LPS-induced BV2 Cells Research Articles

4-Hydroxydictyolactone alleviates cerebral ischemia injury by regulating neuroinflammation and autophagy via AMPK signaling pathway

BackgroundCerebral ischemia (CI), a cerebrovascular disorder, is a major contributor to disability and mortality. Marine-derived compounds are an important source of new neuroprotective drug candidates. Xenicane-type diterpenes from brown algae of the genus Dictyota have exhibited potential neuroprotective effects against CI injury, attributed to their antioxidant properties. However, whether there are other underlying neuroprotective mechanisms of xenicane diterpenes against CI is still ambiguous. PurposeThis study aims to elucidate the neuroprotective efficacy and mechanism of 4-hydroxydictyolactone (HDTL) in the treatment of CI. MethodsThe LPS-induced BV2 cell model was used for anti-neuroinflammatory activity assay. Tandem Mass Tag (TMT)-based quantitative proteomics was employed to identify underlying mechanisms. The OGD/R-induced SH-SY5Y cell model and a MCAO mice model were used to assess the neuroprotective effect of HDTL against CI in vitro and in vivo. ResultsHDTL reduced inflammation in LPS-stimulated BV2 cells by inhibiting the IKK/IκB/NF-κB pathway and by enhancing AMPK phosphorylation. Additionally, in SH-SY5Y cells treated with OGD/R, HDTL facilitated autophagy and reduced apoptosis. The neuroprotective properties of HDTL were abrogated in AMPK- silenced SH-SY5Y cells. In MCAO mice, HDTL ameliorated CI injury as evidenced by decreases in neurological deficit scores and cerebral infarction. HDTL also promoted autophagy and reduced apoptosis in vivo through both the AMPK/mTOR and IKK/IκB/NF-κB pathways. ConclusionHDTL exhibits neuroprotective effects through regulating the AMPK/mTOR and IKK/IκB/NF-κB pathways. These findings suggest that HDTL is a promising therapeutic candidate for CI treatment.

LncRNA MALAT1 facilitates Parkinson's disease progression by increasing SOCS3 promoter methylation.

Long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been shown to be involved in Parkinson's disease (PD) progression, but its mechanism needs to be further explored. Mice were injected with 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to induce PD mice models, and BV2 cells were treated with lipopolysaccharides (LPS) to mimic PD cell models. MALAT1 expression and suppressor of cytokine signaling 3 (SOCS3) protein level were examined using quantitative real-time PCR and western blot, respectively. Cell functions were tested by cell counting kit 8 assay and flow cytometry. The interaction between MALAT1 and SOCS3 was confirmed using RNA pull-down and RIP assays. MALAT1 was upregulated in MPTP-induced PD mice and LPS-induced BV2 cells. Silencing of MALAT1 increased viability, while inhibited apoptosis and inflammation in LPS-induced BV2 cells. Besides, MALAT1 enhanced the SOCS3 promoter methylation to decrease its expression by recruiting DNMT1, DNMT3A and DNMT3B. Furthermore, SOCS3 knockdown eliminated sh-MALAT1-mediated the inhibition effect on LPS-induced BV2 cell injury. In vivo, MALAT1 silencing ameliorated neurological impairment and neuroinflammation in MPTP-induced PD mice. Our data revealed that MALAT1 worsened PD processes via inhibiting SOCS3 expression by increasing its promoter methylation.

The Anti-Inflammatory Activities of Benzylideneacetophenone Derivatives in LPS Stimulated BV2 Microglia Cells and Mice.

A previously reported study highlighted the neuroprotective potential of the novel benzylideneacetophenone derivative, JC3, in mice. In pursuit of compounds with even more robust neuroprotective and anti-inflammatory properties compared to JC3, we synthesized substituted 1,3-diphenyl-2-propen-1-ones based on chalcones. Molecular modeling studies aimed at discerning the chemical structural features conducive to heightened biological activity revealed that JCII-8,10,11 exhibited the widest HOMOLUMO gap within this category, indicating facile electron and radical transfer between HOMO and LUMO in model assessments. From the pool of synthesized compounds, JCII-8,10,11 were selected for the present investigation. The biological assays involving JCII-8,10,11 demonstrated their concentration-dependent suppression of iNOS and COX-2 protein levels, alongside various cytokine mRNA expressions in LPS-induced murine microglial BV2 cells. Furthermore, western blot analyses were conducted to investigate the MAPK pathways and NF-κB/p65 nuclear translocation. These evaluations conclusively confirmed the inflammatory inhibition effects in both in vitro and in vivo inflammation models. These findings establish JCII-8,10,11 as potent anti-inflammatory agents, hindering inflammatory mediators and impeding NF-κB/p65 nuclear translocation via JNK and ERK MAPK phosphorylation in BV2 cells. The study positions them as potential therapeutics for inflammation-related conditions. Additionally, JCII-11 exhibited greater activity compared to other tested JCII compounds.

