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.

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.

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.

Experimental study on effects of astragalus polysaccharide on treating EAE in mice and regulating activation of BV-2 microglial cell line / 中国免疫学杂志

Objective:To investigate the therapeutic effects of astragalus polysaccharide (APS) on experimental autoimmune encephalomyelitis(EAE) and to explore the regulating effects on microglia activation that is associated with the pathogenesis of EAE and its possible mechanisms.Methods:Animal experiments:EAE model was induced by MOG35-55in C57BL/6 mice.APS was given by gavage.EAE was scored according to a 0-5 scale to observe the therapeutic effects of APS.Cell experiments:The effects of lipopolysac-charide (LPS) on cell viability of BV-2 microglial cell line were investigated by MTT assay and then the appropriate concentration of LPS to activate the BV-2 microglial cell line was selected.The microglia activation model was established.The changes in BV-2 microglial cell line morphology were observed with an inverted microscope.The cytokines of TNF-α and IFN-γ in the cell culture supernatant of BV-2 microglial cell line were detected by ELISA.The activated BV-2 microglial cells were treated with APS in different concentrations.The regulatory roles of the APS on the BV-2 microglial cell activation were observed.Western blot and Real-time PCR method were used to measure the protein and mRNA level of the PD-L1 on the cell surface of BV-2 microglial cells treated with APS.Results:APS could effectively ameliorate the symptoms in EAE mice and could suppress neuroinflammation of EAE significantly.The microglia activation model in vitro induced by LPS was successful.APS in certain concentration could inhibit the activation of microglia,increase the viabilily of the active microglia.Meanwhile,it could downregulated the level of the cytokines including IFN-γ and TNF-α and upregulated the expression of protein and mRNA of PD-L1 on activated microglia.Conclusion:APS can effectively inhibit the autoimmune reaction of EAE and effectively suppress the microglia activation induced by LPS,reduce the pro-duction of IFN-γ and TNF-α.APS plays a crucial role in reducing the inflammation induced by microglia activation.The potential mech-anisms might be related to the upregualtion of the PD-1/PD-L1 pathway.

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.

Protosappanin A exerts anti-neuroinflammatory effect by inhibiting JAK2-STAT3 pathway in lipopolysaccharide-induced BV2 microglia / 中国天然药物

Microglial activation and resultant neuroinflammatory response are implicated in various brain diseases including Alzheimer's disease and Parkinson's disease. Treatment with anti-neuroinflammatory agents could provide therapeutic benefits for such disorders. Protosappanin A (PTA) is a major bioactive ingredient isolated from Caesalpinia sappan L.. In this work, the anti-neuroinflammatory effects of PTA on LPS-stimulated BV2 cells were investigated and the underlying mechanisms were explored. Results showed that PTA significantly inhibited the production of TNF-α and IL-1β in LPS-activated BV2 microglia. Moreover, the mRNA expressions of IL-6, IL-1β, and MCP-1 were reduced by PTA in a dose-dependent manner. Furthermore, PTA suppressed JAK2/STAT3-dependent inflammation pathway through down-regulating the phosphorylation of JAK2 and STAT3, as well as STAT3 nuclear translocation against LPS treatment. These observations suggested a novel role for PTA in regulating LPS-induced neuroinflammatory injuries.

A vitro experiment study of role of TLR4/NF-κB signal pathway in pathogenesis of brain injury during deep hypothermia circulatory arrest / 实用医学杂志

Objective To investigate the role of TLR4/NF-κB signal pathway in pathogenesis of brain inju-ry during deep hypothermia circulatory arrest(DHCA). Methods BV2 microglia cells were subjected to oxygen-glucose deprivation/reoxygenation(OGD/R),in vitro model for DHCA. The BV2 were randomly divided into the control group(C group)and the experimental group(O group). BV2 viability was determined by CCK-8 assay. TLR4 and its downstream signaling molecules ,MyD88 and phosphorylated NF-κB (p-p65) expressions were detected by Western blotting. TLR4 mRNA expression in BV2 microglial cells were determined by RT-PCR. Level of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) in culture medium was detected by ELASA. Results Compared with the group C,BV2 microglia cell viability in experiment group was obviously weaker(P<0.05). Expressions of TLR4,MyD88 and phosphorylated NF-κB(p-p65)from the experiment group increased remarkedly than those from the group C (P < 0.05). TLR4 mRNA level was higher significantly in the group O than in the group C (P < 0.01). Production of IL-6 and TNF-α in the group O were up-regulated apparently compared to the group C(P<0.01). Conclusion TLR4/NF-κB signaling pathway contributed to activation of BV2 microglia cells treated by OGD/Reoxygenation ,which was probably the exactly way that involved in pathogenesis of brain injury during deep hypothermia circulatory arrest.

