Chrysin inhibits ferroptosis of cerebral ischemia/reperfusion injury via regulating HIF-1α/CP loop.

Chrysin is a natural flavonoid with powerful neuroprotective capacity. Cerebral ischemia/reperfusion injury (CIRI) is associated with oxidative stress and ferroptosis. Hypoxia-inducible factor 1α (HIF-1α) and ceruloplasmin (CP) are the critical targets for oxidation reactions and iron transport. But the regulatory mechanism between them is still unclear. Transient middle cerebral artery occlusion (tMCAO) model in rats and oxygen and glucose deprivation/re-oxygenation (OGD/R) model in PC12 cells were applied. Pathological tissue staining and biochemical kit were used to evaluate the effect of chrysin. The relationship between HIF-1α and CP was verified by transcriptomics, qRT-PCR and Western blot. In CIRI, HIF-1α/CP loop was discovered to be the regulatory pathway of ferroptosis. CIRI led to activation and nuclear translocation of HIF-1α, which promoted CP transcription and translation, and downstream ferroptosis. Inhibition of HIF-1α had opposite effect on CP and ferroptosis regulation. Overexpression of CP increased the expression of HIF-1α, nevertheless, inhibited the nuclear translocation of HIF-1α and alleviated CIRI. Silencing CP promoted HIF-1α elevation in nucleus and aggravated CIRI. Mechanistically, chrysin restrained HIF-1α nuclear translocation, thereby inhibiting CP transcription and translation, which in turn reduced downstream HIF-1α expression and mitigated ferroptosis in CIRI. Our results highlight chrysin restrains ferroptosis in CIRI through HIF-1α/CP loop.

Ganshuang granule plays a pharmacological role in anti-alcoholic and anti-hangover via regulating alcohol metabolism and affecting neurotransmitters.

BACKGROUND: To explore the effect of Ganshuang granule on anti-alcoholic and anti-hangover and its potential mechanism. METHODS: SPF SD rats' drunken model and SPF Kunming mice's hangover model were used as models. RESULTS: Ganshuang granule could significantly reduce sleep time, the time to climb in mice, and significantly prolong the tolerance time and shorten sleep time in rats (p < 0.05). The blood ethanol concentration of rats in each administration group was lower than that in the model group at each time point (p < 0.05). Compared with the control group, the activities of ADH and ALDH in the liver of the model group were significantly decreased (p < 0.05); the content of DA and 5-HT in the striatum of the model group was significantly increased (p < 0.05); and the activity of AchE in the hippocampus was significantly decreased (p < 0.05). The above processes could be improved and regulated in the drug administration group. Compared with the control group, there was no significant difference between ADH and ALDH in the serum of the model group (p > 0.05). However, the activities of ADH and ALDH in the liver of drunk rats could be upregulated by Ganshuang granule (p < 0.05). CONCLUSION: Ganshuang granule has the pharmacological effects of anti-alcoholic and anti-hangover, which is related to regulating the activities of ADH and ALDH in the liver, the contents of DA and 5-HT in striatum, and the activity of AchE in the hippocampus.

[Chrysin alleviates cerebral ischemia-reperfusion injury by inhibiting ferroptosis in rats].

The purpose of this study is to investigate whether chrysin reduces cerebral ischemia-reperfusion injury(CIRI) by inhi-biting ferroptosis in rats. Male SD rats were randomly divided into a sham group, a model group, high-, medium-, and low-dose chrysin groups(200, 100, and 50 mg·kg~(-1)), and a positive drug group(Ginaton, 21.6 mg·kg~(-1)). The CIRI model was induced in rats by transient middle cerebral artery occlusion(tMCAO). The indexes were evaluated and the samples were taken 24 h after the operation. The neurological deficit score was used to detect neurological function. The 2,3,5-triphenyl tetrazolium chloride(TTC) staining was used to detect the cerebral infarction area. Hematoxylin-eosin(HE) staining and Nissl staining were used to observe the morphological structure of brain tissues. Prussian blue staining was used to observe the iron accumulation in the brain. Total iron, lipid pero-xide, and malondialdehyde in serum and brain tissues were detected by biochemical reagents. Real-time quantitative polymerase chain reaction(RT-qPCR), immunohistochemistry, and Western blot were used to detect mRNA and protein expression of solute carrier fa-mily 7 member 11(SLC7A11), transferrin receptor 1(TFR1), glutathione peroxidase 4(GPX4), acyl-CoA synthetase long chain family member 4(ACSL4), and prostaglandin-endoperoxide synthase 2(PTGS2) in brain tissues. Compared with the model group, the groups with drug intervention showed restored neurological function, decreased cerebral infarction rate, and alleviated pathological changes. The low-dose chrysin group was selected as the optimal dosing group. Compared with the model group, the chrysin groups showed reduced content of total iron, lipid peroxide, and malondialdehyde in brain tissues and serum, increased mRNA and protein expression levels of SLC7A11 and GPX4, and decreased mRNA and protein expression levels of TFR1, PTGS2, and ACSL4. Chrysin may regulate iron metabolism via regulating the related targets of ferroptosis and inhibit neuronal ferroptosis induced by CIRI.

