control, NC, miR-139-5p mimic, miR-139-5p inhibitor, TAK-242, and miR-139-5p inhibitor+TAK-242 groups. The levels of miR-139-5p, proteins related to the TLR4/MyD88/NF-κB signaling pathway (TLR4, MyD88, and p-NF-κB p50), and MPO, SOD, GSH, and MDA in lung tissue were measured. The lung tissue wet-to-dry mass ratio (W/D), arterial oxygen partial pressure (PaO2), and carbon dioxide partial pressure (PaCO2) were measured. RESULTS: A web-based bioinformatic tool predicted that MyD88 was a target of miR-139-5p, which was verified by a dual luciferase reporter assay. Compared with those in the Normal group, the levels of miR-139-5p, PaO2, SOD, and GSH were significantly lower, while those of TLR4, MyD88, p-NF-κB p50, W/D, PaCO2, IL-1β, TNF-α, IL-6, MPO, and MDA were higher in all other groups. Moreover, compared with their levels in the Control group, these indicators exhibited contrasting results in the miR-139-5p mimic and TAK-242 groups, but were similar in the miR-139-5p inhibitor group. In the miR-139-5p inhibitor+TAK-242 group, acute lung injury, aggravated by miR-139-5p inhibitor, was partially rescued by TAK-242. CONCLUSION: miR-139-5p inhibits the TLR4/MyD88/NF-κB signaling pathway to alleviate acute lung injury in septic mice.

Danshen-Chuanxiongqin Injection attenuates cerebral ischemic stroke by inhibiting neuroinflammation via the TLR2/ TLR4-MyD88-NF-κB Pathway in tMCAO mice / 中国天然药物

Danshen-Chuanxiongqin Injection (DCI) is a commonly used traditional Chinese medicine for the treatment of cerebral ischemic stroke in China. However, its underlying mechanisms remain completely understood. The current study was designed to explore the protective mechanisms of DCI against cerebral ischemic stroke through integrating whole-transcriptome sequencing coupled with network pharmacology analysis. First, using a mouse model of cerebral ischemic stroke by transient middle cerebral artery occlusion (tMCAO), we found that DCI (4.10 mL·kg

Relationship between Polymorphism of miR-155 and Its Target Gene MyD88 and Clinicopathological Features of Diffuse Large B-cell Lymphoma / 中国实验血液学杂志

OBJECTIVE@#To investigate the relationship between the polymorphism of miR-155 and its target gene MyD88 and clinicopathological features of diffuse large B-cell lymphoma (DLBCL).@*METHODS@#135 cases of DLBCL patients in our hospital from March 2015 to August 2017 were selected, and 90 cases of reactive hyperplasia of lymph nodes were selected as the control group. The relative expression of miR-155 and MyD88 gene polymorphism were detected in the two groups, and the relationship between miR-155 and MyD88 gene polymorphism and clinicopathological characteristics of DLBCL was analyzed.@*RESULTS@#The relative expression of miR-155 in DLBCL patients was significantly higher than that in the control group (P<0.05). The mutation rate of MyD88 L265P in DLBCL group was significantly higher than that in control group (P<0.05). The relative expression of miR-155 in patients with MyD88 L265P mutation was significantly higher than that in patients with wild-type DLBCL (P<0.05). The relative expression of miR-155 and the polymorphism of MyD88 L265P were associated with lesion location, stage, BCL-2 protein expression and MyD88 protein expression in DLBCL patients (t=7.461、8.804、6.487、10.812； χ@*CONCLUSION@#The abnormal expression of miR-155 and the mutation rate of MyD88 gene in DLBCL patients are increased, and the expression of miR-155 and the mutation of MyD88 gene affect the disease progression and prognosis of patients, which may be potential biological indicators for the diagnosis, treatment and prognosis of DLBCL.

