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.

Effects of MD2 gene silencing on high glucose-induced proliferation inhibition, apoptosis and inflammation in rat cardiomyocytes / 中国应用生理学杂志

OBJECTIVE@#To investigate the effects of myeloid differentiation-2 (MD2) gene silencing on high glucose-induced proliferation inhibition, apoptosis and inflammation in rat cardiomyocytes.@*METHODS@#The immortalized rat cardiomyocyte cell line H9C2 were transfected with MD2 small interfering RNA (si-MD2) and negative control for 24 h, then stimulated with high glucose (HG) for 48 h. RT-qPCR was performed to detect the mRNA levels of MD2 and inflammatory factors TNF-α, IL-1β and IL-6. MTS and flow cytometry were used to evaluate cell proliferation, cell cycle and apoptosis rate. Western blot was used to detect protein expression levels and phosphorylation levels.@*RESULTS@#The mRNA and protein levels of MD2 in H9C2 cells were dramatically decreased after transfected with si-MD2 (P＜0.01). After stimulation of high glucose, the mRNA levels of inflammatory factors, the cells in G0/G1 phase , the cell apoptosis rate and the protein level of cleaved Caspase-3 were significantly increased, while the cell proliferation ability was decreased (P＜0.01). MD2 gene silencing antagonized the effects of high glucose on cell proliferation, cell cycle, cell apoptosis and the mRNA levels of TNF-α, IL-1β , IL-6(P＜0.05). Western blot analysis showed that the phosphorylation levels of extracellular signal-regulated kinase(ERK1/2), P38 mitogen-activated protein kinase(P38 MAPK) and C-Jun N-terminal kinase(JNK) protein were increased significantly in H9C2 cells treated with high glucose, which could be reversed by silencing of MD2 (P＜0.01).@*CONCLUSION@#This study demonstrates that MD2 gene silencing reverses high glucose-induced myocardial inflammation, apoptosis and proliferation inhibition via the mechanisms involving suppression of ERK, P38 MAPK, JNK signaling pathway.

Effect of sophoridine and TLR4/MD-2 blocking agent on pathway of LPS-induced RAW264. 7 macrophage TLR4-NF-kappaB-TNF-alpha / 中国中药杂志

<p><b>OBJECTIVE</b>To study the effect of sophoridine and TLR4/MD-2 blocking agent on pathway of LPS-induced RAW264. 7 macrophage TLR4-NF-kappaB-TNF-alpha in and its pharmacological mechanism of antiendotoxin.</p><p><b>METHOD</b>RAW264. 7 macrophages were cultured and divided into 5 groups, namely the blank control group, the LPS model group, the sophoridine + LPS group, the TLR4/MD-2 blocking agent + LPS group and the anti-TLR4/MD-2 + sophoridine + LPS group. Cells and cell culture fluids were collected at 120 min after the each group was processed. The expression of TLR4 protein was measured by western blot, the distribution and expression of NF-kappaB protein were measured by immunocytochemistry, and the expression of NF-kappaB and TNF-alpha mRNA were measured by western blot and reverse transcriptase polymerase chain reaction (RT-PCR). The content of TNF-alpha in the cell supernatant was detected by using radioimmunoassay.</p><p><b>RESULT</b>Compared with the LPS group, the expression of TLR4 protein, NF-kappaB mRNA, the rate of NF-kappaB entry the nucleus, TNF-alpha mRNA and TNF-alpha content in the cell supernatant were significantly decreased in the sophoridine + LPS group (P < 0.01). The rate of NF-kappaB entry the nucleus and TNF-alpha in the TLR4/MD-2 blocking agent + LPS group and the TLR4/MD-2 blocking agent + sophoridine + LPS group were notablly lower than that of the LPS model group (P < 0.01), close to that of the blank control group. However, there was no statistical significance between the two groups.</p><p><b>CONCLUSION</b>TLR4/MD-2 may be one of sophoridine's targets. Sophordine's inhibitory effect on pathway activity of TLR4-NF-kappaB-TNF-alpha may be one of its antiendotoxin mechanisms.</p>

