Daidzein exerts neuroprotective activity against MPTP-induced Parkinson's disease in experimental mice and lipopolysaccharide-induced BV2 microglial cells.

Parkinson's disease (PD) ranks as the second most neurodegenerative disease characterized by loss of neurons, bradykinesia, anosmia, sleep disorder, and motor deficiency with increased global prevalence. Here, we have analyzed daidzein's neuroprotective functions in in vitro and in vivo models of PD. BV2 microglial cells induced with lipopolysaccharide (LPS) and C57BL6 mice induced with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) were used in this study to investigate neuroprotective functions of daidzein. BV2 cells induced with LPS do not exert and significant (p < 0.05) reduction in cell viability up to concentration range (5-100 µM/ml). Furthermore, LPS exposed BV2 microglia exhibited significantly (p < 0.05) increased NO production, pro-inflammatory mediators PGE2, interleukin-6 (IL6), and interleukin-1ß (IL-1ß) levels. Treatment with daidzein (10, 25, and 50 µM/ml) to LPS-induced BV2 microglia exhibited significantly (p < 0.05) decreased NO, pro-inflammatory mediators PGE2, IL6, and IlL-1ß. Similar to the in vitro results, C57BL6 mice induced with MPTP showed defects in motor functions as observed from altered forelimb and hindlimb footprint analyses, grip strength, and perturbed motor coordination observed via rotarod tests. Additionally, levels of dopamine were significantly reduced, and pro-inflammatory mediators tumor necrosis factor alpha (TNF-α), IL-1ß, IL6 were found to be increased in MPTP-induced C57BL6 PD mice. Administering daidzein significantly restored the functional levels of dopamine and pro-inflammatory mediators TNF-α, IL-1ß, IL6 to near normal physiology as seen in healthy C57BL6 mice controls. Similarly, daidzein treatment to PD mice also restored the histological architecture to near normal levels as in control mice. Together, our results collectively endorse the neuroprotective functions of daidzein as observed from our initial studies, and further studies aimed at investigating daidzein's ability in regulating the catecholamine synthesis pathway to protect substantia nigra pars compacta (SNpc) neurons are in focus.

Anti-inflammatory Effects of Sanhuang-Siwu-Tang in Lipopolysaccharide-Stimulated RAW264.7 Macrophages and BV2 Microglial Cells.

Sanhuang-Siwu-Tang (SST), composed of seven medicinal herbs, is a well-known herbal formula used for the treatment of gynecologic diseases. To expand the clinical use of SST, we explored the anti-inflammatory or anti-neuroinflammatory effects of SST water extract in lipopolysaccharide-stimulated RAW264.7 macrophages and BV2 microglial cells. According to HPLC analysis, the main components of SST were from Scutellariae Radix, Coptidis Rhizoma, and Paeoniae Radix. SST significantly inhibited pro-inflammatory mediators including lipopolysaccharide (LPS)-induced production of nitric oxide (NO) and prostaglandin E2 (PGE2) as well as protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and the production of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in LPS-stimulated RAW264.7 macrophages and BV2 microglial cells. Furthermore, these anti-inflammatory or anti-neuroinflammatory effects of SST were mediated by mitogen-activated protein kinase-related proteins (MAPK) and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB)-related proteins. Overall, this study demonstrated that SST is a potential therapeutic formula for the prevention or treatment of inappropriate inflammation, neuroinflammation, or neurodegenerative diseases.

Bioactive sesquiterpene coumarins from the resin of Ferula sinkiangensis targeted on over-activation of microglia.

Nine undescribed (1-4, 6-10) sesquiterpene coumarins, together with a new natural one (5) and ten known ones (11-20), were isolated from the low polarity fraction of the 95% ethanol extract of the resin of Ferula sinkiangensis. Their structures were elucidated based on the comprehensive analysis of HRESIMS, 1D and 2D NMR data. The absolute configurations were determined by comparison of experimental and calculated ECD spectra. All the identified SCs were evaluated for their anti-neuroinflammatory activities in LPS-induced BV-2 cells. Ferusingensine G (8) displayed a significant inhibitory effect on nitric oxide (NO) production with an IC50 value of 1.2 µM. The results suggested that natural SCs might be served as potential neuroinflammatory inhibitors.

Stilbenes from the tubers of Bletilla striata with potential anti-neuroinflammatory activity.

