N-Cinnamoylpyrrole-derived alkaloids from the genus Piper as promising agents for ischemic stroke by targeting eEF1A1.

BACKGROUND: Ischemic stroke (IS) is a serious cerebrovascular disease characterized by significantly elevated mortality and disability rates, and the treatments available for this disease are limited. Neuroinflammation and oxidative stress are deemed the major causes of cerebral ischemic injury. N-Cinnamoylpyrrole alkaloids form a small group of natural products from the genus Piper and have not been extensively analyzed pharmacologically. Thus, identifying the effect and mechanism of N-cinnamoylpyrrole-derived alkaloids on IS is worthwhile. PURPOSE: The present research aimed to explore the antineuroinflammatory and antioxidative stress effects of N-cinnamoylpyrrole-derived alkaloids isolated from the genus Piper and to explain the effects and mechanism on IS. METHODS: N-cinnamoylpyrrole-derived alkaloids were isolated from Piper boehmeriaefolium var. tonkinense and Piper sarmentosum and identified by various chromatographic methods. Lipopolysaccharide (LPS)-induced BV-2 microglia and a mouse model intracerebroventricularly injected with LPS were used to evaluate the antineuroinflammatory and antioxidative stress effects. Oxygen‒glucose deprivation/reperfusion (OGD/R) and transient middle cerebral artery occlusion (tMCAO) models were used to evaluate the effect of PB-1 on IS. To elucidate the fundamental mechanism, the functional target of PB-1 was identified by affinity-based protein profiling (ABPP) strategy and verified by cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS), and circular dichroism (CD) analyses. The effect of PB-1 on the NF-κB and NRF2 signaling pathways was subsequently evaluated via western blotting and immunofluorescence staining. RESULTS: The results showed that N-cinnamoylpyrrole-derived alkaloids significantly affected neuroinflammation and oxidative stress. The representative compound, PB-1 not only inhibited neuroinflammation and oxidative stress induced by LPS or OGD/R insult, but also alleviated cerebral ischemic injury induced by tMCAO. Further molecular mechanism research found that PB-1 promoted antineuroinflammatory and antioxidative stress activities via the NF-κB and NRF2 signaling pathways by targeting eEF1A1. CONCLUSION: Our research initially unveiled that the therapeutic impact of PB-1 on cerebral ischemic injury might rely on its ability to target eEF1A1, leading to antineuroinflammatory and antioxidative stress effects. The novel discovery highlights eEF1A1 as a potential target for IS treatment and shows that PB-1, as a lead compound that targets eEF1A1, may be a promising therapeutic agent for IS.

Americanin B inhibits pyroptosis in lipopolysaccharide-induced septic encephalopathy mice through targeting NLRP3 protein.

BACKGROUND: Sepsis is considered as a severe illness due to its high mortality. Sepsis can cause septic encephalopathy, thus leading to brain injury, behavioral and cognitive dysfunction. Pyroptosis is a type of regulated cell death (RCD) and takes a crucial part in occurrence and development of sepsis. Americanin B (AMEB) is a lignan compounds, which is extracted from Vernicia fordii. In our previous study, AMEB could inhibit microglial activation in inflammatory cell model. However, the function of AMEB in septic encephalopathy mice is uncertain. It would be worthwhile to ascertain the role and mechanism of AMEB in sepsis. PURPOSE: Current study designs to certify the relationship between pyroptosis and septic encephalopathy, and investigate whether AMEB can restrain NOD-like receptor pyrin domain-containing 3 (NLRP3) inflammasome activation and restrict pyroptosis by targeting NLRP3 in septic mice model. STUDY DESIGN: C57BL/6 mice were utilized to perform sepsis model in vivo experiments. BV-2 cell lines were used for in vitro experiments. METHODS: In vivo sepsis model was established by lipopolysaccharide (LPS) intraperitoneal injection in male C57BL/6 J mice and in vitro model was exposed by LPS plus ATP in BV-2 cells. The survival rate was monitored on the corresponding days. NLRP3, apoptosis associated Speck-like protein (ASC), caspase-1, GasderminD (GSDMD), interleukin-1ß (IL-1ß) and interleukin-18 (IL-18) level were detected by western blotting and immunofluorescence analysis. Molecular docking, cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS) experiments, RNAi transfection and quantitative real-time PCR were applied to confirm the potential target of AMEB. RESULTS: The results suggested that AMEB could rise survival percentage and lighten brain injury in LPS-induced sepsis mice. In addition, AMEB could inhibit pyroptosis and the activiation of NLRP3 inflammasome. The inhibiting function of AMEB on the activiation of NLRP3 inflammasome is weakened following si-NLRP3 transfection. Moreover, AMEB exerted anti-pyroptosis effect via targeting NLRP3 protein. CONCLUSIONS: Our findings first indicate NLRP3 is an effective druggable target for septic encephalopathy related brain injury, and also provide a candidate-AMEB for the treatment of septic encephalopathy. These emerging findings on AMEB in models of sepsis suggest an innovative approach that may be beneficial in the prevention of septic encephalopathy.

