Study on the anti-sepsis mechanism of ursolic acid by targeting myeloid differentiation protein-2 / 中华危重病急救医学

OBJECTIVE@#To explore the mechanism of ursolic acid in treating sepsis using myeloid differentiation protein-2 (MD-2) as the research carrier.@*METHODS@#The affinity of ursolic acid and MD-2 was determined by biofilm interferometry technique, and the bonding mode between ursolic acid and MD-2 was tested with the aid of molecular docking technique. Raw 264.7 cells were cultured in RPMI 1640 medium and subcultured was conducted when the cell density reached 80%-90%. The second-generation cells were used for in the experiment. The effects of 8, 40 and 100 mg/L ursolic acid on cell viability were assessed by methyl thiazolyl tetrazolium (MTT) method. Cells were divided into blank group, lipopolysaccharide (LPS) group (LPS 100 μg/L) and ursolic acid group (100 μg/L LPS treatment after addition of 8, 40 or 100 mg/L ursolic acid). The effect of ursolic acid on the release of cytokines nitric oxide (NO), tumor necrosis factor-α (TNF-α) and interleukins (IL-6, IL-1β) were evaluated by enzyme-linked immunosorbent assay (ELISA). The influence of ursolic acid on the mRNA expressions of TNF-α, IL-6, IL-1β, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were detected by reverse transcription-polymerase chain reaction (RT-PCR). The implication of ursolic acid on the protein expressions of LPS-Toll-like receptor 4 (TLR4)/MD-2-nuclear factor-κB (NF-κB) pathway were tested by Western blotting.@*RESULTS@#Ursolic acid could bind to the hydrophobic cavity of MD-2 through hydrophobic bond with the amino acid residues of the protein. Therefore, ursolic acid showed high affinity with MD-2 [dissociation constant (KD) = 1.43×10-4]. The cell viability were decreased slightly, with the concentration of ursolic acid increasing, and the cell viability of 8, 40 and 100 mg/L ursolic acid were 96.01%, 94.32% and 92.12%, respectively, and there was no significant difference compared with the blank group (100%). Compared with the blank group, the cytokine level of the LPS group was significantly increased. The level of cytokines were significantly reduced by the treatment of 8, 40 and 100 mg/L ursolic acid, and the higher the concentration, the more obvious effect [compared between 100 mg/L ursolic acid group and LPS group: IL-1β (μmol/L): 38.018±0.675 vs. 111.324±1.262, IL-6 (μmol/L): 35.052±1.664 vs. 115.255±5.392, TNF-α (μmol/L): 39.078±2.741 vs. 119.035±4.269, NO (μmol/L): 40.885±2.372 vs. 123.405±1.291, all P < 0.01]. Compared with the blank group, the mRNA expressions of TNF-α, IL-6, IL-1β, iNOS and COX-2 in the LPS group were significantly increased, and the protein expressions of MD-2, myeloid differentiation factor 88 (MyD88), phosphorylation NF-κB p65 (p-NF-κB p65) and iNOS in the LPS-TLR4/MD-2-NF-κB pathway were significantly up-regulated. Compared with the LPS group, the mRNA expressions of TNF-α, IL-6, IL-1β, iNOS and COX-2 were significantly reduced by the treatment of 100 mg/L ursolic acid bound with MD-2 protein [TNF-α (2-ΔΔCt): 4.659±0.821 vs. 8.652±0.787, IL-6 (2-ΔΔCt): 4.296±0.802 vs. 11.132±1.615, IL-1β (2-ΔΔCt): 4.482±1.224 vs. 11.758±1.324, iNOS (2-ΔΔCt): 1.785±0.529 vs. 4.249±0.811, COX-2 (2-ΔΔCt): 5.591±1.586 vs. 16.953±1.651, all P < 0.01], and the proteins expressions of MD-2, MyD88, p-NF-κB p65 and iNOS in the LPS-TLR4/MD-2-NF-κB pathway were significantly down-regulated (MD-2/β-actin: 0.191±0.038 vs. 0.704±0.049, MyD88/β-actin: 0.470±0.042 vs. 0.875±0.058, p-NF-κB p65/β-actin: 0.178±0.012 vs. 0.571±0.012, iNOS/β-actin: 0.247±0.035 vs. 0.549±0.033, all P < 0.01). However, there was no difference in protein expression of NF-κB p65 among the three groups.@*CONCLUSIONS@#Ursolic acid inhibits the release and expression of cytokines and mediators and regulates LPS-TLR4/MD-2-NF-κB signaling pathway by blocking MD-2 protein, and thus plays an anti-sepsis role.