Neuroinflammation are involved in the pathogenesis of many neurodegenerative disorders. In our screening of natural effective neuroinflammatory inhibitors from natural products, stilbenes, such as resveratrol and its analogues, have received considerable attention over the last several decades as anti-neuroinflammatory agents. Then, Bletilla striata attracted our attention due to its abundant stilbenes portion, PE fraction. So, three new stilbenes: dusuanlansin E1 (23a), dusuanlansin E2 (23b), 3-hydroxy-5-methoxybibenzyl-3'-O-ß-d-glucopyranoside (27), and 30 known stilbene compounds were isolated from B. striata. These structures of the compounds were established on the basis of extensive spectroscopic analysis including 1D and 2D NMR and circular dichroism (CD) data. Furthermore, all the isolated components were tested in vitro for their inhibitory effects on the nitric oxide generation in LPS-stimulated BV2 cells. As a result, compounds 2, 5, 6, 16, 17 can greatly inhibit the NO production without cytotoxicity. In addition, SARs between stilbenes and anti-neuroinflammation effects were discussed briefly. In conclusion, stilbenes were characteristic constituents of the tubers of B. striata with potential anti- neuroinflammatory effects.

Anti-neuroinflammatory effects of cudraflavanone A isolated from the chloroform fraction of Cudrania tricuspidata root bark.

CONTEXT: Cudrania tricuspidata Bureau (Moraceae) is an important source of traditional Korean and Chinese medicines used to treat neuritis and inflammation. OBJECTIVE: The anti-neuroinflammatory effects of cudraflavanone A isolated from a chloroform fraction of C. tricuspidata were investigated in LPS-induced BV2 cells. MATERIALS AND METHODS: Cudraflavanone A was isolated from the root of C. tricuspidata, and its structure was determined by MS and NMR data. Cytotoxicity of the compound was examined by MTT assay, indicating no cytotoxicity at 5-40 µM of cudraflavanone A. NO concentration was measured by the Griess reaction, and the levels of PGE2, cytokines and COX-2 enzyme activity were measured by each ELISA kit. The mRNA levels of cytokines were analysed by quantitative-PCR. The expression of iNOS, COX-2, HO-1, NF-κB, MAPKs and Nrf2 was detected by Western blot. RESULTS: Cudraflavanone A had no major effect on cell viability at 40 µM indicating 91.5% viability. It reduced the production of NO (IC50 = 22.2 µM), PGE2 (IC50 = 20.6 µM), IL-1ß (IC50 = 24.7 µM) and TNF-α (IC50 = 33.0 µM) in LPS-stimulated BV2 cells. It also suppressed iNOS protein, IL-1ß and TNF-α mRNA expression. These effects were associated with the inactivation of NF-κB, JNK and p38 MAPK pathways. This compound mediated its anti-neuroinflammatory effects by inducing HO-1 protein expression via increased nuclear translocation of Nrf2. DISCUSSION AND CONCLUSIONS: The present study suggests a potent effect of cudraflavanone A to prevent neuroinflammatory diseases. Further investigation is necessary to elucidate specific molecular mechanism of cudraflavanone A.

Ginsenoside Rg1 relieves tert-Butyl hydroperoxide-induced cell impairment in mouse microglial BV2 cells.

Microglial activation plays an important role in neurodegenerative diseases associated with oxidative stress. tert-Butyl hydroperoxide (t-BHP), an analog of hydroperoxide, mimics the oxidative damage to microglial cells. It has been reported that ginsenoside Rg1 (G-Rg1), an active ingredient of Panax ginseng, has anti-stress and anti-inflammatory properties. The present study aims to investigate the ability of G-Rg1 to decrease the t-BHP-mediated cell damage of BV2 microglial cells. We performed flow cytometry assays to facilitate the detection of reactive oxygen species as well as Western blotting analyses and immunofluorescence assays using specific antibodies, such as antibodies against phospho-mitogen-activated protein kinases (p-MAPKs), phospho-nuclear factor-κB (p-NF-κB), B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X (Bax), Caspase-3, autophagy marker light chain 3 (LC3), and Becline-1. We found that treatment with 50 µM G-Rg1 protected microglial cells against oxidative damage induced by 10 µM t-BHP.

Lindenane sesquiterpenoid dimers from Chloranthus holostegius with anti-neuroinflammatory activities in BV-2 microglia.

Fifteen undescribed lindenane-type sesquiterpenoid dimers, designated chloranholides F-T (1-15), together with twenty-five known analogs (16-40), were isolated from the whole plants of Chloranthus holostegius. The isolate structures were elucidated by analysis of spectroscopic data and chemical methods, and their absolute configurations were determined by X-ray crystallography and electronic circular dichroism spectra. In anti-neuroinflammatory assays, all isolates were evaluated by examination of their inhibitory effect on nitric oxide (NO) in LPS-stimulated BV-2 cells, and the results showed that 21-24, 26, 30, 32 and 36 significantly inhibited the production of the inflammatory mediator NO, with IC50 values ranging from 3.18 to 11.46 µM, which was better than that of quercetin. Structure-activity relationship analysis revealed that two essential functional groups played an indispensable role in the anti-inflammatory effects. Moreover, 22 and 24 inhibited the LPS-induced upregulation of iNOS and COX-2 enzymes in BV-2 microglia at the protein level.

