This study aims to explore the effects and action mechanisms of the active ingredients in Buyang Huanwu Decoction(BYHWD), namely tetramethylpyrazine(TMP) and hydroxy-safflor yellow A(HSYA), on oxygen-glucose deprivation/reglucose-reoxygenation(OGD/R)-induced inflammation and oxidative stress of microglia(MG). Network pharmacology was used to screen the effective monomer ingredients of BYHWD and determine the safe concentration range for each component. Inflammation and oxidative stress models were established to further screen the best ingredient combination and optimal concentration ratio with the most effective anti-inflammatory and antioxidant effects. OGD/R BV2 cell models were constructed, and BV2 cells in the logarithmic growth phase were divided into a normal group, a model group, an HSYA group, a TMP group, and an HSYA + TMP group. Enzyme-linked immunosorbent assay(ELISA) was used to detect the levels of inflammatory cytokines such as interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α), and interleukin-6(IL-6). Oxidative stress markers, including superoxide dismutase(SOD), nitric oxide(NO), and malondialdehyde(MDA), were also measured. Western blot was used to analyze the protein expression of both inflammation-related pathway [Toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB)] and oxidative stress-related pathway [nuclear factor erythroid 2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)]. Immunofluorescence was used to assess the expression of proteins such as inducible nitric oxide synthase(iNOS) and arginase-1(Arg-1). The most effective ingredients for anti-inflammatory and antioxidant effects in BYHWD were TMP and HSYA. Compared to the normal group, the model group showed significantly increased levels of IL-1β, TNF-α, IL-6, NO, and MDA, along with significantly higher protein expression of NF-κB, TLR4, Nrf2, and HO-1 and significantly lower SOD levels. The differences between the two groups were statistically significant. Compared to the model group, both the HSYA group and the TMP group showed significantly reduced levels of IL-1β, TNF-α, IL-6, NO, and MDA, lower expression of NF-κB and TLR4 proteins, higher levels of SOD, and significantly increased protein expression of Nrf2 and HO-1. Additionally, the expression of the M1-type MG marker iNOS was significantly reduced, while the expression of the M2-type MG marker Arg-1 was significantly increased. The results of the HSYA group and the TMP group had statistically significant differences from those of the model group. Compared to the HSYA group and the TMP group, the HSYA + TMP group showed further significant reductions in IL-1β, TNF-α, IL-6, NO, and MDA levels, along with significant reductions in NF-κB and TLR4 protein expression, an increase in SOD levels, and elevated Nrf2 and HO-1 protein expression. Additionally, the expression of the M1-type MG marker iNOS was reduced, while the M2-type MG marker Arg-1 expression increased significantly in the HSYA + TMP group compared to the TMP or HSYA group. The differences in the results were statistically significant between the HSYA + TMP group and the TMP or HSYA group. The findings indicated that the combined use of HSYA and TMP, the active ingredients of BYHWD, can effectively inhibit OGD/R-induced inflammation and oxidative stress of MG, showing superior effects compared to the individual use of either component.
Oxidative Stress/drug effects* ; Drugs, Chinese Herbal/pharmacology* ; Animals ; Mice ; Glucose/metabolism* ; Cell Line ; Inflammation/genetics* ; Oxygen/metabolism* ; Pyrazines/pharmacology* ; Microglia/metabolism* ; NF-E2-Related Factor 2/immunology* ; NF-kappa B/immunology* ; Toll-Like Receptor 4/immunology* ; Anti-Inflammatory Agents/pharmacology* ; Humans