Pterosin B improves cognitive dysfunction by promoting microglia M1/M2 polarization through inhibiting Klf5/Parp14 pathway

BackgroundPterosin B (PB) exhibits strong neuroprotective effects in vitro, but its therapeutic effect and underlying mechanism on Alzheimer's disease (AD) remain elusive. PurposeThis study aimed to investigate the anti-AD effect and mechanism of PB. Study designThe therapeutic effect and mechanism of PB were investigated in APP/PS1 mice and lipopolysaccharide (LPS)-induced BV-2 cells. MethodsAfter 8 weeks of oral administration of PB or donepezil, the cognitive function was assessed using behavioral tests. Pathological damage was evaluated using histological analysis and immunohistochemical staining. Flow cytometry was applied to detect M1/M2 polarization. The expression levels of glycolysis- and oxidative phosphorylation-related proteins as well as enzyme activities were determined using Western blot and biochemical kits, respectively. The levels of inflammatory cytokines and Kruppel-like factor 5 (Klf5) were measured using enzyme-linked immunosorbent assay. AD biomarkers in serum were analyzed using single-molecular array. RNA sequencing identified the downstream molecules of Klf5, and interaction was evaluated using dual-luciferase reporter assay. ResultsOur findings demonstrated that PB effectively ameliorated cognitive impairment and reduced pathological damage in APP/PS1 mice. Furthermore, PB facilitated the transition of the phenotype of LPS-induced BV-2 cells from M1 to M2 by modulating metabolic reprogramming. Additionally, Klf5 had high expression levels in the serum of patients with AD, which strongly correlated with cognitive performance and AD biomarkers. PB downregulated Klf5 expression both in vitro and in vivo. Subsequently, poly-ADP ribosyl polymerase 14 (Parp14) was identified as a downstream molecule of Klf5 involved in regulating metabolic reprogramming, and PB regulated microglia M1/M2 polarization by inhibiting the Klf5/Parp14 pathway. ConclusionThe findings suggested that PB ameliorated cognitive dysfunction in AD by modulating microglia M1/M2 polarization via inhibiting Klf5/Parp14 pathway.

Effects of Leaf Extracts from Genetic Resource of Capsicum spp. on Neuroprotection and Anti-Neuroinflammation in HT22 and in BV2 Cells.

To develop functional varieties of Capsicum spp. leaves, 40 genetic resources were collected and extracted with 30% aqueous-fermented ethanol. We investigated the protective effects of extracts from 40 genetic resources of Capsicum spp. on glutamate-induced HT22 and LPS-induced BV2 cells. The results showed that the five extracts exhibited cell-protective activities. We also investigated the anti-inflammatory effects of these five extracts on LPS-induced BV2 cell neuroinflammation and found that 23OM18 exhibited superior anti-inflammatory effects. We further investigated the protective activity and anti-inflammatory mechanisms of 23OM18 in these two cell models. In addition, the profiles of 16 metabolites were compared between the representative accessions and among the five genetic resources using ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS). The results showed that 23OM18 protected HT22 cells by inhibiting reactive oxygen species generation and regulating the MAPK-JNK signaling pathway, thereby reducing LPS-induced BV2 cell neuroinflammation by regulating the NF-κB and MAPK signaling pathways. Based on these results, 23OM18 has the potential to be developed as a functional food for the treatment of neurodegenerative diseases.

Phytochemical constituents from root barks of Eleutherococcus henryi Oliv. and their anti-neuroinflammatory effect