Leaky Gut Plays a Critical Role in the Pathophysiology of Autism in Mice by Activating the Lipopolysaccharide-Mediated Toll-Like Receptor 4-Myeloid Differentiation Factor 88-Nuclear Factor Kappa B Signaling Pathway / 神经科学通报·英文版

Increased intestinal barrier permeability, leaky gut, has been reported in patients with autism. However, its contribution to the development of autism has not been determined. We selected dextran sulfate sodium (DSS) to disrupt and metformin to repair the intestinal barrier in BTBR T+tf/J autistic mice to test this hypothesis. DSS treatment resulted in a decreased affinity for social proximity; however, autistic behaviors in mice were improved after the administration of metformin. We found an increased affinity for social proximity/social memory and decreased repetitive and anxiety-related behaviors. The concentration of lipopolysaccharides in blood decreased after the administration of metformin. The expression levels of the key molecules in the toll-like receptor 4 (TLR4)-myeloid differentiation factor 88 (MyD88)-nuclear factor kappa B (NF-κB) pathway and their downstream inflammatory cytokines in the cerebral cortex were both repressed. Thus, "leaky gut" could be a trigger for the development of autism via activation of the lipopolysaccharide-mediated TLR4-MyD88-NF-κB pathway.

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.

Formononetin improves cognitive behavior in aging rats with chronic unpredictable mild in hippocampal tissue stress by blocking the NF-κB pathway and inhibiting the release of inflammatory factors / 细胞与分子免疫学杂志

Objective To investigate the effects of formononetin (FMN) on cognitive behavior and inflammation in aging rats with chronic unpredictable mild stress (CUMS). Methods SD rats aged about 70 weeks were divided into healthy control group, CUMS model group, CUMS combined with 10 mg/kg FMN group, CUMS combined with 20 mg/kg FMN group and CUMS combined with 1.8 mg/kg fluoxetine hydrochloride (Flu) group. Except for healthy control group, other groups were stimulated with CUMS and administered drugs for 28 days. Sugar water preference, forced swimming experiment and open field experiment were used to observe the emotional behavior of rats in each group. HE staining was used to observe the pathological injury degree of brain equine area. The contents of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were detected by the kit. The apoptosis was tested by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) in the brain tissue. The levels of tumor necrosis factor α (TNF-α), inducible nitric oxide synthase (iNOS) and interleukin 6 (IL-6) in peripheral blood were measured by ELISA. Western blot analysis was used to detect Bcl2, Bcl2 associated X protein (BAX), cleaved caspase-9, cleaved caspase-3, Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and phosphorylated nuclear factor κB p65 (p-NF-κB p65) in brain tissues. Results Compared with CUMS model group, sugar water consumption, open field activity time, open field travel distance and swimming activity time significantly increased in the CUMS combined with 20 mg/kg FMN group and the CUMS combined with 1.8 mg/kg Flu group. The number of new outarm entry increased significantly, while the number of initial arm entry and other arm entry decreased significantly. The pathological damage of brain equine area was alleviated, and the contents of 5-HT and 5-HIAA were significantly increased. The ratio of BAX/Bcl2 and the expression of cleaved caspase-9 and cleaved caspase-3 protein as well as the number of apoptotic cells were significantly decreased. The contents of TNF-α, iNOS and IL-6 were significantly decreased. The protein levels of TLR4, MyD88 and p-NF-κB p65 were significantly decreased. Conclusion FMN can inhibit the release of inflammatory factors by blocking NF-κB pathway and improve cognitive and behavioral ability of CUMS aged rats.