Anti-inflammatory effects of quinolinyl analog of resveratrol targeting TLR4 in MCAO/R ischemic stroke rat model.

BACKGROUND: Among adults, stroke is the main causes of mortality and permanent disability. Neuroinflammation is one of the main causes of stoke-mediated neuronal death. Our previous study revealed that (E)-5-(2-(Quinolin-4-yl) vinyl) benzene-1, 3-diol (RV01), a quinolinyl analog of resveratrol, inhibits microglia-induced neuroinflammation and safeguards neurons from inflammatory harm. The preventive role of RV01 in ischemic stroke and its underlying cellular mechanisms and molecular targets remain poorly understood. PURPOSE: To investigate whether RV01 alleviates ischemia-reperfusion (I/R) injury by inhibiting microglia-mediated neuroinflammation and determine the potential molecular mechanisms and targets by which RV01 inhibits the I/R-mediated microglia activation. METHODS: Rat middle cerebral artery occlusion and reperfusion (MCAO/R) and BV-2 or primary microglial cells oxygen-glucose deprivation and reperfusion (OGD/R) models were established. The neurological behavior scores, 2, 3, 5-triphenyl tetrazolium chloride staining and immunofluorescence were used to detect the neuroprotective effect of RV01 in the MCAO/R rats. In addition, the mRNA expression levels of IL-6, TNF-α, and IL-1ß were detected to reveal the antineuroinflammatory effect of RV01. Moreover, a western blot assay was performed to explore the protein expression changes in NF-κB-mediated neuroinflammation. Finally, we identified TLR4 as an RV01 target through molecular docking, drug sensitivity target stability analysis, cellular thermal shift analysis, and surface plasmon resonance techniques. RESULTS: RV01 reduced the infarct volume and neurological deficits, increased the rotarod duration, and decreased the number of rightward deflections in the MCAO/R rats. RV01 inhibited the NF-κB signaling pathway in vitro and in vivo, as demonstrated by the reduction in the transcription factor p65-mediated expression of several inflammatory factors including IL-6, TNF-α, and IL-1ß. Further studies showed that its protective effect was associated with targeting the TLR4 protein. Notably, the anti-inflammatory effect of RV01 was markedly reinforced by the TLR4 knockdown, but inhibited by the overexpression of TLR4. Results revealed that the conditioned medium derived from the RV01-treated BV-2 cells significantly decreased the OGD/R-mediated neuronal damage. CONCLUSION: Our results are the first to reveal the protective effects of RV01 on cerebral ischemia, depending on its inhibitory effect on the NF-κB pathway by targeting TLR4. RV01 could be a potential protective agent in ischemic stroke treatment.

Kellerin alleviates cerebral ischemic injury by inhibiting ferroptosis via targeting Akt-mediated transcriptional activation of Nrf2.

BACKGROUND: Ischemic stroke (IS) is characterized as a detrimental cerebrovascular disease with high mortality and disability. Ferroptosis is a novel mechanism involved in neuronal death. There is a close connection between IS and ferroptosis, and inhibiting ferroptosis may provide an effective strategy for treating IS. Our previous investigations have discovered that kellerin, the active compound of Ferula sinkiangensis K. M. Shen, possesses the capability to shield against cerebral ischemia injury. PURPOSE: Our objective is to clarify the relationship between the neuroprotective properties of kellerin against IS and its ability to modulate ferroptosis, and investigate the underlying regulatory pathway. STUDY DESIGN: We investigated the impact and mechanism of kellerin in C57BL/6 mice underwent middle cerebral artery occlusion/reperfusion (MCAO/R) as well as SH-SY5Y cells exposed to oxygen-glucose deprivation/ re-oxygenation (OGD/R). METHODS: The roles of kellerin on neurological severity, cerebral infarction and edema were investigated in vivo. The regulatory impacts of kellerin on ferroptosis, mitochondrial damage and Akt/Nrf2 pathway were explored. Molecular docking combined with drug affinity responsive target stability assay (DARTS) and cellular thermal shift assay (CETSA) were performed to analyze the potential target proteins for kellerin. RESULTS: Kellerin protected against IS and inhibited ferroptosis in vivo. Meanwhile, kellerin improved the neuronal damage caused by OGD/R and suppressed ferroptosis by inhibiting the production of mitochondrial ROS in vitro. Further we found that kellerin directly interacted with Akt and enhanced its phosphorylation, leading to the increase of Nrf2 nuclear translocation and its downstream antioxidant genes expression. Moreover, kellerin's inhibitory effect on ferroptosis and mitochondrial ROS release was eliminated by inhibiting Akt/Nrf2 pathway. CONCLUSIONS: Our study firstly demonstrates that the neuroprotective properties of kellerin against IS are related to suppressing ferroptosis through inhibiting the production of mitochondrial ROS, in which its modulation on Akt-mediated transcriptional activation of Nrf2 plays an important role. This finding shed light on the potential mechanism that kellerin exerts therapeutic effects in IS.