Chrysin protects against cerebral ischemia-reperfusion injury in hippocampus via restraining oxidative stress and transition elements.

Chrysin is a natural flavonoid compound that has antioxidant and neuroprotective effects. Cerebral ischemia reperfusion (CIR) is closely connected with increased oxidative stress in the hippocampal CA1 region and homeostasis disorder of transition elements such as iron (Fe), copper (Cu) and zinc (Zn). This exploration was conducted to elucidate the antioxidant and neuroprotective effects of chrysin based on transient middle cerebral artery occlusion (tMCAO) in rats. Experimentally, sham group, model group, chrysin (50.0 mg/kg) group, Ginaton (21.6 mg/kg) group, Dimethyloxallyl Glycine (DMOG, 20.0 mg/kg) + chrysin group and DMOG group were devised. The rats in each group were performed to behavioral evaluation, histological staining, biochemical kit detection, and molecular biological detection. The results indicated that chrysin restrained oxidative stress and the rise of transition element levels, and regulated transition element transporter levels in tMCAO rats. DMOG activated hypoxia-inducible factor-1 subunit alpha (HIF-1α), reversed the antioxidant and neuroprotective effects of chrysin, and increased transition element levels. In a word, our findings emphasize that chrysin plays a critical role in protecting CIR injury via inhibiting HIF-1α against enhancive oxidative stress and raised transition metal levels.

Zhachong Shisanwei Pill resists ischemic stroke by lysosome pathway based on proteomics and bioinformatics.

ETHNOPHARMACOLOGICAL RELEVANCE: Zhachong Shisanwei Pill (ZSP) is a commonly used Mongolian medicine in treating cerebrovascular diseases and plays a role in the clinical treatment of ischemic stroke (IS). AIM OF THE STUDY: Based on determining the protective effect of ZSP on cerebral ischemia, they adopted the proteomics method to explore the mechanism of ZSP against IS. MATERIALS AND METHODS: Rats with middle cerebral artery occlusion (MCAO) model were prepared by wire embolization method, and divided into sham group, model group, ZSP high-dose group, medium-dose group, low-dose group and positive drug group. We collected the brain tissue of rats for 12 h after modeling. Neurological deficit score and cerebral infarction volume ratio evaluated pharmacodynamics, and we selected the optimal dose for subsequent experiments. Proteomics was used to screen out possible ZSP anti-IS mediated pathways and differentially expression proteins. Network pharmacology was used to verify the correlation between diseases and drugs. Hematoxylin-eosin (HE) staining and transmission electron microscope (TEM) were used to explore further the pharmacodynamic effect of ZSP against IS and its possible mechanism. RESULTS: The cerebral infarction rate and neurological function score in rats showed that the medium-dose ZSP group had the best efficacy. Proteomics results showed that the anti-IS action of ZSP was mainly through lysosome pathway. LAMP2, AP3M1, and SCARB2 were the differentially changed proteins in this pathway. Network pharmacology verified this. HE staining and TEM results showed that ZSP could improve the pathological state of neurons in MCAO rats and reduce the number of lysosomes in MCAO rats. Western blot (WB) results showed that compared with the model group, the protein expression levels of LAMP2 and AP3M1 in the ZSP group were significantly down-regulated, and the protein expression levels of SCARB2 were significantly up-regulated. CONCLUSION: This study confirms that ZSP regulates the lysosomal pathway, which may protect IS by down-regulating LAMP2 and AP3M1 and up-regulating SCARB2.

[Exploration of mechanism of "simultaneous treatment of brain and heart" of Naoxintong Capsules based on Toll-like receptor signaling pathway].

