Proteomic Research on Characteristic Processing Materials of Porcine Cardiac Blood and Porcine Blood from Menghe Medical School and Their Effect on Anti-microglial Inflammatory Damage of Salviae Miltiorrhizae Radix et Rhizoma / 中国实验方剂学杂志

ObjectiveProteomics was used to investigate the protein differences between porcine cardiac blood（PCB） and porcine blood（PB） from Menghe medical school and to compare the effects of both on the microglial inflammation of Salviae Miltiorrhizae Radix et Rhizoma（DS）. MethodNanoliquid chromatography-quadrupole orbitrap mass spectrometry（nLC-MS/MS） and bioinformatics were utilized to compare the proteomic differences of PCB and PB in simulated gastrointestinal digestion. Furthermore， Western blot was used to verify the contents of some shared proteins and differential proteins identified in PCB and PB. In addition， BV2 neuroinflammation model constructed by corticosterone（CORT） and lipopolysaccharide（LPS） was applied to detect the intervention effects of PCB and PB on the levels of tumor necrosis factor（TNF）-α and interleukin（IL）-6 in BV2 inflammatory cells of DS. ResultA total of 69 common proteins and 68 differential proteins were identified in PCB and PB， among which the common proteins included transferrin（Tf） with brain-targeting effect， and the differential proteins in the two were 41 and 27， respectively. Western blot validation showed that the difference in the content of the same protein Tf between PCB and PB was not statistically significant， while the difference in the contents of the specific proteins of creatine kinase M and heart-type fatty acid binding protein（H-FABP） were statistically significant（P<0.05）. Moreover， in vitro experimental studies revealed that compared with the same concentration of DS group， in addition to the 100 mg·L-1 PB-DS group， PCB-DS and PB-DS groups could significantly inhibit the levels of TNF-α and IL-6 in BV2 inflammatory cells（P<0.05， P<0.01）， and PCB-DS group had more significant anti-inflammatory effect than PB-DS group with the same concentration（P<0.05， P<0.01）. ConclusionBoth of PCB and PB can enhance the inhibitory effect of DS on the release of inflammatory factors， thus playing a neuroprotective role， and PCB promotes DS inhibition more significantly， which may be due to the existence of the two involved in energy metabolism-related differential proteins， which can lay a foundation for revealing the scientific connotation of the processing of PCB-DS and PB-DS.

Ginsenoside Rg1 regulating BV2 microglia polarization in lipopolysaccharide-induced inflammatory response via peroxisome proliferator activated receptor γ / 解剖学报

[Abstract] Objective To establish an inflammation model by stimulating BV2 microglia by lipopolysaccharide, and to explore the regulation effect of ginsenoside Rg1 on inflammation by activating peroxisome proliferator activated receptor γ(PPARγ) receptor protein. Methods BV2 microglia were randomly divided into control group, model group, ginsenoside Rg1 group, rosiglitazone group and GW9662 group. The control group did not do any treatment, the model group was treated with 1 mg/ L lipopolysaccharide, and the other groups were treated with lipopolysaccharide added with 0. 4 mmol/ L ginsenoside Rg1, 10 μmol/ L rosiglitazone or 10 μmol/ L respectively. GW9662. The proliferation of BV2 microglia in each group was detected by CCK-8 method; PPAR-γ, phospho-NF-κB p65 (p-NF-κB p65), induced expression of inducible nitric oxide synthase(iNOS) and human arginase 1(ARG-1) proteins. ELISA was used to detect the inflammatory factors interleukin-1β(IL-1β), interleukin-6(IL-6), interleukin-8(IL-8) and the content of tumor necrosis factor-α (TNF-α). Results Compared with the control group, the cell proliferation rate in the model group was significantly increased, and the contents of IL-1β, IL-6, IL-8 and TNF-α increased significantly. The result of immunofluorescence and Western blotting showed that iNOS and p-NF-κB p65 increased significantly, and the positive expressions of PPARγ and ARG-1 decreased significantly(both P<0. 01). The expression level of TNF-α decreased, the positive expressions of iNOS and p-NF-κB p65 decreased significantly, and the positive expressions of PPARγ and ARG-1 increased significantly(all P<0. 01). Conclusion Ginsenoside Rg1 inhibits the inflammatory response of BV2 microglia after lipopolysaccharide stimulation, and its mechanism may be related to the regulation of PPARγ/ NF-κB pathway to promote the M2-type polarization of microglia.

Mechanism of Schisandra Chinensis-mediated microglia phenotypic transformation by regulation of the TLR4 pathway based on miR-124 / 药学学报