Mechanism of Berberis atrocarpa anthocyanin against Alzheimer's disease based on network pharmacology and experimental verification / 中国中药杂志

This study aimed to explore the potential mechanism of Berberis atrocarpa Schneid. anthocyanin against Alzheimer's disease(AD) based on network pharmacology, molecular docking technology, and in vitro experiments. Databases were used to screen out the potential targets of the active components of B. atrocarpa and the targets related to AD. STRING database and Cytoscape 3.9.0 were adopted to construct a protein-protein interaction(PPI) network and carry out topological analysis of the common targets. Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analyses were performed on the target using the DAVID 6.8 database. Molecular docking was conducted to the active components and targets related to the nuclear factor kappa B(NF-κB)/Toll-like receptor 4(TLR4) pathway. Finally, lipopolysaccharide(LPS) was used to induce BV2 cells to establish the model of AD neuroinflammation for in vitro experimental validation. In this study, 426 potential targets of active components of B. atrocarpa and 329 drug-disease common targets were obtained, and 14 key targets were screened out by PPI network. A total of 623 items and 112 items were obtained by GO functional enrichment analysis and KEGG pathway enrichment analysis, respectively. Molecular docking results showed that NF-κB, NF-κB inhibitor(IκB), TLR4, and myeloid differentiation primary response 88(MyD88) had good binding abilities to the active components, and malvidin-3-O-glucoside had the strongest binding ability. Compared with the model group, the concentration of nitric oxide(NO) decreased at different doses of malvidin-3-O-glucoside without affecting the cell survival rate. Meanwhile, malvidin-3-O-glucoside down-regulated the protein expressions of NF-κB, IκB, TLR4, and MyD88. This study uses network pharmacology and experimental verification to preliminarily reveal that B. atrocarpa anthocyanin can inhibit LPS-induced neuroinflammation by regulating the NF-κB/TLR4 signaling pathway, thereby achieving the effect against AD, which provides a theoretical basis for the study of its pharmacodynamic material basis and mechanism.

Clinical characteristics and prognosis of primary and secondary diffuse large B-cell lymphoma of the pancreas / 中华血液学杂志

Objective: To analyze the clinical characteristics and prognosis of primary and secondary pancreatic diffuse large B-cell lymphoma (DLBCL) . Methods: Clinical data of patients with pancreatic DLBCL admitted at Shanghai Rui Jin Hospital affiliated with Shanghai Jiao Tong University School of Medicine from April 2003 to June 2020 were analyzed. Gene mutation profiles were evaluated by targeted sequencing (55 lymphoma-related genes). Univariate and multivariate Cox regression models were used to evaluate the prognostic factors of overall survival (OS) and progression-free survival (PFS) . Results: Overall, 80 patients were included; 12 patients had primary pancreatic DLBCL (PPDLBCL), and 68 patients had secondary pancreatic DLBCL (SPDLBCL). Compared with those with PPDLBCL, patients with SPDLBCL had a higher number of affected extranodal sites (P<0.001) and had higher IPI scores (P=0.013). There was no significant difference in the OS (P=0.120) and PFS (P=0.067) between the two groups. Multivariate analysis indicated that IPI intermediate-high/high risk (P=0.025) and double expressor (DE) (P=0.017) were independent adverse prognostic factors of OS in patients with pancreatic DLBCL. IPI intermediate-high/high risk (P=0.021) was an independent adverse prognostic factor of PFS in patients with pancreatic DLBCL. Targeted sequencing of 29 patients showed that the mutation frequency of PIM1, SGK1, BTG2, FAS, MYC, and MYD88 in patients with pancreatic DLBCL were all >20%. PIM1 (P=0.006 for OS, P=0.032 for PFS) and MYD88 (P=0.001 for OS, P=0.017 for PFS) mutations were associated with poor OS and PFS in patients with SPDLBCL. Conclusion: There was no significant difference in the OS and PFS between patients with PPDLBCL and those with SPDLBCL. IPI intermediate-high/high risk and DE were adverse prognostic factors of pancreatic DLBCL. PIM1, SGK1, BTG2, FAS, MYC, and MYD88 were common mutations in pancreatic DLBCL. PIM1 and MYD88 mutations indicated worse prognosis.