Ethnopharmacological relevanceThe cortex of Eleutherococcus henryi (EH, Araliaceae), also known as "Wu-Jia-Pi", is known for its effects such as dispelling wind and dampness, calming the mind and enhancing intelligence, removing heat and toxin, strengthening muscles and bones, and nourishing the liver and kidneys. Throughout Chinese history and tradition, it has been used for conditions like amnesia, mental fatigue, arthritis, hepatitis, and rheumatism. However, research evaluating its neuroprotective effects and pharmacological properties remains scarce. Aim of the studyThe goal is to explore the anti-neuroinflammatory properties of EH in vitro and to discover precisely the bioactive natural products within the medicinal plant that are relevant to its traditional usage. Materials and methodsUtilizing chromatographic techniques, a phytochemical exploration was conducted. The phytochemical structures of the natural products were then elucidated through an analysis involving comprehensive spectra and a comparison with relevant data from published studies. Network pharmacology combined with molecular dynamics simulations (MDs) and docking were applied to forecast potential anti-neuroinflammatory targets of active compounds. In vitro, the anti-neuroinflammatory efficacy was evaluated via the suppression of inflammatory mediators activated by lipopolysaccharide (LPS) in BV2 microglia. ResultsThe methanol extract of E.henryi (EHME) restrained the NO release in LPS-activated BV2 microglia, demonstrating anti-neuroinflammatory activity. Subsequently, chemical composition analysis revealed the separation and elucidation of 31 secondary metabolites, comprising 7 new compounds (1–7) and 1 new natural product (8). Based on LPS-induced BV2 cell in vitro activity tests, compounds 4–17, 19, 20, 22, 23, 26, 29 and 31 were found to exhibit potential anti-neuroinflammatory activity, with compound 6 showing the highest efficacy. Furthermore, employing network pharmacology in conjunction with both molecular docking and MDs, potential anti-neuroinflammatory targets of compound 6 were predicted to include TLR4, Src, MAPK, and NF-κB. Finally, validation through in vitro experiments confirmed that the anti-neuroinflammatory mechanism of compound 6 is associated with the TLR4/Src/MAPK p38/NF-κB p65 signaling pathways. ConclusionsThe study affirmed the traditional efficacy of E. henryi and unveiled novel lignans as potent agents against neuroinflammation.

Flavaglines with anti-neuroinflammatory activity from Aglaia edulis (Roxb.) Wall. and structure revision of related flavaglines

Eight cyclopenta[b]benzofurans (1, 2, 4, and 5-9) and eight cyclopenta[bc]benzopyrans (3, 10-16), including a revised (4) and three undescribed compounds (1-3), were isolated from the twigs and leaves of Aglaia edulis (Roxb.) Wall. Their structures were determined by a combination of spectral analysis in conjunction with NMR and ECD calculations. Moreover, based on the findings from 13C NMR calculations and DP4+ statistical analysis, an empirical guideline was established to differentiate the structures of cyclopenta[bc]benzopyrans and cyclopenta[b]benzofurans by aggregating chemical shift data from known compounds. This guideline facilitated the proposal of structural revisions for three previously reported analogs (R-1, R-2, R-3). Biological assay indicated that cyclopenta[b]benzofuran flavalines (2, and 4-8) could significantly inhibit NO production in LPS-induced BV-2 cells with IC50 values from 0.002 to 0.05 μM.

Lignans of Schisandra chinensis (Turcz.) Baill inhibits Parkinson's disease progression through mediated neuroinflammation-TRPV1 expression in microglia

BackgroundSchisandra chinensis (Turcz.) Baill (S. chinensis), a member of the Magnoliaceae family, is renowned for its distinctive medicinal attributes and is commonly employed in the treatment of disorders affecting the CNS. PurposeThe potential therapeutic effects of a lignan-enriched extract derived from Schisandra chinensis (Turcz.) Baill (LSC) on PD is assessed, which focuses on its mechanisms of action in addressing neuroinflammation. MethodsThe LSC has been obtained by purifying the ethyl alcohol extract of S. chinensis. The Orbitrap-MS method has been employed to analyze the chemical composition of the LSC. In LPS-induced BV2 cells, LSC-induced changes in M1/M2 type inflammatory cytokines have been examined using the Griess reaction, Elisa, JC-1, flow cytometry, IF, and WB methods. A model of PD has been established by treatment of MPTP in C57BL/6 mice. The effect of LSC on behavioral changes, inflammatory factor levels, expression of TH and IBA-1, and production of autophagy in the midbrain has been investigated by TEM, immunohistochemistry, Elisa, and WB. ResultsLSC has relieved sports injuries and pathological damage, and targeted the TRPV1-AMPK-NLRP3 signaling pathway, which affected neuroinflammation and autophagy in vivo. Furthermore, in vitro investigations demonstrated that LSC has activated M1/M2 transformation, its related inflammatory factors, and protein expressions of the NLRP3-Caspase1 signaling pathway in LPS-BV2 cells. The research notably demonstrated that the LSC promoted autophagy and suppressed inflammation through targeting TRPV1. ConclusionIn the investigation, LSC focused on TRPV1 and controlled neuroinflammation-autophagy by regulating AMPK-NLRP3, which has been proven for the first time. The study has presented molecular data supporting the use of LSC in treating PD and offers references for developing drugs. Remarkably, LSC has the potential to be utilized as a therapeutic or health medication that could significantly decrease PD.