To investigate the mechanism by which Schisandra Chinensis mediates the phenotypic transformation of microglia via microRNA-124 (miR-124)-based regulation of the Toll-like receptor 4 (TLR4) pathway, a model was established using lipopolysaccharide (LPS) stimulation of BV2 cells. Cells were treated with different doses of Schisandra Chinensis extract (SCE). MiR-124 inhibitors and negative control sequences (NC inhibitor) were transfected into LPS-induced BV2 cells and treated with SCE. The MTT assay was used for cell activity detection; an NO kit was used to measure NO release; ELISA kits were used to measure the levels of interleukin-10 (IL-10) and tumor necrosis factor-α (TNF-α). Microglia markers, including ionized calcium binding adapter molecule-1 (IBA-1) and arginase-1 (Arg-1), and the nuclear translocation of nuclear factor-kappa B (NF-κB) were evaluated by immunofluorescent staining. NF-κB p65, IBA-1, Arg-1, TLR4, myeloid differentiation primary factor 88 (MyD88), inhibitor of nuclear factor-kappa B kinases-α (IKK-α), IL-10, TNF-α were detected by immunoblot. SCE at concentrations ranging from 31.25 to 250 μg·mL-1 had no significant effect on cell activity. SCE treatment significantly inhibited NO release induced by LPS (P < 0.001, P < 0.01), increased the level of IL-10 (P < 0.05), and decreased the level of TNF-α (P < 0.001). In addition, SCE significantly reduced the expression of TNF-α, IBA-1, TLR4, and MyD88 (P < 0.01, P < 0.001) and elevated the expression of IL-10, Arg-1, NF-κB P65 and IKK-α (P < 0.001, P < 0.01, P < 0.05). SCE treatment could also promote the expression of miR-124 (P < 0.01). However, transfection with the miR-124 inhibitor increased TNF-α (P < 0.001), decreased the level of IL-10 (P < 0.05), increased the mRNA level and the protein expression of TNF-α and IBA-1 (P < 0.05, P < 0.01, P < 0.001), and decreased the mRNA level and protein expression of IL-10 and Arg-1 (P < 0.001, P < 0.01). In addition, the inhibition of TLR4 and MyD88 was attenuated. In conclusion, SCE appears to inhibit the activation of TLR4 signaling pathway by upregulating miR-124 so as to inhibit microglia M1 polarization and promote microglia M2 polarization.

Protective effect of platelet-rich plasma on LPS-induced neuroinflammation in BV2 microglia through NRF2/HO-1 pathway / 中国输血杂志

【Objective】 To investigate the protective effect and mechanism of platelet-rich plasma (PRP) on lipopolysaccharide (LPS) -induced inflammatory response in BV2 cells. 【Methods】 BV2 microglia were divided into normal control group, 10%PRP control group, LPS group (LPS induction), 3%PRP+ LPS group (LPS induction, 3%PRP pretreatment), 5%PRP+ LPS group (LPS induction, 5%PRP pretreatment), 10%PRP+ LPS group (LPS induction, 10%PRP pretreatment), and the proliferation of BV2 cells was measured by CCK-8. The mitochondrial membrane potential of BV2 cells was measured by confocal microscopy, ROS was measured by fluorescence method, and NO was measured by Griess method. The protein expressions of IL-6, TNF-α, BACH1, GPX4, NRF2 and HO-1 were detected by Western blot. In addition, BV2 microglia were treated with HO-1 inhibitor and divided into normal control group, LPS group, ZnPP+ LPS group, 10%PRP+ LPS group, ZnPP+ LPS+ 10%PRP group, and the protein expressions of HO-1, IL-6 and TNF-α were detected by Western blot. 【Results】 Compared with normal control group, PRP promoted the proliferation of BV2 cells (P<0.01). The mitochondrial membrane potential decreased, ROS production increased, the levels of NO, IL-6, TNF-α and BACH1 increased (P<0.01). However, the expression levels of GPX4, NRF2 and HO-1 decreased (P<0.01) in LPS group. Compared with LPS group, the proliferation activity and mitochondrial membrane potential of BV2 cells in 3%PRP+ LPS, 5%PRP+ LPS and 10%PRP+ LPS groups significantly increased. The levels of ROS, NO, IL-6, TNF-α and BACH1 significantly decreased (P<0.01). The expressions of GPX4, NRF2 and HO-1 in different concentrations of PRP (3%, 5% and 10%) increased (P<0.01). Moreover, the expression of IL-6 and TNF-α in ZnPP+ LPS group was significantly higher than that in LPS group after HO-1 inhibitor treatment. Compared with 10%PRP+ LPS+ ZnPP group, HO-1 inhibitor could reverse the effect of PRP on the expression of IL-6 and TNF-α in LPS-induced BV2 cells (P<0.01). 【Conclusion】 PRP inhibits the inflammatory response of BV2 microglia induced by LPS by activating the NRF2/HO-1 signaling pathway.

Role of JNK/AP-1 signaling pathway in paraquat-induced activation of NLRP3 inflammasome in microglia / 环境与职业医学

Background Paraquat (PQ) is one of the environmental factors that can cause sporadic Parkinson's disease (PD). Microglia-mediated neuroinflammation plays an important role in the occurrence and development of PD. Our previous studies have found that low doses of PQ can activate BV-2 microglia to the M1 phenotype and exert pro-inflammatory effects, but the associated mechanism is not clear yet. Objective To explore the role of c-Jun N-terminal kinase (JNK)/activator protein 1 (AP-1) signaling pathway in PQ-induced activation of the NOD-like receptor thermal protein domain associated protoin 3 (NLRP3) inflammasome in microglia. Methods An in vitro microglia model was established. The cells were treated with 0, 0.03, 0.06,and 0.12 μmol·L−1 PQ for 24 h, the whole cell protein was extracted. The relative expression levels of JNK, AP-1 constituent proteins (c-Jun, c-Fos), NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), caspasse-1 precursor (pro caspase-1), interleukin-18 (IL-18), and interleukin-1β (IL-1β) were evaluated by Western blotting, to observe the effects of PQ exposure on JNK/AP-1 signaling pathway and NLRP3 inflammasome. After the treatment of 20 μmol·L−1 JNK inhibitor SP600125, the above proteins were detected again, to explore the driving effect of JNK/AP-1 signaling pathway on NLRP3 inflammasome activation. Results After PQ exposure, the relative expression levels of key proteins of JNK, c-Jun, and c-Fos, NLRP3, ASC, and pro caspase-1 in the 0.06 μmol·L−1 PQ group and the 0.12 μmol·L−1 PQ group were higher than those in the 0 μmol·L−1 PQ group (P＜0.05), and the relative expression levels of IL-18 and IL-1β increased with higher exposure (P＜0.05). After the treatment of JNK inhibitor SP600125, the relative expression levels of key proteins of JNK/AP-1 signaling pathway (JNK, c-Jun, and c-Fos), NLRP3 inflammasome (NLRP3, ASC, and Pro caspase-1), and inflammatory factors (IL-18 and IL-1β) in the control group, the 20 μmol·L−1 SP600125 group, and the 20 μmol·L−1 SP600125+0.06 μmol·L−1 PQ group were lower than those in the 0.06 μmol·L−1 PQ group (P＜0.05). Conclusion PQ exposure can activate the JNK/AP-1 signaling pathway and subsequently drive the activation of NLRP3 inflammasome in BV-2 microglia to mediate neuroinflammatory responses..