Clinicopathologic characteristics and prognostic analysis of testicular diffuse large B-cell lymphoma / 中华血液学杂志

Objective: To analyze the clinicopathologic characteristics and prognosis of testicular diffuse large B-cell lymphoma (DLBCL) . Methods: A retrospective analysis was performed on 68 patients with testicular DLBCL admitted to Ruijin Hospital affiliated to Shanghai Jiao Tong University School of Medicine from October 2001 to April 2020. The gene mutation profile was evaluated by targeted sequencing (55 lymphoma-related genes) , and prognostic factors were analyzed. Results: A total of 68 patients were included, of whom 45 (66.2% ) had primary testicular DLBCL and 23 (33.8% ) had secondary testicular DLBCL. The proportion of secondary testicular DLBCL patients with Ann Arbor stage Ⅲ-Ⅳ (P<0.001) , elevated LDH (P<0.001) , ECOG score ≥ 2 points (P=0.005) , and IPI score 3-5 points (P<0.001) is higher than that of primary testicular DLBCL patients. Sixty-two (91% ) patients received rituximab in combination with cyclophosphamide, adriamycin, vincristine, and prednisone (R-CHOP) -based first-line regimen, whereas 54 cases (79% ) underwent orchiectomy prior to chemotherapy. Patients with secondary testicular DLBCL had a lower estimated 5-year progression-free survival (PFS) rate (16.5% vs 68.1% , P<0.001) and 5-year overall survival (OS) rate (63.4% vs 74.9% , P=0.008) than those with primary testicular DLBCL, and their complete remission rate (57% vs 91% , P=0.003) was also lower than that of primary testicular DLBCL. The ECOG scores of ≥2 (PFS: P=0.018; OS: P<0.001) , Ann Arbor stages Ⅲ-Ⅳ (PFS: P<0.001; OS: P=0.018) , increased LDH levels (PFS: P=0.015; OS: P=0.006) , and multiple extra-nodal involvements (PFS: P<0.001; OS: P=0.013) were poor prognostic factors in testicular DLBCL. Targeted sequencing data in 20 patients with testicular DLBCL showed that the mutation frequencies of ≥20% were PIM1 (12 cases, 60% ) , MYD88 (11 cases, 55% ) , CD79B (9 cases, 45% ) , CREBBP (5 cases, 25% ) , KMT2D (5 cases, 25% ) , ATM (4 cases, 20% ) , and BTG2 (4 cases, 20% ) . The frequency of mutations in KMT2D in patients with secondary testicular DLBCL was higher than that in patients with primary testicular DLBCL (66.7% vs 7.1% , P=0.014) and was associated with a lower 5-year PFS rate in patients with testicular DLBCL (P=0.019) . Conclusion: Patients with secondary testicular DLBCL had worse PFS and OS than those with primary testicular DLBCL. The ECOG scores of ≥2, Ann Arbor stages Ⅲ-Ⅳ, increased LDH levels, and multiple extra-nodal involvements were poor prognostic factors in testicular DLBCL. PIM1, MYD88, CD79B, CREBBP, KMT2D, ATM, and BTG2 were commonly mutated genes in testicular DLBCL, and the prognosis of patients with KMT2D mutations was poor.

Moxibustion improves experimental colitis in rats with Crohn's disease by regulating bile acid enterohepatic circulation and intestinal farnesoid X receptor / 中西医结合学报

OBJECTIVE@#This 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).@*METHODS@#Sprague-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.@*RESULTS@#Mild 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.@*CONCLUSION@#Mild 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.

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.

Genistein attenuates LPS-induced inflammatory injury of rat dorsal root ganglion neuron via down-regulating HDAC6 / 中南大学学报(医学版)

OBJECTIVES@#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.@*METHODS@#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.@*RESULTS@#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).@*CONCLUSIONS@#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.

Electroacupuncture Attenuates Immune-Inflammatory Response in Hippocampus of Rats with Vascular Dementia by Inhibiting TLR4/MyD88 Signaling Pathway / 中国结合医学杂志