Integrated network pharmacology and experimental validation-based approach to reveal the underlying mechanisms and key material basis of Jinhua Qinggan granules against acute lung injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Jinhua Qinggan granules (JHQG), the traditional Chinese formula come into the market in 2016, has been proved clinically effective against coronavirus disease. Acute lung injury (ALI) is a major complication of respiratory infection such as coronavirus and influenza virus, with a high clinical fatality rate. Macrophage activation-induced inflammatory response plays a crucial role in the pathogenesis of ALI. However, the participation of inflammatory response in the efficacy of JHQG and its material basis against ALI is still unknown. AIM OF THE STUDY: The research aims to investigate the inflammatory response-involved efficacy of JHQG on ALI, explore the "ingredient-target-pathway" mechanisms, and searching for key material basis of JHQG by integrated network pharmacology and experimental validation-based approach. MATERIALS AND METHODS: Lipopolysaccharide (LPS)-induced ALI mice was established to assess the protective impact of JHQG. Network pharmacology was utilized to identify potential targets of JHQG and investigate its action mechanisms related to inflammatory response in treating ALI. The therapeutic effect and mechanism of the primary active ingredient in JHQG was verified through high performance liquid chromatography (HPLC) and a combination of wet experiments. RESULTS: JHQG remarkably alleviated lung damage in mice model via suppressing macrophage activation, and inhibiting pro-inflammatory mediator level, p-ERK and p-STAT3 expression, TLR4/NF-κB activation. Network pharmacology combined with HPLC found luteolin is the main effective component of JHQG, and it could interact with TLR4/MD2 complex, further exerting the anti-inflammatory property and the protective role against ALI. CONCLUSIONS: In summary, our finding clarified the underlying mechanisms and material basis of JHQG therapy for ALI by integrated network pharmacology and experimental validation-based strategy.

Crebanine ameliorates ischemia-reperfusion brain damage by inhibiting oxidative stress and neuroinflammation mediated by NADPH oxidase 2 in microglia.

BACKGROUND: The urgent challenge for ischemic stroke treatment is the lack of effective neuroprotectants that target multiple pathological processes. Crebanine, an isoquinoline-like alkaloid with superior pharmacological activities, presents itself as a promising candidate for neuroprotection. However, its effects and mechanisms on ischemic stroke remain unknown. METHODS: The effects of crebanine on brain damage following ischemic stroke were evaluated using the middle cerebral artery occlusion and reperfusion (MCAO/R) model. Mechanism of action was investigated using both MCAO/R rats and lipopolysaccharide (LPS)-activated BV-2 cells. RESULTS: We initially demonstrated that crebanine effectively ameliorated the neurological deficits in MCAO/R rats, while also reducing brain edema and infarction. Treatment with crebanine resulted in the up-regulation of NeuN+ fluorescence density and down-regulation of FJB+ cell count, and mitigated synaptic damage. Crebanine attenuated the hyperactivation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) by downregulating NADP+ and NADPH levels, suppressing gp91phox and p47phox expressions, and reducing p47phox membrane translocation in Iba-1+ cells. Additionally, crebanine reduced the quantity of Iba-1+ cells and protein expression. Correlation analysis has demonstrated that the inhibition of NOX2 activation in microglia is beneficial for mitigating I/R brain injuries. Moreover, crebanine exhibited significant antioxidant properties by down-regulating the expression of superoxide anion and intracellular reactive oxygen species in vivo and in vitro, and reducing lipid and DNA peroxidation. Crebanine exerted anti-inflammatory effect, as evidenced by the reduction in the expressions of nitric oxide, interleukin 1ß, tumor necrosis factor α, interleukin 6, and inducible nitric oxide synthase. The effect of crebanine was achieved through the suppression of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPK) signaling pathway. This is supported by evidence showing reduced NF-κB p65 promoter activity and nucleus translocation, as well as suppressed IκBα phosphorylation and degradation. Additionally, it inhibited the phosphorylation of ERK, JNK, and p38 MAPKs. Importantly, the anti-oxidative stress and neuroinflammation effects of crebanine were further enhanced after silencing gp91phox and p47phox. CONCLUSION: Crebanine alleviated the brain damages of MCAO/R rats by inhibiting oxidative stress and neuroinflammation mediated by NOX2 in microglia, implying crebanine might be a potential natural drug for the treatment of cerebral ischemia.