MMP2 and MMP9 are Associated with Apical Periodontitis Progression and Might be Modulated by TLR2 and MyD88

Abstract The aim of this study was to evaluate the expression of MMP2 and MMP9 during apical periodontitis (AP) progression in TLR2 (TLR2 KO) and in MyD88 (MyD88 KO) knockout mice compared to wild type (WT) mice. AP was induced in mandibular first molars of TLR2 KO (n= 18), MyD88 KO (n= 18), and WT mice (n= 18). After 7, 21, and 42 days, the animals were euthanized and the jaws were dissected and subjected to histotechnical processing. Subsequent sections were stained by immunohistochemistry and evaluated for detection of MMP2 and MMP9. Statistical analysis of the semi-quantitative analysis of immunohistochemistry was performed using chi-square test (α = 0.05). In the initial periods of AP progression, an increased expression of MMP9 in the TLR2 KO and MyD88 KO mice was observed. In the final periods of AP progression, a reduction of MMP2 expression and an increase of MMP9 expression in the TLR2 KO mice were observed. MMP2 and MMP9 production was modulated for TLR2 and MyD88 during apical periodontitis progression.

Resumo O objetivo deste estudo foi avaliar a expressão de MMP2 e MMP9 durante a progressão da periodontite apical (AP) em camundongos knockout para TLR2 (TLR2 KO) e MyD88 (MyD88 KO) comparados aos camundongos wild type (WT). A AP foi induzida nos primeiros molares inferiores dos camundongos TLR2 KO (n = 18), MyD88 KO (n = 18) e WT (n = 18). Após 7, 21 e 42 dias, os animais foram eutanaziados e as mandíbulas foram dissecadas e submetidas a processamento histotécnico. As lâminas foram coradas por imuno-histoquímica e analisadas para a detecção de MMP2 e MMP9. A análise estatística semi-quantitativa da imuno-histoquímica foi realizada pelo teste qui-quadrado (α = 0,05). Nos períodos iniciais de progressão AP, foi observada uma expressão aumentada de MMP9 nos camundongos TLR2 KO e MyD88 KO. Nos períodos finais de progressão AP, observou-se uma redução da expressão de MMP2 e um aumento da expressão de MMP9 nos camundongos TLR2 KO. A produção de MMP2 e MMP9 foi modulada por TLR2 e MyD88 durante a progressão da periodontite apical.

Efeito da infecção por HPV nas vias de sinalização por toll like receptors / Effects of HPV infection on TLR signaling pathway

As oncoproteínas E6 e E7 do Papilomavírus Humano (HPV) estão envolvidas na desregulação do sistema imune inato, provocando alterações na expressão dos receptores do tipo Toll (TLR). Considerando-se a função da via de sinalização iniciada por TLR, haveria uma vantagem para o vírus capaz de manipular a resposta desta via de modo que possa persistir nas células sem ser detectado pelo sistema imune ou ainda modulando essa resposta e criando um ambiente mais propício à manutenção da infecção. No entanto, muitos dos mecanismos que levam à eliminação da infecção ou persistência do HPV ainda são pouco conhecidos. O objetivo principal desse trabalho é investigar o papel das vias de TLR no processo de carcinogênese mediado por HPV. Inicialmente, foi analisada a expressão de genes da via de TLR em linhagens de tumores cervicais e em células expressando as oncoproteínas virais. Foram identificados vários genes diferencialmente expressos entre linhagens de células tumorais e queratinócitos normais, incluindo moléculas adaptadoras da via de TLR e genes associados à via da MAP quinase, ativação de NFkappaB e resposta imune antiviral. Cerca de 90% destes genes foram regulados negativamente. Entre eles, destacamos HMGB1, que apesar de possuir menos RNAm nas células tumorais possui um nível proteico muito maior, além de ter-se mostrado de grande importância para a viabilidade e proliferação das células tumorais, conforme demonstrado através de experimentos de supressão gênica. Em conjunto, os nossos dados indicam que E6 e E7 de HPVs de alto risco inibem proteínas da via de sinalização de TLR

Previous studies have shown that E6 and E7 HPV oncoproteins are involved in innate immune system dysregulation, causing alterations on Toll-like receptors (TLR) expression. Considering TLR pathway function, it would be advantageous for a virus to manipulate the response of this pathway so it can persist in cells without being detected by the immune system or to modulate this response to create a better environment for persistence of infection. However, many of the mechanisms leading to HPV infection clearance or persistence are still unknown and matter of active investigation. We analyzed in cervical cancer cell lines expression of genes from TLR pathway; several were differentially expressed between tumor cells lines and normal keratinocytes, including TLR adaptors molecules and genes associated with MAP kinase pathway, NFkappaB activation and antiviral immune response. About 90% of these genes were down regulated. Among them, we selected HMGB1 for further characterization due to its interference with tumor cell viability and proliferation. Altogether, our data indicate that high risk HPV E6 and E7 can inhibit TLR signaling pathway