Role of STAT3 activated NLRP3 inflammasomes in BV2 cell inflammatory response induced by maltol aluminum / 环境与职业医学

Background Aluminum activates signal transducer and activator of transcription 3 (STAT3), causing microglial nucleotide-binding and oligomerization domain-like receptors protein 3 (NLRP3) inflammasome activation and inflammatory responses and producing neurotoxicity. Objective To explore the role of STAT3 regulated NLRP3 inflammasomes in the inflammatory response of mouse microglia cell line (BV2) cells induced by maltol aluminum [Al(mal)3]. Methods BV2 cells were assigned to five groups: one control group, three Al(mal)3 exposure groups (low, medium, and high doses at 40, 80, and 160 μmol·L−1 Al(mal)3 respectively), and one C188-9 (STAT3 antagonist) intervention group [10 μmol·L−1 C188-9 +160 μmol·L−1 Al(mal)3]. Cell viability was detected by CCK8. The expression of M1/M2 type markers, i.e. CD68/CD206, STAT3, p-STAT3, NLRP3, cleaved-casepase-1, and apoptosis-associated speck-like protein (ASC) in BV2 cells were detected by Western blotting, and proinflammatory cytokines interleukin (IL)-1β and IL-18, and anti-inflammatory cytokine IL-10 were determined by ELISA. Results The results of cell viability assay showed that cell viability gradually decreased with the increase of Al(mal)3 dose. Compared with the control group, the cell viability of the Al(mal)3 high-dose group was decreased by 18% (P<0.05); compared with the Al(mal)3 high-dose group, the cell viability of the C188-9 intervention group was significantly elevated by 14% (P<0.05). Compared with the control group, the expression levels of CD68 in the Al(mal)3 low-, medium-, and high-dose groups were elevated by 19%, 20%, and 21%, respectively (P<0.05); the expression level of CD206 in the Al(mal)3 high-dose group was decreased by 25% (P<0.05). Compared with the Al(mal)3 high-dose group, the expression level of CD68 in the C188-9 intervention group was reduced by 9% (P<0.05), whereas the expression level of CD206 was elevated by 22% (P<0.05). Compared with the control group, the p-STAT3 protein expression and the p-STAT3/STAT3 ratio in the Al(mal)3 high-dose group increased by 129% and 127%, respectively (P<0.05). Compared with the Al(mal)3 high-dose group, the p-STAT3 protein expression and the p-STAT3/STAT3 ratio in the C188-9 intervention group were decreased by 55% and 54%, respectively (P>0.05). Compared with the control group, the expression level of NLRP3 protein increased by 75% in the Al(mal)3 high-dose group (P<0.05), the expression levels of cleaved-casepase-1 protein increased by 28% and 35% in the Al(mal)3 medium- and high-dose groups (P<0.05), and the expression levels of ASC increased by 22%, 25%, and 53% in the Al(mal)3 low-, medium- and high-dose groups (P<0.05), respectively. Compared with the Al(mal)3 high-dose group, the expression levels of NLRP3, cleaved-casepase-1, and ASC proteins in the C188-9 intervention group decreased by 30%, 19%, and 32%, respectively (P<0.05). Compared with the control group, the levels of IL-1β in the Al(mal)3 medium- and high-dose groups increased by 18% and 21%, respectively (P<0.05), and the level of IL-18 in the Al(mal)3 high-dose group increased by 10% (P<0.05). Compared with the Al(mal)3 high-dose group, the IL-18 levels were reduced by 23% in the C188-9 intervention group (P<0.05). The content of anti-inflammatory factor IL-10 did not differ significantly between groups (P>0.05). Conclusion Aluminum can induce inflammatory responses in BV2 microglia and is predominantly pro-inflammatory, and the mechanism may involve STAT3 regulation of NLRP3 inflammasome secretion of inflammatory factors.

Tetrahydropalmatine alleviated diabetic neuropathic pain by inhibiting activation of microglia via p38 MAPK signaling pathway / 中国中药杂志

Neuropathic pain is one of the common complications of diabetes. Tetrahydropalmatine(THP) is a main active component of Corydalis Rhizoma with excellent anti-inflammatory and pain-alleviating properties. This study aims to investigate the therapeutic effect of THP on diabetic neuropathic pain(DNP) and the underlying mechanism. High-fat and high-sugar diet(4 weeks) and streptozotocin(STZ, 35 mg·kg~(-1), single intraperitoneal injection) were employed to induce type-2 DNP in rats. Moreover, lipopolysaccharide(LPS) was used to induce the activation of BV2 microglia in vitro to establish an inflammatory cellular model. Fasting blood glucose(FBG) was measured by a blood glucose meter. Mechanical withdrawal threshold(MWT) was assessed with von Frey filaments, and thermal withdrawal latency(TWL) with hot plate apparatus. The protein expression levels of OX42, inducible nitric oxide synthase(iNOS), CD206, p38, and p-p38 were determined by Western blot, the fluorescence expression levels of OX42 and p-p38 in the dorsal horn of the rat spinal cord by immunofluorescence, the mRNA content of p38 and OX42 in rat spinal cord tissue by qRT-PCR, and levels of nitric oxide(NO), interleukin-1β(IL-1β), interleukin-6(IL-6), tumor necrosis factor-α(TNF-α), interleukin-10(IL-10), and serum fasting insulin(FINS) by enzyme-linked immunosorbent assay(ELISA). RESULTS:: showed that the mo-del group demonstrated significant decrease in MWT and TWL, with pain symptoms. THP significantly improved the MWT and TWL of DNP rats, inhibited the activation of microglia and p38 MAPK signaling pathway in rat spinal cord, and ameliorated its inflammatory response. Meanwhile, THP promoted the change of LPS-induced BV2 microglia from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype, suppressed the activation of the p38 MAPK signaling pathway, decreased the expression levels of inflammatory factors NO, IL-1β, IL-6, and TNF-α, and increased the expression level of anti-inflammatory factor IL-10. The findings suggested that THP can significantly ameliorate the pain symptoms of DNP rats possibly by inhibiting the inflammatory response caused by M1 polarization of microglia via the p38 MAPK pathway.