Pterostilbene participates in TLR4- mediated inflammatory response and autophagy-dependent Aß1-42 endocytosis in Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD), the most prevalent form of dementia, remains untreatable. One of the factors that contributes to its progression is microglia-mediated inflammation. Pterostilbene, a compound isolated from Chinese dragon's blood, can reduce inflammation caused by overactive microglia. However, its effects on AD transgenic animals and the possible underlying mechanism remain unknown. METHODS: We evaluated the effect of pterostilbene on learning and memory difficulties in transgenic APP/PS1 mice. We used immunofluorescence to detect microglial activation and Aß aggregation. We explored the cellular mechanism of pterostilbene by establishing LPS- stimulated BV2 cells and oAß1-42- exposed HEK 293T cells that overexpress TLR4 and/or MD2 via lentivirus. We applied flow cytometry and immunoprecipitation to examine how pterostilbene regulates TLR4 signaling. RESULTS: Pterostilbene enhanced the learning and memory abilities of APP/PS1 mice and reduced microglial activation and Aß aggregation in their hippocampus. Pterostilbene alleviated oAß1-42-induced inflammation, which required the involvement of MD2. Pterostilbene disrupted the binding between TLR4 and MD2, which may further prevent TLR4 dimerization and subsequent inflammatory response. Moreover, pterostilbene restored the impaired endocytosis of oAß1-42 through an autophagy-dependent mechanism. CONCLUSION: This is the first demonstration that pterostilbene can potentially treat AD by blocking the interaction of TLR4 and MD2, thereby suppressing TLR4-mediated inflammation.

1H NMR-guided isolation of hasubanan alkaloids from the alkaloidal extract of Stephania longa.

1H NMR-guided fractionation led to the isolation of 16 alkaloids from the alkaloidal extract of Stephania longa, including 11 new hasubanan alkaloids (1-11) and five known alkaloids (12-16). Interestingly, compounds 2 and 11 are typically considered protonated tertiary amine compounds, whereas compounds 1 and 10 are regarded as oxidized versions of the corresponding compounds. Their gross structures were determined through an extensive analysis of spectroscopic data (NMR (nuclear magnetic resonance) and HRESIMS (high resolution electrospray ionization mass spectroscopy)), and their absolute configurations were established by comparing their experimental and calculated electronic circular dichroism (ECD) spectra. The new (3) and a known (12) compounds in all isolates displayed stronger antineuroinflammatory effects (IC50 values of 1.8 and 11.1 µM, respectively) than minocycline (IC50 value of 15.5 µM) against NO production on LPS-activated BV2 cells.

Potential inhibitors of microglial activation from the roots of Wikstroemia lichiangensis W. W. Sm.

Research on natural inhibitors of microglial overactivation derived from members of the Wikstroemia genus revealed that the extract of W. lichiangensis W. W. Sm. Has a remarkable inhibitory effect on nitric oxide production in overactivated microglia. In the present study, thirty-four compounds, including five undescribed sesquiterpenoids [wiksdauctins A-B (1-2) and wikscarotins A-C (3-5)] and one undescribed lignan [wikstroeminasin A (8)], were isolated from a 95% EtOH extract of W. lichiangensis roots using bio-guided phytochemical research. The structures of the isolated compounds were elucidated using comprehensive spectroscopic analyses. Furthermore, their anti-neuroinflammatory effects were evaluated in lipopolysaccharide-stimulated BV-2 microglia. Seventeen isolated compounds exhibited stronger inhibitory effects than positive control minocycline (IC50 values of 67.08 ± 1.95 µM), with IC50 values ranging from 7.35 ± 2.51 to 64.49 ± 3.38 µM. The findings of this study imply that the isolated compounds might serve as potential therapeutic agents for neurodegenerative diseases.

Isoamericanin A improves lipopolysaccharide-induced memory impairment in mice through suppression of the nicotinamide adenine dinucleotide phosphateoxidase-dependent nuclear factor kappa B signaling pathway.