Nrf2 Deficiency Exacerbates Parkinson's Disease by Aggravating NLRP3 Inflammasome Activation in MPTP-Induced Mouse Models and LPS-Induced BV2 Cells.

Parkinson's disease (PD) is a movement disorder characterized by the progressive loss of dopamine neurons. Microglia-mediated neuroinflammation drives disease progression and becomes a critical factor in neuronal degeneration. Recent studies have found that nuclear factor-erythroid 2-related-2 (Nrf2) expression levels are reduced during aging and neurodegenerative diseases, but its regulatory mechanism on microglia-induced neuroinflammation has not been fully elucidated. In vivo, we used the intraperitoneal injection of the neurotoxic drug neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to establish an animal model of PD and, at the same time, administered Nrf2 inhibitors ML385 and dimethyl fumarate to regulate Nrf2 protein levels. In vitro, we used si-RNA to knock out the Nrf2 gene to intervene in BV2 cells and used lipopolysaccharide (LPS) to stimulate and induce the cell model. The study found that inhibition of Nrf2 expression aggravated the motor defects of PD mice, accompanied by a significant loss of dopaminergic neurons in the substantia nigra and striatum of the brain. In addition, after inhibition of Nrf2, the malondialdehyde (MDA) level in the substantia nigra of the midbrain of mice increased, and the levels of superoxide dismutase (SOD) and heme oxygenase-1 (HO-1) decreased, accompanied by the proliferation of microglia and astrocytes. In addition, the activation of the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome, the assembly of apoptosis-associated speck-like protein containing a CARD (ASC) protein in microglia, and the release of downstream inflammatory factors caspase-1 and interleukin (IL)-1β, were aggravated. At the cellular level, it was found that knocking out the expression of Nrf2 would aggravate the activation of NLRP3 inflammasomes and the assembly of ASC in LPS-induced BV2 cells. Inhibited Nrf2 activity can reduce the downstream antioxidant enzyme HO-1 and antioxidant levels, induce NLRP3 inflammasome activation and ASC protein assembly in microglia, and ultimately aggravate PD inflammatory response and dopamine neuron degeneration.

Systematic Characterization of Sesquiterpenes from Dendrobium nobile through Offline Two-Dimensional Chromatography Tandem Mass Spectrometry and Target Isolation.

Dendrobium nobile is a species of the genus Dendrobium that can be used as both a medicinal herb and healthy food. The sesquiterpenes in D. nobile have attracted extensive attention in recent years. In this study, Amide × RP offline two-dimensional chromatography separation tandem high-resolution mass spectrometry combined with GNPS (Global Natural Product Social Molecular Networking) was developed for the characterization of sesquiterpenes in D. nobile. After first-dimensional amide separation, the 70% ethanol extract of D. nobile was divided into 40 fractions, which were analyzed by second-dimensional reverse-phase system separation and LTQ-Orbitrap detection. The raw data was imported into the GNPS, resulting in the efficient clustering of similar substances. Finally, 594 sesquiterpene compounds were characterized, and 25 compounds were isolated based on molecular network analysis, including six new compounds. In vitro bioassays, the isolated compounds decreased NO production in the LPS-induced microglial BV-2 cell model and the content of MDA in PC12 cells, demonstrating neuroprotective activity. These findings unraveled the underlying material and provided valuable insights into the quality control of D. nobile.

Jiao-tai-wan and its effective component-coptisine alleviate cognitive impairment in db/db mice through the JAK2/STAT3 signaling pathway

