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.

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.

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.

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.

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.

Physicochemical, Antibacterial Properties, and Compatibility of ZnO-NP/Chitosan/ß-Glycerophosphate Composite Hydrogels.

In this study we aimed to develop novel ZnO-NP/chitosan/ß-glycerophosphate (ZnO-NP/CS/ß-GP) antibacterial hydrogels for biomedical applications. According to the mass fraction ratio of ZnO-NPs to chitosan, mixtures of 1, 3, and 5% ZnO-NPs/CS/ß-GP were prepared. Using the test-tube inversion method, scanning electron microscopy and Fourier-transform infrared spectroscopy, the influence of ZnO-NPs on gelation time, chemical composition, and cross-sectional microstructures were evaluated. Adding ZnO-NPs significantly improved the hydrogel's antibacterial activity as determined by bacteriostatic zone and colony counting. The hydrogel's bacteriostatic mechanism was investigated using live/dead fluorescent staining and scanning electron microscopy. In addition, crystal violet staining and MTT assay demonstrated that ZnO-NPs/CS/ß-GP exhibited good antibacterial activity in inhibiting the formation of biofilms and eradicating existing biofilms. CCK-8 and live/dead cell staining methods revealed that the cell viability of gingival fibroblasts (L929) cocultured with hydrogel in each group was above 90% after 24, 48, and 72 h. These results suggest that ZnO-NPs improve the temperature sensitivity and bacteriostatic performance of chitosan/ß-glycerophosphate (CS/ß-GP), which could be injected into the periodontal pocket in solution form and quickly transformed into hydrogel adhesion on the gingiva, allowing for a straightforward and convenient procedure. In conclusion, ZnO-NP/CS/ß-GP thermosensitive hydrogels could be expected to be utilized as adjuvant drugs for clinical prevention and treatment of peri-implant inflammation.

Prevention of Retinal Degeneration in a Rat Model of Smith-Lemli-Opitz Syndrome.

Smith-Lemli-Opitz Syndrome (SLOS) is a recessive human disease caused by defective cholesterol (CHOL) synthesis at the level of DHCR7 (7-dehydrocholesterol reductase), which normally catalyzes the conversion of 7-dehydrocholesterol (7DHC) to CHOL. Formation and abnormal accumulation of 7DHC and 7DHC-derived oxysterols occur in SLOS patients and in rats treated with the DHCR7 inhibitor AY9944. The rat SLOS model exhibits progressive and irreversible retinal dysfunction and degeneration, which is only partially ameliorated by dietary CHOL supplementation. We hypothesized that 7DHC-derived oxysterols are causally involved in this retinal degeneration, and that blocking or reducing their formation should minimize the phenotype. Here, using the SLOS rat model, we demonstrate that combined dietary supplementation with CHOL plus antioxidants (vitamins E and C, plus sodium selenite) provides better outcomes than dietary CHOL supplementation alone with regard to preservation of retinal structure and function and lowering 7DHC-derived oxysterol formation. These proof-of-principle findings provide a translational, pre-clinical framework for designing clinical trials using CHOL-antioxidant combination therapy as an improved therapeutic intervention over the current standard of care for the treatment of SLOS.

Large-Scale Structural Characterization of Drug and Drug-Like Compounds by High-Throughput Ion Mobility-Mass Spectrometry.

Ion mobility-mass spectrometry (IM-MS) can provide orthogonal information, i.e., m/z and collision cross section (CCS), for the identification of drugs and drug metabolites. However, only a small number of CCS values are available for drugs, which limits the use of CCS as an identification parameter and the assessment of structure-function relationships of drugs using IM-MS. Here, we report the development of a rapid workflow for the measurement of CCS values of a large number of drug or drug-like molecules in nitrogen on the widely available traveling wave IM-MS (TWIM-MS) platform. Using a combination of small molecule and polypeptide CCS calibrants, we successfully determined the nitrogen CCS values of 1425 drug or drug-like molecules in the MicroSource Discovery Systems' Spectrum Collection using flow injection analysis of 384-well plates. Software was developed to streamline data extraction, processing, and calibration. We found that the overall drug collection covers a wide CCS range for the same mass, suggesting a large structural diversity of these drugs. However, individual drug classes appear to occupy a narrow and unique space in the CCS-mass 2D spectrum, suggesting a tight structure-function relationship for each class of drugs with a specific target. We observed bimodal distributions for several antibiotic species due to multiple protomers, including the known fluoroquinolone protomers and the new finding of cephalosporin protomers. Lastly, we demonstrated the utility of the high-throughput method and drug CCS database by quickly and confidently confirming the active component in a pharmaceutical product.

Antioxidant supplementation ameliorates molecular deficits in Smith-Lemli-Opitz syndrome.

BACKGROUND: Smith-Lemli-Opitz syndrome (SLOS) is an inborn error of cholesterol biosynthesis characterized by diminished cholesterol and increased 7-dehydrocholesterol (7-DHC) levels. 7-Dehydrocholesterol is highly reactive, giving rise to biologically active oxysterols. METHODS: 7-DHC-derived oxysterols were measured in fibroblasts from SLOS patients and an in vivo SLOS rodent model using high-performance liquid chromatography tandem mass spectrometry. Expression of lipid biosynthesis genes was ascertained by quantitative polymerase chain reaction and Western blot. The effects of an antioxidant mixture of vitamin A, coenzyme Q10, vitamin C, and vitamin E were evaluated for their potential to reduce formation of 7-DHC oxysterols in fibroblast from SLOS patients. Finally, the effect of maternal feeding of vitamin E enriched diet was ascertained in the brain and liver of newborn SLOS mice. RESULTS: In cultured human SLOS fibroblasts, the antioxidant mixture led to decreased levels of the 7-DHC-derived oxysterol, 3ß,5α-dihydroxycholest-7-en-6-one. Furthermore, gene expression changes in SLOS human fibroblasts were normalized with antioxidant treatment. The active ingredient appeared to be vitamin E, as even at low concentrations, it significantly decreased 3ß,5α-dihydroxycholest-7-en-6-one levels. In addition, analyzing a mouse SLOS model revealed that feeding a vitamin E enriched diet to pregnant female mice led to a decrease in oxysterol formation in brain and liver tissues of the newborn Dhcr7-knockout pups. CONCLUSIONS: Considering the adverse effects of 7-DHC-derived oxysterols in neuronal and glial cultures and the positive effects of antioxidants in patient cell cultures and the transgenic mouse model, we believe that preventing formation of 7-DHC oxysterols is critical for countering the detrimental effects of DHCR7 mutations.