Alzheimer's disease (AD) is the most frequent cause of dementia in the elderly. Isoamericanin A (ISOA) is a natural lignan possessing great potential for AD treatment. This study investigated the efficacy of ISOA on memory impairments in the mice intrahippocampal injected with lipopolysaccharide (LPS) and the underlying mechanism. Y-maze and Morris Water Maze data suggested that ISOA (5 and 10 mg/kg) ameliorated short- and long-term memory impairments, and attenuated neuronal loss and lactate dehydrogenase activity. ISOA exerted anti-inflammatory effect demonstrating by the reduction of ionized calcium-binding adapter molecule 1 positive cells and suppression of marker protein and pro-inflammation cytokines expressions induced by LPS. ISOA suppressed the nuclear factor kappa B (NF-κB) signaling pathway by inhibiting IκBα phosphorylation and NF-κB p65 phosphorylation and nuclear translocation. ISOA inhibited superoxide and intracellular reactive oxygen species accumulation by reducing nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation, demonstrating by suppressing NADP+ and NADPH contents, gp91phox expression, and p47phox expression and membrane translocation. These effects were enhanced in combination with NADPH oxidase inhibitor apocynin. The neuroprotective effect of ISOA was further proved in the in vitro models. Overall, our data revealed a novel pharmacological activity of ISOA: ameliorating memory impairment in AD via inhibiting neuroinflammation.

YY1 complex in M2 macrophage promotes prostate cancer progression by upregulating IL-6.

BACKGROUND: Tumor-associated macrophages are mainly polarized into the M2 phenotype, remodeling the tumor microenvironment and promoting tumor progression by secreting various cytokines. METHODS: Tissue microarray consisting of prostate cancer (PCa), normal prostate, and lymph node metastatic samples from patients with PCa were stained with Yin Yang 1 (YY1) and CD163. Transgenic mice overexpressing YY1 were constructed to observe PCa tumorigenesis. Furthermore, in vivo and in vitro experiments, including CRISPR-Cas9 knock-out, RNA sequencing, chromatin immunoprecipitation (ChIP) sequencing, and liquid-liquid phase separation (LLPS) assays, were performed to investigate the role and mechanism of YY1 in M2 macrophages and PCa tumor microenvironment. RESULTS: YY1 was highly expressed in M2 macrophages in PCa and was associated with poorer clinical outcomes. The proportion of tumor-infiltrated M2 macrophages increased in transgenic mice overexpressing YY1. In contrast, the proliferation and activity of anti-tumoral T lymphocytes were suppressed. Treatment targeting YY1 on M2 macrophages using an M2-targeting peptide-modified liposome carrier suppressed PCa cell lung metastasis and generated synergistic anti-tumoral effects with PD-1 blockade. IL-4/STAT6 pathway regulated YY1, and YY1 increased the macrophage-induced PCa progression by upregulating IL-6. Furthermore, by conducting H3K27ac-ChIP-seq in M2 macrophages and THP-1, we found that thousands of enhancers were gained during M2 macrophage polarization, and these M2-specific enhancers were enriched in YY1 ChIP-seq signals. In addition, an M2-specific IL-6 enhancer upregulated IL-6 expression through long-range chromatin interaction with IL-6 promoter in M2 macrophages. During M2 macrophage polarization, YY1 formed an LLPS, in which p300, p65, and CEBPB acted as transcriptional cofactors. CONCLUSIONS: Phase separation of the YY1 complex in M2 macrophages upregulated IL-6 by promoting IL-6 enhancer-promoter interactions, thereby increasing PCa progression.

Loureirin C inhibits ferroptosis after cerebral ischemia reperfusion through regulation of the Nrf2 pathway in mice.

BACKGROUND: Ischemic stroke (IS) is considered as a serious cerebral vascular disease. Ferroptosis is a novel type of regulated cell death (RCD), that closely related to the occurrence and progress of IS. Loureirin C, a type of dihydrochalcone compound derived from the Chinese Dragon's blood (CDB). The effective components extracted from CDB have shown neuroprotective effects in ischemia reperfusion models. However, the role of Loureirin C in mice after IS is not well understood. Thus, it is worth to identify the effect and mechanism of Loureirin C on IS. PURPOSE: The present research aims to prove the existence of ferroptosis in IS and explore whether Loureirin C can inhibit ferroptosis by regulating nuclear factor E2 related factor 2 (Nrf2) pathway in mice and exert neuroprotective effects on IS models. METHODS: Middle cerebral artery occlusion and reperfusion (MCAO/R) model was established to evaluate the occurrence of ferroptosis and the potential Loureirin C brain-protective effect in vivo. The analysis of free iron, glutamate content, reactive oxygen species (ROS) and lipid peroxidation levels, along with transmission electron microscope (TEM) was applied to prove the existence of ferroptosis. The function of Loureirin C on Nrf2 nuclear translocation was verified by immunofluorescence staining. In vitro, primary neurons and SH-SY5Y cells were processed with Loureirin C after oxygen and glucose deprivation-reperfusion (OGD/R). ELISA kits, western blotting, co-immunoprecipitation (Co-IP) analysis, immunofluorescence, and quantitative real-time PCR were devoted to proving the neuroprotective effects of Loureirin C on IS via regulating ferroptosis and Nrf2 pathways. RESULTS: The results showed that Loureirin C not only dramatically alleviated brain injury and inhibited neurons ferroptosis in mice after MCAO/R, but also dose-dependently reduce ROS accumulation in ferroptosis after OGD/R. Further, Loureirin C inhibits ferroptosis by activating Nrf2 pathway, and promoting nuclear translocation of Nrf2. Besides, Loureirin C increases heme oxygenase 1 (HO-1), quinone oxidoreductase 1 (NQO1) and glutathione peroxidase 4 (GPX4) content after IS. Intriguingly, the anti-ferroptosis effect of Loureirin C is weakened by Nrf2 knockdown. CONCLUSION: Our discoveries first revealed that the inhibitory action of Loureirin C on ferroptosis may greatly depend on its adjusting effect on the Nrf2 pathway, suggesting that Loureirin C could act as a novel anti-ferroptosis candidate and play a therapeutic role in IS. These novel discoveries on the role of Loureirin C on IS models reveal an innovative method that may contribute to neuroprotection for the prevention of IS.