Protective Mechanism of Banxia Houputang on Lipopolysaccharide-induced Neuroinflammatory Injury / 中国实验方剂学杂志

ObjectiveTo study the inhibitory effect of Banxia Houputang （BHT） on lipopolysaccharide （LPS）-induced inflammation of microglia （BV2） cells and the neuroprotective effect on human neuroblastoma （SH-SY5Y） cells. MethodAfter the neuroinflammatory model was constructed by LPS inducing BV2 cells， model group （LPS 100 µg·L-1）， administration groups （LPS+1 g·L-1 BHT， LPS+2 g·L-1 BHT， LPS+5 g·L-1 BHT， LPS+10 g·L-1 BHT）， and blank group were given DEME medium at the same volume. In addition， neuronal apoptosis model was established by co-culture of LPS-induced BV2 cell inflammation medium and SH-SY5Y cells （LPS-DMEM） and was administrated according to the above grouping. Cell viability was detected by Cell Counting Kit-8 （CCK-8） assay. The content of nitric oxide （NO） and that of tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， and interleukin-6 （IL-6） were determined by Griess aasay and enzyme-linked immunosorbent assay （ELISA）， respectively. The mRNA levels of TNF-α， IL-1β， interleukin-4 （IL-4）， nitric oxide synthase （iNOS）， and interleukin-10 （IL-10） were measured by real-time polymerase chain reaction （Real-rime PCR）. Western blot was used to detect the expression levels of signal transducer and activator of transcription 3 （STAT3）， Janus kinase 2 （JAK2） and nuclear factor kappa-B （NF-κB p65）， protein kinase B （Akt）， inhibitor of nuclear factor κB α （IκBα）， B-cell lymphoma-2 （Bcl-2）， and Bcl-2 associated X protein （Bax）. ResultCompared with blank group， LPS increased the NO release， levels of TNF-α， IL-1β， IL-6， and iNOS and protein expression of Akt， NF-κB p65， IκBα， JAK2 and STAT3， decreased the content of IL-4 and IL-10 in BV2 cells， and induced apoptosis of co-cultured SH-SY5Y cells （P<0.01）. Compared with model group， BHT reduced the content of NO， TNF-α， IL-1β， and iNOS （P<0.01） and protein expression of Akt， NF-κB p65， IκBα， JAK2 and STAT3 （P<0.01）， elevated the content of IL-4 and IL-10 （P<0.01）， and inhibited the apoptosis of SH-SY5Y cells induced by LPS-DMEM （P<0.01）. ConclusionThis experiment reveals that BHT inhibited LPS-induced inflammation in BV2 cells by regulating Akt/NF-κB/JAK2/STAT3 signaling pathway and showed neuroprotective effects on SH-SY5Y cells.

Mechanisms of ferroptosis in microglial cell line BV-2 cells after lead acetate exposure / 环境与职业医学

Background Lead exposure induces microglial cell death, of which the mechanism is unclear. Ferroptosis is a new death form and its role in microglia death has not been reported. Objective To investigate the role of ferroptosis in microglia following lead exposure in order to provide a theoretical basis for the mechanism of lead neurotoxicity. Methods Microglial cell line BV-2 cells were co-cultured with 0, 10, 20 and 40 μmol·L−1 lead acetate for 24 h. The 40 μmol·L−1 lead acetate group with iron chelator (DFO) was named the 40+DFO group. Changes in BV-2 cell morphology after lead exposure were observed under an inverted microscope; tissue iron kit and glutathione kit were used to detect intracellular iron and glutathione (GSH) respectively; flow cytometry was applied to detect lipid reactive oxygen species (lipid ROS) immunofluorescence intensity. Western blotting and qPCR were adopted to detect the expressions of glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11), transferrin receptor 1 (TFR-1), divalent metal transporter 1 (DMT1), ferroportin 1 (FPN1) protein and mRNA. Results Compared with the control group, the number of BV-2 cells decreased with increasing doses of lead and the cells showed a large, round amoeboid shape. The intracellular levels of iron of BV-2 cells were (1.08±0.04), (1.29±0.03), and (1.72±0.10) mg·g−1 (calculated by protein, thereafter) in the 10, 20, and 40 μmol·L−1 lead acetate groups, respectively, significantly higher than that in the control group (P<0.05), and the intracellular level of iron in the 40+DFO group, (1.34±0.10) mg·g−1, was lower than that in the 40 μmol·L−1 lead acetate group, (1.72±0.03) mg·g−1 (P<0.05). Compared with the control group, the TFR-1 and DMT1 protein and mRNA expressions were increased in BV-2 cells in the 10, 20, 40 μmol·L−1 lead acetate groups (P<0.05), especially in the 40 μmol·L−1 lead acetate group; the FPN1 protein expression did not change significantly, but the FPN1 mRNA expressions in BV-2 cells in the 10, 20, 40 μmol·L−1 lead acetate groups were significantly decreased (P<0.05). Compared with the control group, the intracellular GSH level decreased and the lipid ROS content increased in all three lead acetate groups; compared with the 40 μmol·L−1 lead acetate group, the GSH level increased by 12.30% and the lipid ROS content decreased by 13.00% in the 40+DFO group (P<0.05). The expressions of GPX4 protein were reduced to 50.00%, 35.00%, and 17.00% of that of the control group in the 10, 20, and 40 μmol·L−1 lead acetate groups respectively, while the expressions of GPX4 mRNA were also significantly reduced; the expressions of SLC7A11 protein and mRNA in the 20 and 40 μmol·L−1 lead acetate groups were lower than that in the control group, with the most significant decrease in the 40 μmol·L−1 lead acetate group (P<0.05). Conclusion Lead exposure could induce ferroptosis in BV-2 cells, in which iron transport imbalance and oxidative damage might be involved.