Stem-leaf saponins from Panax notoginseng (SLSP) comprise numerous PPD-type saponins with diverse pharmacological properties; however, their role in Parkinson's disease (PD), characterized by microglia-mediated neuroinflammation, remains unclear. This study evaluated the effects of SLSP on suppressing microglia-driven neuroinflammation in experimental PD models, including the 1-methyl-4-phenylpyridinium (MPTP)-induced mouse model and lipopolysaccharide (LPS)-stimulated BV-2 microglia. Our findings revealed that SLSP mitigated behavioral impairments and excessive microglial activation in models of PD, including MPTP-treated mice. Additionally, SLSP inhibited the upregulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX2) and attenuated the phosphorylation of PI3K, protein kinase B (AKT), nuclear factor-κB (NFκB), and inhibitor of NFκB protein α (IκBα) both in vivo and in vitro. Moreover, SLSP suppressed the production of inflammatory markers such as interleukin (IL)-1β, IL-6, and tumor necrosis factor alpha (TNF-α) in LPS-stimulated BV-2 cells. Notably, the P2Y2R agonist partially reversed the inhibitory effects of SLSP in LPS-treated BV-2 cells. These results suggest that SLSP inhibit microglia-mediated neuroinflammation in experimental PD models, likely through the P2Y2R/PI3K/AKT/NFκB signaling pathway. These novel findings indicate that SLSP may offer therapeutic potential for PD by attenuating microglia-mediated neuroinflammation.
Animals ; Panax notoginseng/chemistry* ; Saponins/pharmacology* ; Microglia/immunology* ; Mice ; NF-kappa B/immunology* ; Signal Transduction/drug effects* ; Proto-Oncogene Proteins c-akt/immunology* ; Phosphatidylinositol 3-Kinases/genetics* ; Male ; Parkinson Disease/immunology* ; Mice, Inbred C57BL ; Disease Models, Animal ; Plant Leaves/chemistry* ; Neuroinflammatory Diseases/drug therapy* ; Humans

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.
Animals ; Anti-Inflammatory Agents ; pharmacology ; Humans ; Inflammation ; drug therapy ; genetics ; immunology ; Interleukin-1beta ; genetics ; immunology ; Lipopolysaccharides ; pharmacology ; Mice ; Microglia ; drug effects ; immunology ; Nitric Oxide ; genetics ; immunology ; Phenols ; pharmacology ; STAT3 Transcription Factor ; genetics ; immunology ; Signal Transduction ; drug effects ; Tumor Necrosis Factor-alpha ; genetics ; immunology

Excessive microglial cell activation is related to the progression of chronic neuro-inflammatory disorders. Heme oxygenase-1 (HO-1) expression mediated by the NFE2-related factor (Nrf-2) pathway is a key regulator of neuro-inflammation. Nardostachys chinensis is used as an anti-malarial, anti-nociceptive, and neurotrophic treatment in traditional Asian medicines. In the present study, we examined the effects of an ethyl acetate extract of N. chinensis (EN) on the anti-neuro-inflammatory effects mediated by HO-1 up-regulation in Salmonella lipopolysaccharide (LPS)- or Staphylococcus aureus lipoteichoic acid (LTA)-stimulated BV2 microglial cells. Our results indicated that EN suppressed pro-inflammatory cytokine production and induced HO-1 transcription and translation through Nrf-2/antioxidant response element (ARE) signaling. EN markedly inhibited LPS- and LTA-induced activation of nuclear factor-kappa B (NF-κB) as well as phosphorylation of mitogen-activated protein kinases (MAPKs) and signal transducer and activator of transcription (STAT). Furthermore, EN protected hippocampal HT22 cells from indirect neuronal toxicity mediated by LPS- and LTA-treated microglial cells. These results suggested that EN impairs LPS- and LTA-induced neuro-inflammatory responses in microglial cells and confers protection against indirect neuronal damage to HT22 cells. In conclusion, our findings indicate that EN could be used as a natural anti-neuro-inflammatory and neuroprotective agent.
Anti-Inflammatory Agents ; pharmacology ; Cell Line ; Heme Oxygenase-1 ; genetics ; immunology ; Humans ; Lipopolysaccharides ; adverse effects ; Microglia ; cytology ; drug effects ; immunology ; Mitogen-Activated Protein Kinases ; genetics ; immunology ; NF-kappa B ; genetics ; immunology ; Nardostachys ; chemistry ; Neuroprotective Agents ; pharmacology ; Plant Extracts ; pharmacology ; Teichoic Acids ; adverse effects