Overexpression of myeloid differentiation protein 88 in mice induces mild cardiac dysfunction, but no deficit in heart morphology

Cardiac remodeling involves changes in heart shape, size, structure, and function after injury to the myocardium. The proinflammatory adaptor protein myeloid differentiation protein 88 (MyD88) contributes to cardiac remodeling. To investigate whether excessive MyD88 levels initiate spontaneous cardiac remodeling at the whole-organism level, we generated a transgenic MyD88 mouse model with a cardiac-specific promoter. MyD88 mice (male, 20-30 g, n=∼80) were born at the expected Mendelian ratio and demonstrated similar morphology of the heart and cardiomyocytes with that of wild-type controls. Although heart weight was unaffected, cardiac contractility of MyD88 hearts was mildly reduced, as shown by echocardiographic examination, compared with wild-type controls. Moreover, the cardiac dysfunction phenotype was associated with elevation of ANF and BNP expression. Collectively, our data provide novel evidence of the critical role of balanced MyD88 signaling in maintaining physiological function in the adult heart.

Cloning, expression and functional analysis of the duck Toll-like receptor 5 (TLR5) gene

Toll-like receptor 5 (TLR5) is responsible for the recognition of bacterial flagellin in vertebrates. In the present study, the first TLR5 gene in duck was cloned. The open reading frame (ORF) of duck TLR5 (dTLR5) cDNA is 2580 bp and encodes a polypeptide of 859 amino acids. We also cloned partial sequences of myeloid differentiation factor 88, 2'-5'-oligoadenylate synthetase (OAS), and myxovirus resistance (Mx) genes from duck. dTLR5 mRNA was highly expressed in the bursa of Fabricius, spleen, trachea, lung, jejunum, rectum, and skin; moderately expressed in the muscular and glandular tissues, duodenum, ileum, caecum, and pancreas; and minimally expressed in the heart, liver, kidney, and muscle. DF-1 or HeLa cells transfected with DNA constructs encoding dTLR5 can activate NF-kappaB leading to the activation of interleukin-6 (IL-6) promoter. When we challenged ducks with a Herts33 Newcastle disease virus (NDV), mRNA transcription of the antiviral molecules Mx, Double stranded RNA activated protein kinase (PKR), and OAS was up-regulated in the liver, lung, and spleen 1 and 2 days post-inoculation.

Myeloid differentiation primary response protein 88 blockade upregulates indoleamine 2,3-dioxygenase expression in rheumatoid synovial fibroblasts

Indoleamine 2,3-dioxygenase (IDO) is a key negative regulator of immune responses and has been implicated in tumor tolerance, autoimmune disease and asthma. IDO was detected in the joint synovial tissue in the inflammatory microenvironment of rheumatoid arthritis (RA), but IDO expression in joint synovial tissue is not sufficient to overcome the inflamed synovial environment. This study aimed to unravel the mechanisms involving the failure to activate tolerogenic IDO in the inflamed joint. We demonstrate that both poly (I:C) and lipopolysaccharide (LPS) induce expression of IDO in synovial fibroblasts. However, inflammatory cytokines such as IL-17, TNF-alpha, IL-12, IL-23 and IL-16 did not induce IDO expression. Poly (I:C) appeared to induce higher IDO expression than did LPS. Surprisingly, toll-like receptor (TLR)4-mediated IDO expression was upregulated after depletion of myeloid differentiation primary response protein 88 (MyD88) in synovial fibroblasts using small interfering RNA (siRNA). IDO, TLR3 and TLR4 were highly expressed in synovial tissue of RA patients compared with that of osteoarthritis patients. In addition, RA patients with severe disease activity had higher levels of expression of IDO, TLR3 and TLR4 in the synovium than patients with mild disease activity. These data suggest that upregulation of IDO expression in synovial fibroblasts involves TLR3 and TLR4 activation by microbial constituents. We showed that the mechanisms responsible for IDO regulation primarily involve MyD88 signaling in synovial fibroblasts, as demonstrated by siRNA-mediated knockdown of MyD88.