Mechanisms of sodium butyrate inhibition of microglia inflammatory activation in hippocampus via Toll-like receptor 4/nuclear factor-κB p65 pathway / 中华危重病急救医学

Objective:To investigate the effects of sodium butyrate (NaB) on long-term anxiety like behavior and inflammatory activation of microglia in the hippocampus of sepsis-associated encephalopathy (SAE) mice.Methods:① Animal experiment: fifty C57BL/6 mice aged 6-8 weeks were randomly divided into Sham group (only the cecum was found by laparotomy without perforation or ligation), and SAE model group caused by cecal ligation and puncture (CLP; SAE model group, the cecum was found by laparotomy and perforated after ligation. The open field test indicated that the ability of independent exploration decreased and showed anxiety like behavior, which proved that the SAE model was successfully replicated) and NaB pretreatment group was established (NaB was administered at a dose of 500 mg·kg -1·d -1 for 3 days before modeling, and the same dose once a day for 3 days after modeling). Open field test was used to detect the anxiety like behavior of mice at 7 days. The protein expressions and content changes of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in hippocampus of mice at 1 day and 3 days after operation were detected by Western blotting and enzyme linked immunosorbent assay (ELISA). Immunofluorescence staining was used to observe microglia labeled protein ionized calcium bindingadaptor molecule-1 (Iba-1) and TNF-α protein co localization. ② Cell experiment: mouse microglia cell line BV-2 microglia were divided into blank control group, lipopolysaccharide (LPS) group (cells were treated with 1 mg/L LPS), and NaB treatment group (cells were treated with 1 mg/L LPS+5 mmol/L NaB). The protein expressions of IL-1β, TNF-α, Toll-like receptor 4 (TLR4), phosphorylated nuclear factor-κB p65 (p-NF-κB p65), nuclear factor-κB p65 (NF-κB p65) and NF-κB inhibitor protein-α (IκB-α) were detected by Western blotting. The expressions of Iba-1 and TNF-α in each group were observed by immunofluorescence. Results:① Animal experiment: compared with the Sham group, the distance and duration of movement in the central area, the total distance moved of mice decreased 7 days after the establishment of SAE model group were decreased [distance of movement in the central area (mm): 13.45±3.97 vs. 161.44±27.00, duration of movement in the central area (s): 1.82±0.58 vs. 13.45±2.17, the total distance moved (mm): 835.01±669.67 vs. 2 254.51±213.45, all P < 0.05]. In the hippocampus tissues of mice, a large number of nerve nuclei were pyknotic and deeply stained, and the arrangement of nerve cells was disordered. The cell bodies of microglia in mouse hippocampus increased significantly. The number of positive cells of Iba-1/TNF-α (Iba-1 +/TNF-α +) increased significantly. The contents and protein expression of proinflammatory factors TNF-α, IL-1β in hippocampal homogenate supernatant 3 days after operation in SAE model group were significantly higher than those in Sham group [TNF-α (ng/L): 119.17±18.40 vs. 90.18±21.17, IL-1β (ng/L): 407.89±70.64 vs. 313.69±34.63; TNF-α/GAPDH: 1.42±0.50 vs. 0.80±0.08, IL-1β/GAPDH: 1.27±0.22 vs. 0.85±0.25, all P < 0.05]. After intragastric administration of NaB, the distance and duration of movement in the central area of mice were significantly higher than those in SAE model group [distance of movement in the central area (mm): 47.39±15.63 vs. 13.45±3.97, duration of movement in the central area (s): 6.12±1.87 vs. 1.82±0.58, all P < 0.05]. There was no significant change in the total distance moved (mm: 1 550.59±1 004.10 vs. 835.01±669.67, P > 0.05). The pyknosis and deep staining of nerve nuclei in mice were significantly less than those in SAE model group. The number of Iba-1 +/TNF-α + positive cells decreased significantly. The contents and protein expression levels of proinflammatory factors TNF-α, IL-1β in hippocampal homogenate supernatant 3 days after operation were significantly lower than those in SAE model group [TNF-α (ng/L): 64.95±9.10 vs. 119.17±18.40, IL-1β (ng/L): 311.94±69.92 vs. 407.89±70.64; TNF-α/GAPDH: 1.02±0.36 vs. 1.42±0.50, IL-1β/GAPDH: 0.86±0.20 vs. 1.27±0.22, all P < 0.05]. ② Cell experiment: after LPS intervention, the fluorescence intensity of TNF-α in BV-2 cells was significantly enhanced, the protein expression levels of TNF-α, IL-1β, TLR4 and p-NF-κB p65 protein increased (TNF-α/GAPDH: 0.39±0.06 vs. 0.20±0.02, IL-1β/GAPDH: 0.27±0.03 vs. 0.19±0.01, TLR4/GAPDH: 0.55±0.12 vs. 0.33±0.09, p-NF-κB p65/NF-κB p65: 0.55±0.05 vs. 0.29±0.04, all P < 0.05), the expression level of IκB-α was lower than that in the control group(IκB-α/GAPDH: 0.54±0.06 vs. 0.81±0.03, P < 0.05). After NaB treatment, the fluorescence intensity of TNF-α in BV-2 cells was decreased. The protein expression levels of TNF-α, IL-1β, TLR4 and p-NF-κB p65 protein were significantly lower than that of LPS model group (TNF-α/GAPDH: 0.26±0.02 vs. 0.39±0.06, IL-1β/GAPDH: 0.11±0.04 vs. 0.27±0.03, TLR4/GAPDH: 0.28±0.14 vs. 0.55±0.12, p-NF-κB p65/NF-κB p65: 0.29±0.01 vs. 0.55±0.05, all P < 0.05), the protein expression level of IκB-α was significantly higher than that in the LPS group (IκB-α/GAPDH: 0.75±0.01 vs. 0.54±0.06, P < 0.05). Conclusion:NaB could antagonism the TLR4 activation induced by LPS, thus inhibiting p-NF-κB p65 nuclear transcription and IκB-α degradation. It can reduce microglia activation and secretion of inflammatory factors, and finally improve the inflammation in the hippocampus of septic mice and long-term anxiety like behavior.