BackgroundCognitive impairment (CI) is now well-accepted as a complication and comorbidity of diabetes mellitus (DM), becoming a serious medical and social problem. Jiao-tai-wan (JTW), one of noted traditional Chinese medicine (TCM), showed dual therapeutic effects on DM and CI. Nevertheless, the potential mechanism is unclear. PurposeThis study sought to investigate the mechanism how JTW protected against DM and CI and screen the active component in JTW. MethodsDb/db mice were used as mouse models. Mice were treated by gavage with 0.9 % saline (0.1 mL/10g/d), low dose of JTW (2.4 g/kg/d) or high dose of JTW (4.8 g/kg/d) for 8 weeks separately. To access the effects of JTW, the levels of OGTT, HOMA-IR, blood lipids, inflammatory cytokines in serum and hippocampus were measured, behavioral tests were conducted, and histopathological changes were observed. The mechanism exploration was performed via network pharmacology, RT-qPCR, western blot, and immunofluorescence staining (IF). The impact and mechanism of coptisine in vitro were investigated using BV2 cells induced by LPS as cellular models. In vitro experiments were conducted in two parts. The first part comprised four groups: Control group, LPS group, LPS+LCOP group and LPS+HCOP group. The second part consisted of four groups: Control group, LPS group, LPS+HCOP group, and LPS+ Fed group. The western blot and RT-qPCR methods were used to examine the changes in biomarkers of the JAK2/STAT3 signaling pathways in BV2 cells. ResultsThe results demonstrated that JTW could improve OGTT and HOMA-IR, reduce the serum levels of LDL-C, HDL-C, TG, and TC, restore neuronal dysfunction and synaptic plasticity, and decrease the deposition of Aβ in the hippocampus. The findings from ELISA, IF, and RT-qPCR revealed that JTW could alleviate microglial activation and inflammatory status in vivo and coptisine could play the same role in vitro. Moreover, the changes of the JAK2/STAT3 signaling pathway in LPS-induced BV2 cells or hippocampus of db/db mice were distinctly reversed by coptisine or JTW, respectively. ConclusionOur study suggested that JTW and its effective component coptisine could alleviate diabetes mellitus-related cognitive impairment, closely linked to the JAK2/STAT3 signaling pathway.

MiR-223 accelerates lipid droplets clearance in microglia following spinal cord injury by upregulating ABCA1

BackgroundSpinal cord injury (SCI) is characterized by extensive demyelination and inflammatory responses. Facilitating the clearance of lipid droplets (LDs) within microglia contributes to creating a microenvironment that favors neural recovery and provides essential materials for subsequent remyelination. Therefore, investigating MicroRNAs (miRNAs) that regulate lipid homeostasis after SCI and elucidating their potential mechanisms in promoting LDs clearance in microglia have become focal points of SCI research.MethodsWe established a subacute C5 hemicontusion SCI model in mice and performed transcriptomic sequencing on the injury epicenter to identify differentially expressed genes and associated pathways. Confocal imaging was employed to observe LDs accumulation. Multi-omics analyses were conducted to identify differentially expressed mRNA and miRNA post-SCI. Pathway enrichment analysis and protein-protein interaction network construction were performed using bioinformatics methods, revealing miR-223-Abca1 as a crucial miRNA-mRNA pair in lipid metabolism regulation. BV2 microglia cell lines overexpressing miR-223 were engineered, and immunofluorescence staining, western blot, and other techniques were employed to assess LDs accumulation, relevant targets, and inflammatory factor expression, confirming its role in regulating lipid homeostasis in microglia.ResultsHistopathological results of our hemicontusion SCI model confirmed LDs aggregation at the injury epicenter, predominantly within microglia. Our transcriptomic analysis during the subacute phase of SCI in mice implicated ATP-binding cassette transporter A1 (Abca1) as a pivotal gene in lipid homeostasis, cholesterol efflux and microglial activation. Integrative mRNA-miRNA multi-omics analysis highlighted the crucial role of miR-223 in the neuroinflammation process following SCI, potentially through the regulation of lipid metabolism via Abca1. In vitro experiments using BV2 cells overexpressing miR-223 demonstrated that elevated levels of miR-223 enhance ABCA1 expression in myelin debris and LPS-induced BV2 cells. This promotes myelin debris degradation and LDs clearance, and induces a shift toward an anti-inflammatory M2 phenotype.ConclusionsIn summary, our study unveils the critical regulatory role of miR-223 in lipid homeostasis following SCI. The mechanism by which this occurs involves the upregulation of ABCA1 expression, which facilitates LDs clearance and myelin debris degradation, consequently alleviating the lipid burden, and inhibiting inflammatory polarization of microglia. These findings suggest that strategies to enhance miR-223 expression and target ABCA1, thereby augmenting LDs clearance, may emerge as appealing new clinical targets for SCI treatment.

Brozopine ameliorates cognitive impairment via upregulating Nrf2, antioxidation and anti-inflammation activities.