OBJECTIVE@#To investigate whether electroacupuncture (EA) alleviates cognitive impairment by suppressing the toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88) signaling pathway, which triggers immune-inflammatory responses in the hippocampus of rats with vascular dementia (VaD).@*METHODS@#The experiments were conducted in 3 parts and in total the Sprague-Dawley rats were randomly divided into 8 groups by a random number table, including sham, four-vessel occlusion (4-VO), 4-VO+EA, 4-VO+non-EA, sham+EA, 4-VO+lipopolysaccharide (LPS), 4-VO+LPS+EA, and 4-VO+TAK-242 groups. The VaD model was established by the 4-VO method. Seven days later, rats were treated with EA at 5 acupoints of Baihui (DV 20), Danzhong (RN 17), Geshu (BL 17), Qihai (RN 6) and Sanyinjiao (SP 6), once per day for 3 consecutive weeks. Lymphocyte subsets, lymphocyte transformation rates, and inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor α(TNF-α) were measured to assess immune function and inflammation in VaD rats. Transmission electron microscopy was used to observe the ultrastructure of nerve cells in the hippocampus. The levels of TLR4, MyD88, IL-6, and TNF-α were detected after EA treatment. TLR4/MyD88 signaling and cognitive function were also assessed after intracerebroventricular injection of TLR4 antagonist TAK-242 or TLR4 agonist LPS with or without EA.@*RESULTS@#Compared with the 4-VO group, EA notably improved immune function of rats in the 4-VO+EA group, inhibited the protein and mRNA expressions of TLR4 and MyD88 in the hippocampus of rats, reduced the expressions of serum IL-6 and TNF-α (all P0.05).@*CONCLUSIONS@#EA attenuated cognitive impairment associated with immune inflammation by inhibition of the TLR4/MyD88 signaling pathway. Thus, EA may be a promising alternative therapy for the treatment of VaD.

Butyl alcohol extract of Baitouweng Decoction alleviates vulvovaginal candidiasis in mice by downregulating NLRP3 inflammasome and related signal pathways / 中国中药杂志

This study aims to explore the effect of butyl alcohol extract of Baitouweng Decoction(BAEB) on vulvovaginal candidiasis(VVC) in mice and to clarify the mechanism from Toll-like receptors(TLRs)/MyD88 and Dectin-1/Syk signal pathways and NLRP3 inflammasome. To be specific, female KM mice were randomized into control group(i.g., normal saline), model group, fluco-nazole group(i.g., 20 mg·kg~(-1)), and low-dose, medium-dose, and high-dose BAEB groups(i.g., 20, 40, and 80 mg·kg~(-1), respectively). VVC was induced in mice except the control group. After the modeling, administration began and lasted 7 days. The ge-neral conditions and body weight of mice were recorded every day. On the 1 st, 3 rd, 7 th, and 14 th after vaginal infection by Candida albicans, the fungal load in the vaginal lavage fluid of the mice was measured with the plate method, and the morphology of C. albicans in vaginal lavage fluid was observed based on Gram staining. After the mice were killed, vaginal tissues were subjected to hematoxylin-eosin(HE) staining and periodic acid-Schiff(PAS) staining for vaginal histopathological analysis. The content of cytokines in vaginal lavage fluid, such as interleukin(IL)-1β, IL-18, tumor necrosis factor-α(TNF-α), IL-6, and S100 a8, was determined by enzyme-linked immunosorbent assay(ELISA), and content of reactive oxygen species(ROS) in vaginal tissues by tissue ROS detection kit. The protein expression of NLRP3, ASC, caspase-1, Dectin-1, Syk, MyD88, TLR2, TLR4, and nuclear factor-κB(NF-κB) in vaginal tissues was detected by Western blot, and the levels and distribution of NLRP3, Dectin-1, Syk, MyD88, TLR2, and TLR4 in vaginal tissues were determined with the immunohistochemical method. The results show that BAEB can improve the general conditions of VVC mice, reduce the fungal load and C. albicans hyphae in vaginal secretion, decrease ROS content in vaginal tissues and content of cytokines in vaginal lavage fluid, and down-regulate the expression of NLRP3, ASC, caspase-1, Dectin-1, Syk, MyD88, TLR2, TLR4, and NF-κB in vaginal tissues. The above results indicate that BAEB exerts therapeutic effect on VVC mice by down-regulating the key proteins in the TLRs/MyD88 and Dectin-1/Syk signal pathways and NLRP3 inflammasome.