Loureirin C ameliorates ischemia and reperfusion injury in rats by inhibiting the activation of the TLR4/NF-κB pathway and promoting TLR4 degradation.

Ischemic stroke is a leading cause of death and disability worldwide. Post-ischemia, microglia respond immediately to the alternations in neuronal activity and mediate inflammation. Toll-like receptor 4 (TLR4) plays a key role in this phenomenon. To explore the effect of loureirin C, an effective compound from Chinese Dragon's blood, on ischemic stroke, Sprague-Dawley rats were subjected to middle cerebral artery occlusion/reperfusion (MCAO/R) with/without intragastric administration of loureirin C (7, 14, and 28 mg/kg). Loureirin C alleviated MCAO/R-induced brain impairment evaluated by neurological scores (p < 0.001), brain water content (p < 0.001), and cerebral infarct volume (p = 0.001). The neuroprotective (p < 0.001) and inhibitory effects on microglial activation (p < 0.001) of loureirin C were revealed by immunofluorescence. Rescue studies with TLR4 overexpression in BV-2 microglia showed that the antiinflammatory effect of loureirin C was attributable to the inhibition of TLR4 protein expression. Moreover, co-immunoprecipitation assays showed that the binding of Triad3A, an E3 ubiquitin ligase of TLR4, was increased by loureirin C (p = 0.003). Our study demonstrates that loureirin C could be a promising therapeutic agent for the management of ischemic stroke by inhibiting microglial activation, potentially by Triad3A-mediated promotion of TLR4 ubiquitination and degradation.

Study on the Changes and Significance of Immune State and Cycokines in Children with Adenovirus Pneumonia.

Objective: To observe the difference between peripheral blood T lymphocytes subsets and cycokines in children with severe adenovirus pneumonia and nonsevere adenovirus pneumonia, and to investigate their clinical value in the prognosis of severe pneumonia. Methods: 215 children with adenovirus pneumonia and 30 healthy volunteers (which was set as the control group) in our hospital from January 2017 to May 2019 were enrolled in the study. There were 47 children with severe pneumonia in the severe group and 168 nonsevere pneumonia children in the nonsevere group. The flow cytometry and ELISA methods were used to detect the serum levels of CD3 +, CD4 +, CD8 + T cells and interleukin-2 (IL-2), IL-4, IL-6, IL-10, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ). Results: (1) The levels of CD3 (%) T cells, CD4 (%) T cells, and CD4/CD8 ratio values of children with adenovirus pneumonia were lower than these of normal children (P < 0.05). And the levels of CD3 (%) T cells, CD4 (%) T cells, and CD4/CD8 ratio values of children in the severe group were lower than these of children in the nonsevere group (P < 0.05). (2) The levels of IL-2, IL-6, IL-10, TNF-α, and IFN-γ values of children with adenovirus pneumonia were lower than these of normal children (P < 0.05). And the levels of IL-2, IL-6, IL-10, TNF-α, and IFN-γ of children in the severe group were higher than these of children in the nonsevere group (P < 0.05). (3) Among the 47 children with severe adenoviral pneumonia, 39 received systematic treatment in our hospital. According to the treatment effect, 39 children were divided into the effective group (n = 25) and the ineffective group (n = 14). (4) The CD3 (%), CD4 (%), and CD4/CD8 ratios of the children in the effective group were higher than those in the ineffective group (P < 0.05). (5) The levels of IL-2, IL-6, IL-10, TNF-α, and IFN-γ in the effective group were lower than those in the ineffective group (P < 0.05). Conclusion: The immunophenotype of peripheral blood T lymphocytes and cycokines could be helpful to judge the severity of adenovirus pneumonia, which could be used as the objective indexes to evaluate the prognosis of children with severe adenovirus pneumonia.