A vitro experiment study of role of TLR4/NF-κB signal pathway in pathogenesis of brain injury during deep hypothermia circulatory arrest / 实用医学杂志

Objective To investigate the role of TLR4/NF-κB signal pathway in pathogenesis of brain inju-ry during deep hypothermia circulatory arrest(DHCA). Methods BV2 microglia cells were subjected to oxygen-glucose deprivation/reoxygenation(OGD/R),in vitro model for DHCA. The BV2 were randomly divided into the control group(C group)and the experimental group(O group). BV2 viability was determined by CCK-8 assay. TLR4 and its downstream signaling molecules ,MyD88 and phosphorylated NF-κB (p-p65) expressions were detected by Western blotting. TLR4 mRNA expression in BV2 microglial cells were determined by RT-PCR. Level of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) in culture medium was detected by ELASA. Results Compared with the group C,BV2 microglia cell viability in experiment group was obviously weaker(P<0.05). Expressions of TLR4,MyD88 and phosphorylated NF-κB(p-p65)from the experiment group increased remarkedly than those from the group C (P < 0.05). TLR4 mRNA level was higher significantly in the group O than in the group C (P < 0.01). Production of IL-6 and TNF-α in the group O were up-regulated apparently compared to the group C(P<0.01). Conclusion TLR4/NF-κB signaling pathway contributed to activation of BV2 microglia cells treated by OGD/Reoxygenation ,which was probably the exactly way that involved in pathogenesis of brain injury during deep hypothermia circulatory arrest.

Effect of brain-derived neurotrophic factor on BV2 microglia activated by ATP / 重庆医学

Objective To study the effect of brain‐derived neurotrophic factor (BDNF) in the process of adenosine triphos‐phate (ATP) activating BV2 microglia .Methods BV2 microglia was cultured by adding different concentrations of ATP .Then the expression level of intracellular CD11b and BDNF and the secretion level of TNF‐α in the supernatant were quantitatively deter‐mined by Western blot .BV2 microglia was treated by different concentrations of BDNF scavenger tyrosine kinase receptors B (TrkB)/Fc and incubated by ATP .The expression level of intracellular CD11b and BDNF and the secretion level of TNF‐αin the supernatant were measured .Finally adding exogenous recombinant BDNF into cultured BV 2 microglia ,intracellular changes of CD11b and supernatant TNF‐αlevels were detected .Results After adding ATP for cultivating BV2 microglia ,intracellular CD11b and BDNF expression levels and supernatant TNF‐αlevel were increased with a dose‐and time‐dependent manner in some ranges . After adding TrkB/Fc ,the levels of intracellular CD11b and BDNF expression and supernatant TNF‐αlevel were decreased with a dose‐and time‐dependent manner in some range .CD11b and BDNF expression levels was decreased in a dose‐and time‐dependent manner .Adding exogenous BDNF ,the levels of intracellular CD11b and BDNF expression and supernatant TNF‐α level were in‐creased again .Conclusion Intracellular BDNF expression is increased when BV2 microglia is activated and replenishing exogenous BDNF can activate microglia .Therefore BDNF may play an important role in the microglia activation process .

Ghrelin Inhibits Oligodendrocyte Cell Death by Attenuating Microglial Activation

BACKGROUND: Recently, we reported the antiapoptotic effect of ghrelin in spinal cord injury-induced apoptotic cell death of oligodendrocytes. However, how ghrelin inhibits oligodendrocytes apoptosis, is still unknown. Therefore, in the present study, we examined whether ghrelin inhibits microglia activation and thereby inhibits oligodendrocyte apoptosis. METHODS: Using total cell extracts prepared from BV-2 cells activated by lipopolysaccharide (LPS) with or without ghrelin, the levels of p-p38 phosphor-p38 mitogen-activated protein kinase (p-p38MAPK), phospho-c-Jun N-terminal kinase (pJNK), p-c-Jun, and pro-nerve growth factor (proNGF) were examined by Western blot analysis. Reactive oxygen species (ROS) production was investigated by using dichlorodihydrofluorescein diacetate. To examine the effect of ghrelin on oligodendrocyte cell death, oligodendrocytes were cocultured in transwell chambers of 24-well plates with LPS-stimulated BV-2 cells. After 48 hours incubation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and terminal deoxynucleotidyl transferase 2'-deoxyuridine, 5'-triphosphate nick end labeling staining were assessed. RESULTS: Ghrelin treatment significantly decreased levels of p-p38MAPK, p-JNK, p-c-Jun, and proNGF in LPS-stimulated BV-2 cells. ROS production increased in LPS-stimulated BV-2 cells was also significantly inhibited by ghrelin treatment. In addition, ghrelin significantly inhibited oligodendrocyte cell death when cocultured with LPS-stimulated BV-2 cells. CONCLUSION: Ghrelin inhibits oligodendrocyte cell death by decreasing proNGF and ROS production as well as p38MAPK and JNK activation in activated microglia as an anti-inflammatory hormone.

Inhibitory effects of caffeic acid phenethyl ester on inflammatory responses of microglial cells (BV-2) induced by lipopolysaccharide / 中国药学杂志

OBJECTIVE: To investigate the inhibitory effects of caffeic acid phenethyl ester (CAPE) on LPS-induced inflammatory responses in BV-2 microglial cells and its mechanism.