Exploring the detection of MYD88 mutation in patients with Waldenström macroglobulinemia by different methods and specimens / 中华血液学杂志

Objective: To improve the positivity rate and accuracy of MYD88 mutation detection in patients with Waldenström macroglobulinemia (WM) . Methods: MYD88 mutation status was retrospectively evaluated in 66 patients diagnosed with WM in Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine from June 2017 to June 2021. The positivity rate and accuracy of the different methods and specimens for MYD88 mutation detection were analyzed. Results: MYD88 mutations were detected in 51 of 66 patients with WM, with an overall positivity rate of 77%. The positivity rate of the next-generation sequencing (NGS) or allele-specific polymerase chain reaction (AS-PCR) was significantly higher than that of the first-generation Sanger sequencing (84% vs 71% vs 46%, P<0.05) . For the different specimens, the positivity rate for the lymph nodes or bone marrow was significantly higher than that of peripheral blood (79% vs 84% vs 52%, P<0.05) . The positivity rate of the MYD88 mutation in the lymph nodes, bone marrow, and peripheral blood determined by NGS was 86%, 90%, and 67%, respectively. The positivity rate in the lymph nodes, bone marrow, and peripheral blood detected by AS-PCR was 78%, 81%, and 53%, respectively. Thirty-nine patients with WM underwent ≥ 2 MYD88 mutation detections. The final MYD88 mutational status for each patient was used as the standard to determine the accuracy of the different methods and in different specimens. The accuracy of MYD88 mutation detection in the lymph nodes (n=18) and bone marrow (n=13) by NGS was significantly higher than that in the peripheral blood (n=4) (100% vs 100% vs 75%, P<0.05) . There was no statistically significant difference in the accuracy of MYD88 mutation detection by AS-PCR in the lymph nodes (n=15) , bone marrow (n=11) , or peripheral blood (n=16) (93% vs 91% vs 88%, P>0.05) . Conclusions: In the detection of the MYD88 mutation in patients diagnosed with WM, NGS or AS-PCR is more sensitive than Sanger sequencing. Lymph nodes and bone marrow specimens are better than peripheral blood specimens.

Anti-inflammatory effects of Abelmoschus manihot (L.) Medik. on LPS-induced cystitis in mice: potential candidate for cystitis treatment based on classic use / 中国天然药物

Abelmoschus manihot (L.) Medik. (A. manihot) is a traditional Chinese herbal medicine with a variety of pharmacological properties. It was first recorded in Jiayou Materia Medica dating back to the Song dynasty to eliminate urinary tract irritation by clearing away heat and diuretic effect. However, its pharmacological action on urinary tract infections has not been investigated. The present study aims to evaluate the anti-inflammatory activity of A. manihot on a mouse model of lipopolysaccharide (LPS)-induced cystitis. The results showed that A. manihot decreased white blood cell (WBC) count in urine sediments of the cystitis mice, alleviated bladder congestion, edema, as well as histopathological damage, reduced the expression levels of tumor necrosis factor-α, interleukin-6, and interleukin-1β simultaneously. Moreover, A. manihot administration significantly downregulated the expression levels of TLR4, MYD88, IκBα, p-IκBα, NF-κB p65, and p-NF-κB p65 in LPS-induced cystitis mice. These findings demonstrated the protective effect of A. manihot against LPS-induced cystitis, which is attributed to its anti-inflammatory profile by suppressing TLR4/MYD88/NF-κB pathways. Our results suggest that A. manihot could be a potential candidate for cystitis treatment.

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.

Inhibitory effect of Xiaochuan pill on obstinate cough and its role in regulation of TLR4MyD88NFκBp65 signaling pathway / 中南大学学报(医学版)

OBJECTIVES@#Cough variant asthma (CVA) is the main cause of obstinate cough. This study aimed to observe the therapeutic effect of Xiaochuan pill on CVA in a rat model, and to explore the mechanisms.@*METHODS@#The rats were sensitized and challenged with 4% ovaibumin (OA) and 2% Al(OH) to establish the CVA models. They were treated with Xiaochuan pill (at the dose of 0.9, 1.8, 3.6 g/kg) or montelukast sodium once a day for 14 days. After 7 and 14 days of intervention, 5 and 10 rats were randomly selected from each group to collect bronchoalveolar lavage fluid (BALF), trachea, and lungs. The number of white blood cells (WBC) and eosinophils (EOS), and the levels of IL-1β, TNF- α, and IFN-γ in BALF were detected. Histopathological examination of lung tissue was performed to observe the histomorphological changes. The expressions of TLR4, MyD88, NF-κBp65, and p-p65 in lung tissue were detected by Western blotting.@*RESULTS@#The numbers of WBC and EOS in BALF of CVA rats were significantly decreased by Xiaochuan pill (<0.05 or <0.01). The hyperplasia of tracheal, bronchial mucosa and the infiltration of inflammatory cells in lung were alleviated obviously. After 14 d of intervention, high dose of Xiaochuan pill significantly increased the level of IFN- γ (<0.01), reduced the levels of IL-1β (<0.05) and TNF-α (<0.05), and decreased the expressions of TLR4, MyD88, p65, and p-p65 (<0.05 or <0.01).@*CONCLUSIONS@#Xiaochuan pill exerts the significant therapeutic effect on obstinate cough in rats. The mechanism of action may be related to the regulation of TLR4-MyD88-NF-κBp65 signaling pathway as well as the inflammation and immune response.