This study examined the neuroprotective effect of cluster of differentiation molecule 200 (CD200) against methamphetamine (METH)-induced neurotoxicity. In the in vitro experiment, neuron-microglia cultures were treated with METH (20 μmol/L), METH (20 μmol/L)+CD200-Fc (10 μg/mL) or CD200-Fc (10 μg/mL). Those untreated served as control. Microglia activation expressed as the ratio of MHC-II/CD11b was assessed by flow cytometry. The cytokines (IL-1β, TNF-α) secreted by activated microglia were detected by enzyme-linked immunosorbent assay (ELISA). In the in vivo experiment, 40 SD rats were divided into control, METH, METH+CD200-Fc and CD200-Fc groups at random. Rats were intraperitoneally injected with METH (15 mg/kg 8 times at 12 h interval) in METH group, with METH (administered as the same dose and time as the METH group) and CD200-Fc (1 mg/kg at day 0, 2, 4 after METH injection) in METH+CD200-Fc group, with CD200-Fc (1 mg/kg injected as the same time as the METH+CD200-Fc group) or with physiological saline solution in the control group. The level of striatal dopamine (DA) in rats was measured by high-performance liquid chromatography (HPLC). The microglial cells were immunohistochemically detected for the expression of Iba-1, a marker for microglial activation. The results showed that METH could increase the microglia activation in the neuron-microglia cultures and elevate the secretion of IL-1β and TNF-α, which could be attenuated by CD200-Fc. Moreover, CD200-Fc could partially reverse the striatal DA depletion induced by METH and reduce the number of activated microglia, i.e. Iba-1-positive cells. It was concluded that CD200 may have neuroprotective effects against METH-induced neurotoxicity by inhibiting microglial activation and reversing DA depletion in striatum.
Animals ; Animals, Newborn ; Antigens, CD ; administration & dosage ; Cells, Cultured ; Coculture Techniques ; Corpus Striatum ; cytology ; drug effects ; immunology ; Cytokines ; immunology ; Dopamine ; immunology ; Drug Interactions ; Male ; Methamphetamine ; toxicity ; Microglia ; drug effects ; immunology ; Neurons ; metabolism ; Rats ; Rats, Sprague-Dawley

Microglial cells are the resident innate immune cells that sense pathogens and tissue injury in the central nervous system (CNS). Microglial activation is critical for neuroinflammatory responses. The synthetic compound 2-hydroxy-3',5,5'-trimethoxychalcone (DK-139) is a novel chalcone-derived compound. In this study, we investigated the effects of DK-139 on Toll-like receptor 4 (TLR4)-mediated inflammatory responses in BV2 microglial cells. DK-139 inhibited lipopolysaccharide (LPS)-induced TLR4 activity, as determined using a cell-based assay. DK-139 blocked LPS-induced phosphorylation of IkappaB and p65/RelA NF-kappaB, resulting in inhibition of the nuclear translocation and trans-acting activity of NF-kappaB in BV2 microglial cells. We also found that DK-139 reduced the expression of NF-kappaB target genes, such as those for COX-2, iNOS, and IL-1beta, in LPS-stimulated BV2 microglial cells. Interestingly, DK-139 blocked LPS-induced Akt phosphorylation. Inhibition of Akt abrogated LPS-induced phosphorylation of p65/RelA, while overexpression of dominant-active p110CAAX enhanced p65/RelA phosphorylation as well as iNOS and COX2 expression. These results suggest that DK-139 exerts an anti-inflammatory effect on microglial cells by inhibiting the Akt/IkappaB kinase (IKK)/NF-kappaB signaling pathway.
Animals ; Binding Sites ; Cell Line ; Chalcones/chemistry/*pharmacology ; Cyclooxygenase 2/metabolism ; I-kappa B Kinase/metabolism ; Inflammation/*drug therapy ; Interleukin-1beta/metabolism ; Lipopolysaccharides/immunology ; Microglia/*drug effects/immunology/metabolism ; Molecular Dynamics Simulation ; NF-kappa B/*antagonists & inhibitors ; Nitric Oxide Synthase Type II/metabolism ; Phosphorylation/drug effects ; Protein Binding ; Proto-Oncogene Proteins c-akt/*antagonists & inhibitors ; Rats ; Signal Transduction ; Toll-Like Receptor 4/*antagonists & inhibitors/metabolism ; Transcription Factor RelA/metabolism

OBJECTIVETo prepare and identify a polyclonal antibody (pAb) against (mouse) cysteinyl leukotriene receptor 1 (CysLT(1)) and to investigate the changes of CysLT(1) receptor expression in BV2 microglial cells after rotenone treatment.