Oxidative stress and inflammation are crucial factors contributing to the occurrence and development of vascular dementia (VD). In a previous study, we demonstrated that brozopine (BZP) is an anti-ischemic drug. In this study, a model of VD in rats with modified permanent bilateral common carotid artery occlusion (2-VO) was established in vivo, a model of cellular excitotoxicity/oxidative stress was established via L-glutamate-induced PC12 cell injury, a model of neuroinflammation was established in LPS-induced BV2 cells in vitro, and the ameliorative effect of BZP on cognitive impairment was assessed. BZP treatment improved memory deficit in VD rats through inhibiting Ca2+overload and the levels of oxidative stress, ferroptosis, and inflammatory markers (IL-1β, IL-6, and COX-2) in different brain regions. Additionally, we found that the levels of inflammatory markers in the plasma were also reduced in the VD rats. BZP was further found to have antioxidative stress, antiferroptosis (ferroptosis markers: GPX4, P53, and ACSL4), and antineuroinflammatory effects in PC12 and BV2 cells. Its mechanisms of action were found to be related to the activation of the Nrf2/TLR4/NF-κB pathway; the protective effect of BZP was partially inhibited after using Nrf2-specific inhibitors. Thus, BZP has therapeutic properties for the potential mitigation of cognitive impairment.

Pongamol Alleviates Neuroinflammation and Promotes Autophagy in Alzheimer's Disease by Regulating the Akt/mTOR Signaling Pathway.

Alzheimer's disease (AD), one of the neurodegenerative disorders, is highly correlated with the abnormal hyperphosphorylation of Tau and aggregation of β-amyloid (Aβ). Oxidative stress, neuroinflammation, and abnormal autophagy are key drivers of AD and how they contribute to neuropathology remains largely unknown. The flavonoid compound pongamol is reported to possess a variety of pharmacological activities, such as antioxidant, antibacterial, and anti-inflammatory. This study investigated the neuroprotective effect and its mechanisms of pongamol in lipopolysaccharide (LPS)-induced BV2 cells, d-galactose/sodium nitrite/aluminum chloride (d-gal/NaNO2/AlCl3)-induced AD mice, and Caenorhabditis elegans models. Our research revealed that pongamol reduced the release of inflammatory factors IL-1β, TNF-α, COX-2, and iNOS in LPS-induced BV2 cells. Pongamol also protected neurons and significantly restored memory function, inhibited Tau phosphorylation, downregulated Aβ aggregation, and increased oxidoreductase activity in the hippocampus of AD mice. In addition, pongamol reversed the nuclear transfer of NF-κB and increased the levels of Beclin 1 and LC3 II/LC3 I. Most importantly, the anti-inflammatory and promoter autophagy effects of pongamol may be related to the regulation of the Akt/mTOR signaling pathway. In summary, these results showed that pongamol has a potential neuroprotective effect, which greatly enriched the research on the pharmacological activity of pongamol for improving AD.

Modulation of choline and lactate metabolism by basic fibroblast growth factor mitigates neuroinflammation in type 2 diabetes: Insights from 1H-NMR metabolomics analysis

BackgroundType 2 diabetes (T2D), a chronic metabolic disease, occurs brain dysfunction accompanied with neuroinflammation and metabolic disorders. The neuroprotective effects of the basic fibroblast growth factor (bFGF) have been well studied. However, the mechanism underlying the anti-inflammatory effects of bFGF remains elusive. MethodsIn this study, db/db mice were employed as an in vivo model, while high glucose (HG)-induced SY5Y cells and LPS-induced BV2 cells were used as in vitro models. Liposomal transfection of MyD88 DNA plasmid was used for MyD88-NF-κB pathway studies. And western blotting, flow cytometry and qPCR were employed. 1H-NMR metabolomics was used to find out metabolic changes. ResultsbFGF mitigated neuroinflammatory and metabolic disorders by inhibiting cortical inflammatory factor secretion and microglia hyperactivation in the cortex of db/db mice. Also, bFGF was observed to inhibit the MyD88-NF-κB pathway in high glucose (HG)-induced SY5Y cells and LPS-induced BV2 cells in in vitro experiments. Moreover, the 1H-NMR metabolomics results showed that discernible disparities between the cortical metabolic profiles of bFGF-treated db/db mice and their untreated counterparts. Notably, excessive lactate and choline deficiency attenuated the anti-inflammatory protective effect of bFGF in SY5Y cells. ConclusionbFGF ameliorates neuroinflammation in db/db mice by inhibiting the MyD88-NF-kB pathway. This finding expands the potential application of bFGF in the treatment of neuroinflammation-related cognitive dysfunction.