Effect of programmed necrosis inhibitor Nec-1 on lead-induced BV2 cell injury / 环境与职业医学

Background Programmed necrosis is closely related to the occurrence and development of neurodegenerative diseases, but whether lead causes programmed cell necrosis has not been reported. Objective This experiment is designed to probe into the function of programmed necrosis and the effect of its inhibitor on lead-induced microglia (BV2 cell) injury. Methods The BV2 cells at logarithmic growth phase were treated with 0, 1, 5, 10, 25, 50, 100, and 200 μmol·L−1 lead acetate for 12, 24, 36, and 48 h, respectively, and methylthiazolyldiphenyl-tetrazolium bromide (MTT) was used to determine cell viability. After treatment with 0, 25, 50, and 100 μmol·L−1 lead acetate for 24 h, enzyme-linked immunosorbent assay, Western blotting, and flow cytometry were used to determine the expressions of tumor necrosis factor-α (TNF-α), receptor-interacting protein kinase 3 (RIPK3), receptor-interacting protein kinase 1 (RIPK1), and mixed lineage kinase domain-like protein (MLKL) in the cells, and the effect of RIPK1 inhibitor Nec-1 pretreatment on lead-induced BV2 cell injury . Results The BV2 cell viability decreased with higher lead concentration (r12 h=−0.995, r24 h=−0.984, r36 h=−0.983, r48 h=−0.981, all P<0.01) and time extension (only for 5 μmol·L−1 lead acetate, r=−0.994, P<0.01). Compared with the control group, the BV2 cell viability decreased at the same exposure time when the concentration of lead was above 10 μmol·L−1 (P<0.01). Compared with the control group, the expressions of RIPK1 and MLKL were increased in the 25, 50, and 100 μmol·L−1 lead groups (P<0.05 or 0.01), accompanied by an increase in the contents of inflammatory cytokine TNF-α, especially in the 100 μmol·L−1 lead group, the increment was the highest (P<0.01). The expression levels of p-RIPK1 and p-MLKL in BV2 cells were both increased when the concentration of lead acetate was above 50 μmol·L−1 (P<0.01). In addition, pretreatment with Nec-1 increased the cell viability rate and decreased the necrosis and late apoptosis rate of BV2 cells exposed to lead compared with corresponding lead exposure groups (P<0.05). Conclusions Lead can reduce BV2 cell viability, increase necrosis rate, and up-regulate the expressions of RIPK1, RIPK3, amd MLKL, and the phosphorylation levels of RIPK1 and MLKL. The RIPK1 inhibitor Nec-1 has an intervention effect on lead-induced damage in BV2 cells, indicating that programmed necrosis may play a role in lead neurotoxicity.

Effect of metformin hydrochloride on the expressions of inflammatory cytokines in Aβ1-42-induced BV-2 cells and its mechanisms / 西安交通大学学报(医学版)

【Objective】 To study the effect of metformin (Met) hydrochloride on the expressions of inflammatory cytokines in Aβ1-42-induced BV-2 cells and its mechanisms. 【Methods】 BV-2 cells were induced by Aβ1-42 to mimic the neuroinflammatory cell model, and after treatment with Met, the contents of cytokines, including TNF-α, IL-1β, IL-6 and IL-18, in cell supernatant were examined by ELISA; the mRNA levels of TNF-α, IL-1β, IL-6 and IL-18 were examined by RT-PCR; and the expressions of NLRP3, ASC, pro-Caspase-1, Caspase-1, NF-κB, p-IκBα and IκBα were examined by Western blotting. 【Results】 Aβ1-42 at 1.25, 2.5 and 5 μmol/L could decrease the BV-2 cell viability in a dose-dependent manner, while Met at 0.5, 1 and 2 mmol/L could increase the cell viability in 5 μmol/L Aβ1-42-induced BV-2 cells. After treatment with Met, the transcription and secretion of TNF-α, IL-1β, IL-6 and IL-18, and the protein expressions of NLRP3, ASC, Caspase-1(p20), NF-κB and p-IκBα were significantly decreased in Aβ1-42-induced BV-2 cells. 【Conclusion】 Met attenuates inflammatory responses in Aβ1-42-induced BV-2 cells, which may be associated with the inhibition of NLRP3 inflammasome activation via NF-κB pathway.