METHODSRabbits were immunized with KLH-coupled CysLT(1) peptide to prepare the pAb. The titer of the pAb in rabbit plasma was detected by ELISA method, and the specificity of the pAb was tested by antigen blockade. After BV2 cells were treated with rotenone (0.01-1 μmol/L) for 24 h, the expression of CysLT(1) was determined by immunostaining, Western blotting and RT-PCR.

RESULTThe pAb showed a titer of 1/32728, and was not cross-reacted with antigens of CysLT(2) receptor and GPR17. Immunostaining, Western blotting and RT-PCR analysis showed the expression of CysLT(1) receptor in BV2 microglia. Rotenone at 1μmol/L significantly induced an increased expression of CysLT(1) receptor.

CONCLUSIONThe prepared CysLT(1) receptor polyclonal antibody has a high titer and high specificity to meet testing requirements of Western blotting and immunostaining; CysLT(1) is associated with rotenone-induced injury of BV2 microglial cells.

Animals ; Cells, Cultured ; Male ; Mice ; Microglia ; drug effects ; metabolism ; pathology ; Rabbits ; Receptors, Leukotriene ; immunology ; metabolism ; Rotenone ; pharmacology

The immunocytes microglia in the central nervous system (CNS) were reported to play a crucial role in neurodegeneration. As a member of P2 receptors family, purinoceptor P2Y6 has attracted much attention recently. Previous studies showed that purinoceptor P2Y6 mainly contributed to microglia activation and their later phagocytosis in CNS, while in immune system, it participated in the secretion of interleukin (IL)-8 from monocytes and macrocytes. So there raises a question: whether purinoceptor P2Y6 also takes part in neuroinflammation? Thus, this review mainly concerns about the properties and roles of purinoceptor P2Y6, including (1) structure of purinoceptor P2Y6; (2) distribution and properties of purinoceptor P2Y6; (3) relationships between purinoceptor P2Y6 and microglia; (4) relationships between purinoceptor P2Y6 and immunoinflammation. Itos proposed that purinoceptor P2Y6 may play a role in neuroinflammation in CNS, although further research is still required.
Animals ; Humans ; Inflammation ; immunology ; metabolism ; Microglia ; drug effects ; metabolism ; Monocytes ; metabolism ; Phagocytosis ; physiology ; Receptors, Purinergic P2 ; chemistry ; genetics ; metabolism

OBJECTIVETo identify the protective effect of lipopolysaccharide (LPS) preconditioning against LPS-induced inflammatory damage in dopaminergic neurons of midbrain slice culture and the possible mechanisms.

METHODSAfter cultured in vitro for 14 d, the rat organotypic midbrain slices were pretreated with different concentrations (0, 1, 3, 6 or 10 ng/mL) of LPS for 24 h followed by treatment with 100 ng/mL LPS for 72 h. The whole slice viability was determined by measurement of the activity of lactic acid dehydrogenase (LDH). Tyrosine hydroxylase-immunoreactive (TH-IR) neurons and CD11b/c equivalent-immunoreactive (OX-42-IR) microglia in the slices were observed by immunohistochemical method, and tumor necrosis factor-alpha (TNF-alpha) levels in the culture media were detected by enzyme-linked immunosorbent assays (ELISA).

RESULTSIn the slices treated with 100 ng/mL LPS for 72 h, the number of TH-IR neurons reduced from 191+/-12 in the control slices to 46+/-4, and the LDH activity elevated obviously (P < 0.01), along with remarkably increased number of OX-42-IR cells and production of TNF-alpha (P < 0.01). Preconditioning with 3 or 6 ng/mL LPS attenuated neuron loss (the number of TH-IR neurons increased to 126+/-12 and 180+/-13, respectively) and markedly reduced LDH levels (P < 0.05), accompanied by significant decreases of OX-42-IR microglia activation and TNF-alpha production (P < 0.05).