Erjingpill bionic cerebrospinal fluid alleviates LPS-induced inflammatory response in BV2 cells by inhibiting glycolysis via mTOR

Ethnopharmacological relevanceErjingpill, a well-known prescription documented in the classic Chinese medical text “Shengji Zonglu,” has been proven to have effective alleviating effects on neuroinflammation in Alzheimer's disease (AD). Although the alterations in microglial cell glycolysis are known to play a crucial role in the development of neuroinflammation, it remains unclear whether the anti-neuroinflammatory effects of Erjingpill are associated with its impact on microglial cell glycolysis. Aim of the studyThis study aims to determine whether Erjingpill exerts anti-neuroinflammatory effects by influencing microglial cell glycolysis. Materials and methodsFirstly, Erjingpill decoction was prepared into an Erjingpill bionic cerebrospinal fluid (EBCF) through a process of in vitro intestinal absorption, hepatocyte incubation, and blood-brain barrier (BBB) transcytosis. Subsequently, UPLC/Q-TOF-MS/MS technology was used to analyze the compounds in Erjingpill and EBCF. Next, an in vitro neuroinflammation model was established by LPS-induced BV2 cells. The impact of EBCF on BV2 cell proliferation activity was evaluated using the CCK-8 assay, while the NO release was assessed using the Griess assay. Additionally, mRNA levels of pro-inflammatory factors (IL-1β, IL-6, TNF-α, and COX-2), anti-inflammatory factors (IL-10, IL-4, Arg-1, and TGF-β), M1 microglial markers (iNOS, CD86), M2 microglial markers (CD36, CD206), and glycolytic enzymes (HK2, GLUT1, PKM, and LDHA) were measured using qPCR. Furthermore, protein expression of microglial activation marker Iba-1, M1 marker iNOS, and M2 marker CD206 were identified through immunofluorescence, while concentrations of pro-inflammatory cytokines IL-1β and TNF-α were measured using ELISA. Enzymatic activity of glycolytic enzymes (HK, PK, and LDH) was assessed using assay kits, and the protein levels of pro-inflammatory factors (IL-1β, iNOS, and COX-2), anti-inflammatory factors (IL-10 and Arg-1), and key glycolytic proteins GLUT1 and PI3K/AKT/mTOR were detected by Western blot. ResultsThrough the analysis of Erjingpill and EBCF, 144 compounds were identified in Erjingpill and 40 compounds were identified in EBCF. The results demonstrated that EBCF effectively inhibited the elevation of inflammatory factors and glycolysis levels in LPS-induced BV2 cells, promoted polarization of M1 microglial cells towards the M2 phenotype, and suppressed the PI3K/AKT/mTOR inflammatory pathway. Moreover, EBCF alleviated LPS-induced BV2 cell inflammatory response by modulating mTOR to inhibit glycolysis. ConclusionsEBCF exhibits significant anti-neuroinflammatory effects, likely attributed to its modulation of mTOR to inhibit microglial cell glycolysis. This study furnishes experimental evidence supporting the clinical utilization of Erjingpill for preventing and treating AD.

A comprehensive study on the regulation of Compound Zaoren Granules on cAMP/CREB signaling pathway and metabolic disorder in CUMS-PCPA induced insomnia rats

Ethnopharmacological relevanceCompound Zaoren Granules (CZG), an optimized herbal formulation based on the traditional Chinese medicine prescription Suanzaoren decoction, are designed specifically for insomnia treatment. However, the mechanisms underlying its efficacy in treating insomnia are not yet fully understood. Aim of the studyThe research investigated the mechanisms of CZG's improvement in insomnia by regulating cAMP/CREB signaling pathway and metabolic profiles. MethodsThe main components of CZG were characterized by liquid chromatography-mass spectrometry (LC-MS). Subsequently, these validated components were applied to network pharmacological analysis to predict signaling pathways associated with insomnia. We evaluated the effect of CZG on BV-2 cells in vitro. We also evaluated the behavioral indexes of CUMS combined with PCPA induced insomnia in rats. HE staining and Nissl staining were used to observe the pathological damage of hippocampus. ELISA was used to detect the levels of various neurotransmitters, orexins, HPA axis, and inflammatory factors in insomnia rats. Then we detected the expression of cAMP/CREB signaling pathway through ELISA, WB, and IHC. Finally, the metabolomics was further analyzed by using UHPLC-QTOF-MS/MS to investigate the changes in the hippocampus of insomnia rats and the possible metabolic pathways were also speculated. ResultsThe results of CZG in vitro experiments showed that CZG has protective and anti-inflammatory effects on LPS induced BV-2 cells. A total of 161 chemical components were identified in CZG. After conducting network pharmacology analysis through these confirmed components, we select the cAMP/CREB signaling pathway for further investigate. The behavioral research results on insomnia rats showed that CZG significantly prolonged sleep time, mitigated brain tissue pathological damage, and exhibited liver protective properties. CZG treats insomnia by regulating the content of various neurotransmitters, reducing levels of orexin, HPA axis, and inflammatory factors. It can also treat insomnia by upregulating the expression of the cAMP/CREB signaling pathway. Hippocampus metabolomics analysis identified 69 differential metabolites associated with insomnia. The metabolic pathways related to these differential metabolites have also been predicted. ConclusionThese results indicate that CZG can significantly prolong sleep time. CZG is used to treat insomnia by regulating various neurotransmitters, HPA axis, inflammatory factors, cAMP/CREB signaling pathways, and metabolic disorders.