Sesquiterpene coumarins isolated from Ferula bungeana and their anti-neuroinflammatory activities.

This is the first study to profile natural sesquiterpene coumarins (SCs) in Ferula bungeana, a medicinal plant of the genus Ferula in China. Eight undescribed sesquiterpene coumarins (1-8), along with six known ones (9-14) were obtained from the whole plant of F. bungeana. These unreported SCs (1-8) enriched the structural diversity of natural SCs, especially these with the hydroxy or carbonyl group at C-7' and a hydroperoxy group at C-7' or C-8'. Compounds (9-14) were reported for the first time from this plant. The in vitro anti-neuroinflammatory activity assay showed that compounds 2 and 9 showed stronger inhibitory effect on nitric oxide (NO) production in lipopolysaccharide (LPS)-induced BV-2 microglia, compared with positive control minocycline, and compounds 5 and 10 showed moderate inhibitory effects.

Composite hydrolytic acidification - aerobic MBBR process for treating traditional Chinese medicine wastewater.

Traditional Chinese medicine (TCM) wastewater is characterized by high organic content, unstable water quality and quantity and low biodegradability. In this paper, the hydrolytic acidification reactor-aerobic moving bed biofilm (MBBR) process was used to degrade TCM wastewater. Besides, a small pilot study was conducted. The appropriate operating parameters: hydraulic retention time (HRT) of the hydrolytic reactor was 16 h, HRT of MBBR was 30 h, dissolved oxygen of MBBR was 6 mg/L, sludge return ratio of MBBR was 100%. The hydrolytic reactor was started for 25 days. MBBR was run in series with the hydrolytic reactor after 24 days of separate operation. The start-up of the composite reactor was completed after another 26 days. The average removal efficiencies of chemical oxygen demand and ammonia nitrogen were 92% and 70%. The hydrolytic reactor was effective in decomposing macromolecules and MBBR had a strong ability to degrade pollutants through the excitation-emission-matrix spectra. The evolution pattern of the dominant bacterial genera and the surface morphology of sludge were studied by scanning electron microscopy and high-throughput sequencing analysis. It could be seen that the surface morphology of the biological filler was suitable for the growth and reproduction of microorganisms.

Lignanamides from the stems of Piper hancei maxim. and their anti-inflammatory and cytotoxic activities.

Four new lignanamides, hancamides A - D (1-4), together with four known analogs (5-8), were isolated from the stems of Piper hancei Maxim. Their structures were determined based on 1D and 2D NMR, IR, UV, and HR-ESIMS spectroscopic analysis as well as by comparison with the reported data. All the isolates exhibited potential inhibitory effects on NO production in LPS-induced BV-2 microglial cells, with IC50 values of 4.26-40.68 µM. Moreover, compounds 2 and 8 displayed moderate cytotoxic activities against MGC-803, HepG2, SKOV-3, T24, and HeLa cells, with IC50 values ranging from 13.57 to 34.20 µM, respectively.

Biochanin A alleviates cognitive impairment and hippocampal mitochondrial damage in ovariectomized APP/PS1 mice.

BACKGROUND: Estrogen deficiency leads to mitochondrial defects that precede Alzheimer's disease (AD)-associated pathological changes in a postmenopausal mouse model. Biochanin A (BCA) is a phytoestrogen isolated from Trifolium pratense L. used to relieve postmenopausal problems in women. In previous work, we observed that oral BCA treatment led to neuroprotection in an ovariectomized rat model. The objective of this study was to investigate whether and how BCA protects against hippocampal mitochondrial damage in a postmenopausal model of AD. METHOD: APP/PS1 mice underwent bilateral ovariectomy and then, seven days later, received oral BCA at 20 or 40 mg/kg, or oral estradiol at 0.5 mg/kg, daily for 90 days. Sham animals were not ovariectomized and received no additional treatments. Cognitive function was examined using the passive avoidance task, novel object recognition test, and Morris water maze test. The level of circulating estrogen in vivo was assessed indirectly by measuring the wet weight of the uterus. We detected Aß deposition and PGC-1α in brain by immunohistochemistry; p62, by immunofluorescence; and ERα, ERß, PGC-1α, NRF1, mtTFA, Drp1, OPA1, Mfn2, Beclin1, LC3B, Pink1, and Parkin by immunoblotting. RESULTS: BCA treatment rescued cognitive decline and reduced Aß deposition and BACE1 expression in the hippocampus of ovariectomized APP/PS1 mice. BCA reversed the imbalance of mitochondrial dynamics caused by ovariectomy by increasing the expression of phospho-Drp1 (ser637), OPA1, and Mfn2. BCA reversed abnormal mitophagy induced by ovariectomy by increasing the expression of Beclin1, LC3B, Pink1, and Parkin, as well as by reducing the expression of p62. CONCLUSIONS: BCA treatment enhances learning and memory abilities and alleviates AD symptoms in a postmenopausal model of AD. A possible mechanism is that BCA rescues the reduction of mitochondrial biogenesis, imbalance of mitochondrial dynamics, and abnormal mitophagy caused by ovariectomy. This study supports further research on BCA to develop treatments for postmenopausal women with AD.