Over-expression of miR-144 inhibits invasion of liver cancer SMMC-7721 cells by suppressing TLR/MyD88 pathway / 南方医科大学学报

OBJECTIVE@#To investigate the effects of over-expression of miR-144 on invasion of SMMC-7721 cells and Toll-like receptor (TLR)/myeloid differentiation factor 88 (MyD88) pathway in hepatocellular carcinoma cells.@*METHODS@#The expressions of miR-144 was examined in normal human hepatocyte line HL-7702 and hepatocarcinoma cell line SMMC-7721 using realtime quantitative PCR (qRT-PCR). SMMC-7721 cells were divided into blank group, miR-144 NC group and miR-144 mimics group, and the expressions of miR-144 in each group were detected with qRT-PCR. Cell count kit-8 (CCK8) was used to assess the survival of SMMC-7721 cells, and the cell invasion was evaluated using Transwell assay. The expressions of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) and TLR/MyD88 pathway-related proteins in the cells were detected with Western blotting; the effect of 40 μ mol/L MyD88 inhibitor on TLR/MyD88 pathway-related proteins was examined in SMMC-7721 cells.@*RESULTS@#Compared with normal human hepatocytes, SMMC-7721 cells expressed a significantly lower level of miR-144 ( < 0.05). CCK-8 assay showed that test showed that miR-144 over-expression significantly decreased the cell survival rate ( < 0.05), lowered the number of invasive cells, and decreased the expression of MMP-2 and MMP-9 in SMMC-7721 cells ( < 0.05). The expressions of Toll-like receptor 4 (TLR4), MyD88, phosphorylated nuclear factor-kappa B (pNF-κB) and NF-κB protein decreased significantly in miR-144 mimics group and TJ-M2010-2 group ( < 0.05) and were comparable between the two groups ( > 0.05).@*CONCLUSIONS@#Overexpression of miR-144 decreases SMMC-7721 cell survival and invasion by inhibiting TLR/MyD88 pathway.

Prognostic impact of MYD88 mutation, proliferative index and cell origin in diffuse large B cell lymphoma

Abstract Background Diffuse large B-cell lymphoma, among non-Hodgkin lymphomas, is one of the most frequent subtypes. Clinical laboratory data and post-treatment outcomes are scarce in the Brazilian population. Objective The main objective of this retrospective study was to assess the impact of tumor markers, including the Myeloid differentiation primary response 88 (MYD88) mutation. Method Eighty-three patients were included and treated with R-CHOP or R-CHOP-like regimens. Results Median age was 64-years old and 58% were female patients. The median follow-up was 42 months. The progression free survival (PFS) at this time was 63% and overall survival (OS), 66%. In the patients with tumors expressing Myc proto-oncogene protein (MYC) and B-cell lymphoma 2 (BCL2), assessed by immunohistochemistry (IHC), known as dual protein expressers, median post-progression survival was 31 (15-45) months. An increased proliferative index were associated with a high rate of progression (hazard ratio 2.31 [95% confidence interval [1.05-5.12]; p = 0.04). The cell of origin (COO), identified by IHC, was not able to predict PFS (p = 0.76). The MYD88 L265P mutation was present in 10.8% (9/83) of patients and did not show a prognostic correlation. Conclusion In conclusion, the MYD88 mutation, although an important tool for diagnosis and a possible target drug, presented at a low frequency and was not a prognostic marker in this population.