Temporin-GHaR Peptide Alleviates LPS-Induced Cognitive Impairment and Microglial Activation by Modulating Endoplasmic Reticulum Stress.

Neuroinflammation is considered an important factor that leads to cognitive impairment. Microglia play a crucial role in neuroinflammation, which leads to cognitive impairment. This study aimed at determining whether temporin-GHaR peptide (GHaR) could improve cognitive function and at uncovering the underlying mechanisms. We found that GHaR treatment alleviated LPS-induced cognitive impairment and inhibited activation of microglia in LPS-induced mice. Furthermore, GHaR inhibited activation of endoplasmic reticulum stress (ERS) and the NF-κB signaling pathway in LPS-induced mice. In vitro, GHaR inhibited M1 polarization of BV2 cells and suppressed TNF-α and IL-6 secretion. Additionally, GHaR neuronal cell viability and apoptosis were induced by LPS-activated microglia-conditioned medium. Moreover, in LPS-induced BV2 cells, GHaR inhibited activation of ERS and the NF-κB signaling pathway. In summary, GHaR improved LPS-induced cognitive and attenuated inflammatory responses via microglial activation reversal. In conclusion, the neuroprotective effects of GHaR were mediated via the ERS signaling pathway.

SLC15A3 is transcriptionally regulated by HIF1α and p65 to worsen neuroinflammation in experimental ischemic stroke.

Systemic inflammatory stimulus is a risk factor for the incidence of ischemic stroke and contributes to poorer clinical outcomes. Solute carrier 15A3 (SLC15A3) is a peptide/histidine transporter that is implicated in regulating inflammatory responses. However, whether SLC15A3 affects the progression of ischemic stroke associated with systemic inflammation is unclear. The transient middle cerebral artery occlusion (tMCAO) mice with LPS administration (LPS/tMCAO) were prepared as an in vivo model, and LPS-induced BV2 cells under oxygen-glucose deprivation (OGD) exposure were utilized as an in vitro model. We found that SLC15A3 was highly expressed in the ischemic penumbra of LPS/tMCAO mice, and its inhibition reduced infarct area, attenuated neurological deficit, recovered motor function, and mitigated apoptotic neurons. Knockdown of SLC15A3 suppressed the proinflammatory M1-type markers and promoted the levels of M2-associated genes. The in vitro results confirmed that SLC15A3 overexpression promoted microglia polarizing towards M1 subtypes, while SLC15A3 inhibition exerted an opposite effect. In addition, we demonstrated that the p65 signaling pathway and HIF1α were activated by LPS/OGD. Luciferase reporter assay showed that inhibiting p65 using its specific inhibitor BAY 11-7082 or silencing HIF1α using siRNAs reduced the transcriptional activity of SLC15A3in LPS/OGD-induced BV2 cells. Results in NIH 3T3 cells also confirmed that p65 and HIF1α directly bound to the SLC15A3 promoter to activate SLC15A3 transcription. In conclusion, this work shows that SLC15A3, transcriptionally activated by p65 and HIF1α, contributes to poor outcomes in ischemic stroke associated with systemic inflammation by promoting microglial cells polarizing towards M1 types.

Characteristic cembrane-type diterpenoids with nitric oxide inhibitory activity from the gum resin of Boswellia carterii Birdw

Five new characteristic cembrane-type diterpenoids (olibacartiols A–E, 1–5) were acquired from the gum resin of Boswellia carterii. The structures of these diterpenoids were characterized by detailed spectroscopic analysis, and compounds 1–3 were unambiguously confirmed by single-crystal X-ray diffraction experiments. The anti-inflammatory activities of the isolated compounds were evaluated using LPS-induced BV2 cell model and compounds 2–5 showed moderate NO inhibitory effects with IC50 values of 8.84 ± 1.02, 9.82 ± 1.95, 9.75 ± 2.24, and 7.39 ± 1.24 μM, respectively.