Acutissimalignan B from traditional herbal medicine Daphne kiusiana var. atrocaulis (Rehd.) F. Maekawa inhibits neuroinflammation via NF-κB Signaling pathway.

BACKGROUND: Emerging evidence indicates the important role of herbal medicine for neuroinflammation, which is closely associated with neurodegenerative diseases. OBJECTIVE: To clarify the characteristics and primary mechanisms of action of the traditional herbal medicine Daphne kiusiana var. atrocaulis (Rehd.) F. Maekawa in neuroinflammation by phytochemistry and bioassays using both in vitro and in vivo assays. METHODS: The chemical composition of D. kiusiana var. atrocaulis was clarified using multiple chromatography technologies and spectroscopic analysis. The anti-neuroinflammatory effects of the identified components were evaluated in LPS-induced BV-2 cells by monitoring the production of nitric oxide. C57BL/6 mice were used to construct a neuroinflammatory model by injecting LPS into the lateral ventricle of the brain. The most promising component was evaluated in vivo by measuring the number of Iba-1 cells and expression of inflammatory factors. Furthermore, the anti-neuroinflammatory mechanism involved in the activation of the NF-κB pathway was investigated using western blot and immunofluorescence. RESULTS: Thirty-two constituents (1-32), including five new compounds, were successfully identified from D. kiusiana var. atrocaulis. Compounds 3, 5, 12-15, and 20 (IC50 values from 5.41 to 57.27 µM) could considerably inhibit the LPS-induced production of NO in BV-2 cells, displaying stronger anti-neuroinflammatory activities than that of minocycline (IC50 = 67.08 µM). The concentration of the most potential compound 13 (IC50 5.41 µM) was 5.4% of the ethyl acetate fraction. Acutissimalignan B (13) could reduce the mRNA expression of iNOs, TNF-α, IL-1ß, and IL-6, inhibit the phosphorylation of IκBα, and inhibit the nuclear translocation of NK-κB p65 in BV-2 cells induced by LPS. Moreover, in the LPS-induced mouse model, compound 13 was found to exert anti-neuroinflammatory activity by attenuating the activation of microglia in the cortex and hippocampus, repressing the phosphorylation of IκBα, inhibiting the nuclear translocation of NK-κB p65, and decreasing the mRNA expression of iNOs, TNF-α, IL-1ß, and IL-6 in the cortex. CONCLUSION: We found that D. kiusiana var. atrocaulis had an inhibitory activity on neuroinflammation. In addition, the main active component (-)-acutissimalignan B (13) showed anti-neuroinflammatory effects in both in vivo and in vitro assays. Its mechanism of action may be associated with the inhibition of the NF-κB signaling pathway. Our current findings provide new information on D. kiusiana var. atrocaulis in the treatment of neuroinflammation.

Natural potential neuroinflammatory inhibitors from Stephania epigaea H.S. Lo.

Stephania epigaea H. S. Lo is a folk medicine widely distributed in the south of China, especially in Yunnan and Guangxi province. An in vitro anti-neuroinflammatory study showed that total alkaloids of it can potently inhibit LPS-induced NO releasing of BV2 cells with an IC50 value of 10.05 ± 2.03 µg/mL (minocycline as the positive drug, IC50 15.49 ± 2.14 µM). The phytochemical investigation of the total alkaloids afforded three new phenanthrene (1-3), two lactams (4a, 4b), and nine aporphine derivatives (5-13). The final structure of 1 was identified by computer-assisted structure elucidation (ACD/Structure Elucidator software and the 13C NMR calculation with GIAO method) due to many possibilities of the substituent pattern. All isolates were evaluated for their anti-neuroinflammatory effects, and as a result, 5, 8, 10, and 11 exhibited stronger inhibitory activities than the minocycline. The results suggested S. epigaea could provide potential therapeutic agents for neurodegenerative diseases.