Relationship of myeloid differentiation-2 gene promoter polymorphisms with susceptivity of complications after severe trauma in Chinese Han population / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the polymorphisms of myeloid differentiation-2 (MD-2) gene promoters, and to explore whether such polymorphisms are associated with the susceptibility to multiple organ dysfunction syndrome (MODS) and sepsis in Chinese Han population.</p><p><b>METHODS</b>Using polymerase chain reaction-restriction fragment length polymorphism method, the authors detected the single nucleotide polymorphisms of the promoter region of MD-2 gene at position - 1625C/G in 105 severe trauma patients (42 with sepsis). The organ function was scored.</p><p><b>RESULTS</b>The frequency of CC genotype in MD-2 gene promoter region at position - 1625 was 0.5 (21/42) in septic patients and 0.7 (44/63) in non-septic patients. The frequency of CG genotype was 0.38 (16/42) in septic patients and 0.27 (17/63) in non-septic patients. The frequency of GG genotype was 0.12 (5/42) in septic patients and 0.03 (2/63) in non-septic patients. The MODS scores in trauma patients carrying G allele at position - 1625 were significantly higher than those carrying C allele (P<0.001 for dominant effect, and P>0.05 for recessive effect). Moreover, trauma patients carrying G allele appeared to have higher risk of sepsis comparing to those carrying C allele (OR 0.477, 95% CI 0.266-0.855, P<0.05). Sepsis morbidity was significantly different between subjects with C and G alleles (P<0.05 for dominant effect, P>0.05 for recessive effect).</p><p><b>CONCLUSIONS</b>The polymorphisms of the promoter region of MD-2 gene at position - 1625 C/G is correlated with MODS and sepsis after severe trauma in Chinese Han population. The people with - 1625 G allele in the promoter region of MD-2 gene may be a risk factor of severe complications.</p>

Analysis of Toll-like receptor 4 and myeloid differentiation protein-2 interaction with fluorescence resonance energy transfer / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the interaction between Toll-like receptor (TLR) 4 and myeloid differentiation protein-2 (MD-2) in living cells using fluorescence resonance energy transfer (FRET) technology.</p><p><b>METHODS</b>The coding sequences of TLR4 and MD-2 (without the signal peptide sequence) were amplified by PCR and cloned into enhanced cyan fluorescence protein (CFP) and enhanced yellow fluorescence protein (YFP) expression vectors carrying TLR4 signal peptides (pECFP-C1-SP and pEYFP-C1-SP). HEK293 cells were transfected respectively or together with the reconstructed plasmids verified by enzyme digestion and sequence analysis, and the expression and sublocalization of these fluorescence proteins in the cells were observed using fluorescence microscope. FRET in the cells coexpressing CFP-TLR4 and YFP-MD-2 was detected using routine and acceptor photobleaching method.</p><p><b>RESULTS</b>The reconstructed plasmids were expressed in HEK293 cells. The cyan or yellow fluorescence was located in the cytoplasm, mainly around the nucleus in the cells transfected with pECFP/TLR4 or pEYFP/MD-2, and both the cyan and yellow fluorescence located mainly in the membrane and occasional in the cytoplasm of cells cotransfected with pECFP/TLR4 and pEYFP/MD-2. Routine or acceptor photobleaching detected FRET phenomena in cells coexpressing CFP-TLR4 and YFP-MD-2, suggesting direct interaction between TLR4 and MD-2.</p><p><b>CONCLUSION</b>This study provides direct evidence of the interaction between TLR4 and MD-2 in living cells.</p>

Novel multi-probe RNase protection assay set for detection of endotoxin associated receptors gene expression / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To construct the multi-probe ribonuclease protection assay (RPA) template set to be used for detecting expression patterns of MD-2, TLR4, CD14 mRNAs in human peripheral blood mononuclear cells.</p><p><b>METHODS</b>The designed cDNA fragments of the three genes were generated by polymerase chain reaction (PCR) using specific primers and directionally cloned into EcoR I and Hind III sites of expression plasmid pSP72 containing the T7 promoter, the linearized plasmids was used as template to synthesize anti-sense RNA probes. Then we extracted total RNA from peripheral blood mononuclear cells (PBMC) and detected the dynamic expression patterns of the three genes with RPA method.</p><p><b>RESULTS</b>The proper sequence and orientation of the template set were confirmed by sequencing and the template set was successfully used to assay TLR4, MD-2 and CD14 mRNAs in human PBMC. The results showed that the three detected genes decreased transiently 1-3 hours after 100 ng/ml LPS stimulation.</p><p><b>CONCLUSIONS</b>These new RPA multi-probe set provided valuable tool for the simultaneous quantitative determination of expression of TLR4, CD14 and MD-2 mRNAs in both constitutive and inducible types.</p>