Anti-neuroinflammatory activity of 6,7-dihydroxy-2,4-dimethoxy phenanthrene isolated from Dioscorea batatas Decne partly through suppressing the p38 MAPK/NF-κB pathway in BV2 microglial cells.

ETHNOPHARMACOLOGICAL RELEVANCE: The rhizome of Dioscorea batatas Decne (called Chinses yam) widely distributed in East Asian countries including China, Japan, Korea and Taiwan has long been used in oriental folk medicine owing to its tonic, antitussive, expectorant and anti-ulcerative effects. It has been reported to have anti-inflammatory, antioxidative, cholesterol-lowering, anticholinesterase, growth hormone-releasing, antifungal and immune cell-stimulating activities. AIM OF THE STUDY: Neuroinflammation caused by activated microglia contributes to neuronal dysfunction and neurodegeneration. In the present study, the anti-neuroinflammatory activity of 6,7-dihydroxy-2,4-dimethoxy phenanthrene (DHDMP), a phenanthrene compound isolated from Dioscorea batatas Decne, was examined in microglial and neuronal cells. MATERIALS AND METHODS: A natural phenanthrene compound, DHDMP, was isolated from the peel of Dioscorea batatas Decne. The anti-neuroinflammatory capability of the compound was examined using the co-culture system of BV2 murine microglial and HT22 murine neuronal cell lines. The expression levels of inflammatory mediators and cytoprotective proteins in the cells were quantified by enzyme-linked immunosorbent assay and Western blot analysis. RESULTS: DHDMP at the concentrations of ≤1 µg/mL did not exhibit a cytotoxic effect for BV2 and HT22 cells. Rather DHDMP effectively restored the growth rate of HT22 cells, which was reduced by co-culture with lipopolysaccharide (LPS)-treated BV2 cells. DHDMP significantly decreased the production of proinflammatory mediators, such as nitric oxide, tumor necrosis factor-α, interleukin-6, inducible nitric oxide synthase, and cyclooxygenase-2 in BV2 cells. Moreover, DHDMP strongly inhibited the nuclear translocation of nuclear factor κB (NF-κB) and phosphorylation of p38 mitogen-activated protein kinase (MAPK) in BV2 cells. The compound did not affect the levels and phosphorylation of ERK and JNK. Concurrently, DHDMP increased the expression of heme oxygenase-1 (HO-1), an inducible cytoprotective enzyme, in HT22 cells. CONCLUSIONS: Our findings indicate that DHDMP effectively dampened LPS-mediated inflammatory responses in BV2 microglial cells by suppressing transcriptional activity of NF-κB and its downstream mediators and contributed to HT22 neuronal cell survival. This study provides insight into the therapeutic potential of DHDMP for inflammation-related neurological diseases.

LncRNA AK148321 alleviates neuroinflammation in LPS-stimulated BV2 microglial cell through regulating microRNA-1199-5p/HSPA5 axis.

AIMS: Dysregulated long non-coding RNA (lncRNA) expression is closely related to neuroinflammation, leading to multiple neurodegenerative diseases. In this study, we investigated the function and regulation of lncRNA AK148321 in neuroinflammation using an in vitro lipopolysaccharide (LPS)-stimulated BV2 microglial cell system. METHODS: Expression of AK148321 was analyzed by qPCR. Inflammatory cytokine expression levels were determined by ELISA assay. The interaction between AK148321, microRNA (miRNA), and its target gene was validated by luciferase reporter assay and RNA immunoprecipitation (RIP). Cell apoptosis was analyzed by Annexin V/PI staining. RESULTS: LPS treatment suppressed AK148321 expression in BV2 cells. Overexpression of AK148321 inhibited LPS-induced BV2 microglial cell activation and decreased the expression of inflammatory cytokine TNF-α and IL-1ß. AK148321 function as a competing endogenous RNA (ceRNA) by sponging microRNA-1199-5p (MiR-1199-5p). In LPS-stimulated BV2 cells, AK148321 exerted its inhibitory function via negatively modulating miR-1199-5p expression. Moreover, we identified that Heat Shock Protein Family A Member 5 (HSPA5) was a direct target of miR-1199-5p. RIP assay using the anti-Ago2 antibody further validated the relationship among AK148321, miR-1199-5p and HSPA5. The AK148321/miR-1199-5p/HSPA5 axis regulated the neuroinflammation in LPS-induced BV2 microglial cells. Microglial cell culture supernatant from LPS-stimulated, AK148321-overexpressing BV2 cells suppressed the cell apoptosis of mouse hippocampal neuronal cell HT22, while HSPA5 knockdown abrogated the suppression effect. CONCLUSION: Our findings suggest that AK148321 alleviates neuroinflammation in LPS-stimulated BV2 microglial cells through miR-1199-5p/HSPA5 axis.