CONCLUSIONLow-dose LPS preconditioning could protect dopaminergic neurons against inflammatory damage in rat midbrain slice culture, and inhibition of microglial activation and reduction of the proinflammatory factor TNF-alpha production may contribute to this protective effect. Further understanding the underlying mechanism of LPS preconditioning may open a new window for treatment of Parkinson's disease.

Animals ; CD11 Antigens ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Immunohistochemistry ; Inflammation ; chemically induced ; pathology ; L-Lactate Dehydrogenase ; metabolism ; Lipopolysaccharides ; administration & dosage ; toxicity ; Mesencephalon ; drug effects ; immunology ; pathology ; Microglia ; drug effects ; immunology ; pathology ; Nerve Degeneration ; metabolism ; pathology ; prevention & control ; Neurons ; drug effects ; immunology ; pathology ; Organ Culture Techniques ; Rats ; Tumor Necrosis Factor-alpha ; metabolism ; Tyrosine 3-Monooxygenase ; metabolism

OBJECTIVENeuroinflammation with microglial activation has been implicated to have a strong association with the progressive dopaminergic neuronal loss in Parkinson's disease (PD). The present study was undertaken to evaluate the activation profile of microglia in 1-methyl-4-phenyl pyridinium (MPP+)-induced hemiparkinsonian rats. Triptolide, a potent immunosuppressant and microglia inhibitor, was then examined for its efficacy in protecting dopaminergic neurons from injury and ameliorating behavioral disabilities induced by MPP+.

METHODSThe rat model of PD was established by intranigral microinjection of MPP+. At baseline and on day 1, 3, 7, 14, 21 following MPP+ injection, the degree of microglial activation was examined by detecting the immunodensity of OX-42 (microglia marker) in the substantia nigra (SN). The number of viable dopaminergic neurons was determined by measuring tyrosine hydroxylase (TH) positive neurons in the SN. Behavioral performances were evaluated by counting the number of rotations induced by apomorphine, calculating scores of forelimb akinesia and vibrissae-elicited forelimb placing asymmetry.

RESULTSIntranigral injection of MPP+ resulted in robust activation of microglia, progressive depletion of dopaminergic neurons, and ongoing aggravation of behavioral disabilities in rats. Triptolide significantly inhibited microglial activation, partially prevented dopaminergic cells from death and improved behavioral performances.

CONCLUSIONThese data demonstrated for the first time a neuroprotective effect of triptolide on dopaminergic neurons in MPP+-induced hemiparkinsonian rats. The protective effect of triptolide may, at least partially, be related to the inhibition of MPP+-induced microglial activation. Our results lend strong support to the use of immunosuppressive agents in the management of PD.

1-Methyl-4-phenylpyridinium ; antagonists & inhibitors ; toxicity ; Animals ; Biomarkers ; metabolism ; CD11b Antigen ; analysis ; metabolism ; Cell Count ; Cell Survival ; drug effects ; physiology ; Disability Evaluation ; Diterpenes ; pharmacology ; therapeutic use ; Dopamine ; metabolism ; Encephalitis ; drug therapy ; immunology ; prevention & control ; Epoxy Compounds ; pharmacology ; therapeutic use ; Gliosis ; drug therapy ; immunology ; prevention & control ; Herbicides ; antagonists & inhibitors ; toxicity ; Immunosuppression ; methods ; Immunosuppressive Agents ; pharmacology ; therapeutic use ; Male ; Microglia ; drug effects ; immunology ; Neurons ; drug effects ; immunology ; pathology ; Parkinsonian Disorders ; drug therapy ; immunology ; physiopathology ; Phenanthrenes ; pharmacology ; therapeutic use ; Rats ; Rats, Sprague-Dawley ; Substantia Nigra ; drug effects ; immunology ; physiopathology ; Treatment Outcome ; Tyrosine 3-Monooxygenase ; analysis ; metabolism