Lipid-rich fraction of the sclerotium of Tiger Milk Mushroom Lignosus rhinocerotis (Agaricomycetes) attenuates LPS-induced inflammation in BV2 cells via Nrf2 pathway

Lignosus rhinocerotis (tiger milk mushroom) is widely used by the indigenous people of Malaysia as a traditional remedy. The present study was carried out in order to evaluate the antioxidant, cytotoxic and anti-neuroinflammatory activities of L. rhinocerotis extract on brain microglial cells (BV2). The antioxidant activity was evaluated by 2,2-diphenyl-1-picryhydrazyl (DPPH•), 2,2'-azinobis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS•+) scavenging assays, and ferric reducing antioxidant power (FRAP). The FRAP, DPPH and ABTS•+ scavenging capacities of the TE3 fraction were 420.77 mg FE/g, 58.01%, and 7%, respectively. The cytotoxic activity was determined by MTS assay. The in vitro model of anti-neuroinflammatory property was evaluated by measuring the production of nitric oxide (NO) in lipopolysaccharide (LPS)-induced BV2 cells. The TE3 fraction showed a significant NO reduction at 1 to 100 µg/mL. The TE3 fraction down-regulated inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX2) genes while it upregulated heme oxygenase (HO-1) and NADPH quinone acceptor oxidoreductase-1 (NQO-1) genes. The nuclear factor (erythroid-derived 2)-like 2 (Nrf2) transcription was also activated. The chemical component of the active fraction (TE3) was identified by gas chromatography-mass spectrometry (GCMS). Overall, the BV2 in vitro model anti-neuroinflammatory activity of L. rhinocerotis may be caused by the lipid constituents identified in the fraction

Two new polyacetylene glycosides from rhizomes of Atractylodes lancea / 中草药

Objective: To investigate the chemical components from the 80% EtOH extract of Atractylodes lancea, as well as the inhibitory activities of the isolated compounds on LPS-induced NO production of microglia BV2 cells. Methods: The n-BuOH-soluble fraction of the crude extract was successively chromatographed with Diaion HP-20, Sephadex LH-20, and preparative HPLC C18-column. At last, the planar and stereochemical structures of these obtained compounds were established on the basis of extensive spectroscopic data (HRESIMS, NMR, and ECD, etc). Results: Ten glycosides were isolated from the n-BuOH-soluble fraction of the 80% EtOH extract of A. lancea, including (2E,8R)-decene-4,6-diyne-1,8-diol-1-O-β-D- apiofuranosyl-(1→6)-β-D-glucopyranoside (1), (8S)-decane-4,6-diyne-1,8-diol-8-O-β-D-glucopyranoside (2), (2E,8R)-decene-4,6- diyne-1,8-diol-8-O-β-D-glucopyranoside (3), (2E,8S)-decene-4,6-diyne-1,8-diol-8-O-β-D-glucopyranoside (4), (2E,8E)-2,8- decadiene-4,6-diyne-1,10-diol-1-O-β-D-glucopyranoside (5), (7R,8S)-3',9,9'-trihydroxyl-3-methoxyl-1'-propanol-7,8-dihydrobenzo- funanneoligan-4-O-β-D-glucopyranoside (6), (7'R*,8S*,8'S*)-lyoniresinol 9'-O-β-D-glucopyranoside (7), (7S,8R)-4,9,9'-trihydroxy- 3'-methoxy-8-O-4'-neolignan 7-O-β-D-glucopyranoside (8), methyl salicylate 2-O-α-L-xylopyranosyl-(1→6)-β-D-glucopyranoside (9), and phenylmethanol 7-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside (10). Conclusion: Compounds 1 and 2 are named atractyeneyneglycoside A and atractyeneyneglycoside B, while compounds 6, 8-10 are first isolated from the rhizomes of A. lancea. At the concentration of 10 μmol/L, compound 10 exhibited the strongest inhibitory effects on LPS-induced NO production of microglia BV2 cells with the value of 31.18%, while compounds 1 and 2 just showed weaker inhibitory effects with values of 22.01% and 14.09%, respectively.

Attenuation of lipopolysaccharide-induced neuroinflammatory events in BV-2 microglial cells by Moringa oleifera leaf extract

Objective: To determine the anti-neuroinflammatory activity of Moringa oleifera leaf extract (MLE) under lipopolysaccharide stimulation of mouse murine microglia BV2 cells in vitro. Methods: The cytotoxicity effect of MLE was investigated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyl-tetrazolium bromide assay. The inflammatory response of BV-2 cells were induced with lipopolysaccharide. The generation of nitric oxide levels was determined by using Griess assay and the level of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) was evaluated by ELISA kit. The expression of iNOS, COX-2 as well as IκB-α was carried out by immunoblot analysis. Results: MLE reduced the nitric oxide production in concentration-dependent manner, and maintained the viability of BV-2 microglial cells which indicated absence of toxicity. In addition, MLE repressed the activation of nuclear factor kappa B by arresting the deterioration of IκB-α, consequently resulted in suppression of cytokines expression such as COX-2 and iNOS. Conclusions: MLE inhibitory activities are associated with the inhibition of nuclear factor kappa B transcriptional activity in BV2 microglial cells. Thus MLE may offer a substantial treatment for neuroinflammatory diseases.

Attenuation of lipopolysaccharide-induced neuroinflammatory events in BV-2 microglial cells by Moringa oleifera leaf extract

Objective: To determine the anti-neuroinflammatory activity of Moringa oleifera leaf extract (MLE) under lipopolysaccharide stimulation of mouse murine microglia BV2 cells in vitro. Methods: The cytotoxicity effect of MLE was investigated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide assay. The inflammatory response of BV-2 cells were induced with lipopolysaccharide. The generation of nitric oxide levels was determined by using Griess assay and the level of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) was evaluated by ELISA kit. The expression of iNOS, COX-2 as well as IκB-α was carried out by immunoblot analysis. Results: MLE reduced the nitric oxide production in concentration-dependent manner, and maintained the viability of BV-2 microglial cells which indicated absence of toxicity. In addition, MLE repressed the activation of nuclear factor kappa B by arresting the deterioration of IκB-α, consequently resulted in suppression of cytokines expression such as COX-2 and iNOS. Conclusions: MLE inhibitory activities are associated with the inhibition of nuclear factor kappa B transcriptional activity in BV2 microglial cells. Thus MLE may offer a substantial treatment for neuroinflammatory diseases.

Polygalasaponin F inhibits secretion of IL-1β and its molecular mechanisms / 中国药理学通报

Aim To investigate the effects of polyga-lasaponin F( PS-F) on LPS-induced inflammatory cytokine IL-1β expression in BV2 cells and explore the potential anti-inflammatory mechanism of PS-F. Methods Five groups were included in the experiment, including the control group, the model group and PS-F treatment groups(0. 10, 1.00, 10.00 μmol • L-1). ELISA, quantitative real-time PCR and Western blot were used to assay the mRNA and protein expression of IL-1β. The levels of NLRP3, caspase-1, ASC, caspase-11 protein were examined by Western blot. Results Compared with model group, PS-F treatment groups effectively reduced IL-1β expression in LPS-in-duced inflammatory model in a dose-dependent manner ( P <0. 01 or P <0. 05). Moreover, PS-F significantly inhibited ASC and caspase-11 expression (P < 0. 05 ). Conclusions PS-F inhibited the expression of inflammatory mediator IL-1β by suppressing the activation of NLRP3 inflammasome and inhibiting caspase-11 in LPS-stimulated BV2 cells.

Anti-inflammatory effects of salidroside on lps-induced bv2 microglia cells via pi3k/akt signaling pathway / 中国药理学通报

 Aim To investigate the anti-inflammatory effect of salidroside on lipopolysaccharide (LPS)-induced BV2 microglia and its mechanism. Methods The model of LPS-induced injury in BV2 microglia was established. The expression of cytokines IL-6, IL-1β and TNF-α mRNA was detected by qPCR, and the expression of Akt, p-Akt, nuclear protein NF-κB p50 protein was dectected by Western blot with different concentrations of salidroside treatment. The model was sequentially treated with PI3K inhibitor LY294002 for 30 min, and after the treatment of salidroside, the expressions of Akt, p-Akt, nuclear protein NF-κB p50, IL-6, IL-1β, TNF-α and other indicators were dectected by Western blot. Results Compared with model, salidroside reduced the expression of IL-6, IL-1β and TNF-α mRNA in BV2 microglia, improved the expression of p-Akt protein, and reduced the nuclear protein NF-κB p50. After the treatment of LY294002, salidroside had no obvious effect on the expression of p-Akt, nuclear protein NF-κB p50, IL-6, IL-1β. Conclusions Salidroside can inhibit LPS-induced BV2 microglia inflammatory reaction by activating PI3K/Akt signaling pathway, promoting Akt phosphorylation, inhibiting NF-κB p50 nuclear transcription, and inhibiting cytokines.

The effects of catechin on inflammatory response induced by lipopolysaccharide in BV-2 cells / 药学学报

The effects of catechin on inflammatory response of BV-2 cells were investigated using the lipopolysaccharide (LPS) model. BV-2 cells were incubated with LPS (1 mg·L-1) for 12 h in the microglia inflammatory model in vitro. After catechin and LPS co-incubation for 12 h, MTT, ELISA and Western blot were used to detect cell viability, cytokines, cell migration and protein expression. In addition, transwell assay was conducted to investigate the effect of catechin on cell chemokaxis. Catechin did not show any cytotoxicity effect on BV-2 cells, but reversed the change in cell morphology and inhibited the release of TNF-α and IL-1β, cell chemotaxis and phosphorylation of NF-κB/p65. In conclusion, Catechin could inhibit the LPS-induced inflammatory response in BV-2 cells.

Synthetic 3′,4′-Dihydroxyflavone Exerts Anti-Neuroinflammatory Effects in BV2 Microglia and a Mouse Model

Neuroinflammation is an immune response within the central nervous system against various proinflammatory stimuli. Abnormal activation of this response contributes to neurodegenerative diseases such as Parkinson disease, Alzheimer’s disease, and Huntington disease. Therefore, pharmacologic modulation of abnormal neuroinflammation is thought to be a promising approach to amelioration of neurodegenerative diseases. In this study, we evaluated the synthetic flavone derivative 3′,4′-dihydroxyflavone, investigating its anti-neuroinflammatory activity in BV2 microglial cells and in a mouse model. In BV2 microglial cells, 3′,4′-dihydroxyflavone successfully inhibited production of chemokines such as nitric oxide and prostaglandin E2 and proinflammatory cytokines such as tumor necrosis factor alpha, interleukin 1 beta, and interleukin 6 in BV2 microglia. It also inhibited phosphorylation of mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB activation. This indicates that the anti-inflammatory activities of 3′,4′-dihydroxyflavone might be related to suppression of the proinflammatory MAPK and NF-κB signaling pathways. Similar anti-neuroinflammatory activities of the compound were observed in the mouse model. These findings suggest that 3′,4′-dihydroxyflavone is a potential drug candidate for the treatment of microglia-related neuroinflammatory diseases.