This study aims to explore the mechanism of "simultaneous treatment of the brain and the heart" of Naoxintong Capsules(NXT) under cerebral ischemia based on Toll-like receptor(TLR) signaling pathway.Male SD rats were randomized into sham operation group, model group, NXT group, and positive drug group.Middle cerebral artery occlusion(MCAO) model rats were used in model group, NXT group, and positive drug group, respectively.Neurological function was scored with the Bederson scale, and brain infarct rate was measured by 2,3,5-triphenyltetrazolium chloride(TTC) staining.Brain edema was detected with wet-dry weight method.Hematoxylin-eosin(HE) staining and TdT-mediated dUTP nick-end labeling(TUNEL) staining were used to observe and count apoptotic cardiocytes.In addition, serum myocardial enzymes were measured.The expression of 8 TLR signaling pathway-related proteins interferon-α(IFN-α), interferon regulatory factor-3(IRF3), interferon regulatory factor-7(IRF7), TLR2, TLR4, TLR7, TLR9, and tumor necrosis factor-α(TNF-α) in the cerebral cortex and heart of rats was detected by Western blot. Brain infarct rate, neurological function score, and brain water content in NXT group decreased significantly compared with those in the model group. At the same time, the reduction in apoptosis rate of cardiocytes and the content of serum aspartate aminotransferase(AST), alanine aminotransferase(ALT), creatine kinase(CK), and lactate dehydrogenase(LDH) were decreased in the NXT group.Systems pharmacological results and previous research showed that TLR signaling pathway played an important role in immune inflammatory response.The study of TLR signaling pathway and related proteins is helpful to elucidate the mechanism of "simultaneous treatment of the brain and the heart". Western blot results showed that NXT significantly inhibited the expression of IRF3, IRF7, TLR2, TLR7, and TNF-α in cerebral cortex and heart under cerebral ischemia.Cerebral ischemia influences cardiac functions, and TLR signaling pathway is one of the pathways for "simultaneous treatment of the brain and the heart" of NXT.

The Differences of Metabolites in Different Parts of the Brain Induced by Shuxuetong Injection against Cerebral Ischemia-Reperfusion and Its Corresponding Mechanism.

Ischemic stroke is often associated with a large disease burden. The existence of ischemia-reperfusion injury brings great challenges to the treatment of ischemic stroke. The purpose of this study was to explore the differences of metabolites in different parts of the brain induced by Shuxuetong injection against cerebral ischemia-reperfusion and to extend the corresponding mechanism. The rats were modeled by transient middle cerebral artery occlusion (t-MCAO) operation, and the success of modeling was determined by neurological function score and TTC staining. UPLC-Q/TOF-MS metabolomics technique and multivariate statistical analysis were used to analyze the changes and differences of metabolites in the cortex and hippocampus of cerebral ischemia-reperfusion rats. Compared with the model group, the neurological function score and cerebral infarction volume of the Shuxuetong treatment group were significantly different. There were differences and changes in the metabolic distribution of the cortex and hippocampus in each group, the distribution within the group was relatively concentrated. The separation trend between the groups was obvious, and the distribution of the Shuxuetong treatment group was similar to that of the sham operation group. We identified 13 metabolites that were differentially expressed in the cortex, including glutamine, dihydroorotic acid, and glyceric acid. We also found five differentially expressed metabolites in the hippocampus, including glutamic acid and fumaric acid. The common metabolic pathways of Shuxuetong on the cortex and hippocampus were D-glutamine and D-glutamate metabolism and nitrogen metabolism, which showed inhibition of cortical glutamine and promotion of hippocampal glutamic acid. Specific pathways of Shuxuetong enriched in the cortex included glyoxylate and dicarboxylate metabolism and pyrimidine metabolism, which showed inhibition of glyceric acid and dihydroorotic acid. Specific pathways of Shuxuetong enriched in the hippocampus include arginine biosynthesis and citrate cycle (TCA cycle), which promotes fumaric acid. Shuxuetong injection can restore and adjust the metabolic disorder of the cortex and hippocampus in cerebral ischemia-reperfusion rats. The expression of Shuxuetong in different parts of the brain is different and correlated.

Systems pharmacology, proteomics and in vivo studies identification of mechanisms of cerebral ischemia injury amelioration by Huanglian Jiedu Decoction.

ETHNOPHARMACOLOGICAL RELEVANCE: Huanglian Jiedu Decoction (HLJDD) has the effect of clearing heat and detoxifying, and has been considered as an effective prescription for cerebral ischemia (CI) for thousands of years in traditional Chinese medicine (TCM). It can improve the quality of life of patients with ischemic stroke, but its pharmacological mechanism remains unclear. AIM OF THE STUDY: The study aimed to explore the pharmacological action and potential mechanism of HLJDD against CI by systems pharmacology, proteomics and in vivo experiments. MATERIALS AND METHODS: In this study, databases such as TCMIP V2.0 and Genecards were used to predict compounds, targets and CI related targets, and network topology criteria of protein-protein interaction (PPI) network was used to screen core targets. The Database for Annotation, Visualization and Integrated Discovery database (DAVID) was used to discover biological processes and pathways. In addition, molecular docking was performed between the screened core biological active compounds and targets to verify the binding activity. Finally, proteomics and Western blot were performed on cerebral cortex tissues of middle cerebral artery occlusion (MCAO) model rats with HLJDD intervention to further verify the predicted results. RESULTS: 77 compounds and 308 targets of HLJDD were identified, 54 of which were predicted to be associated with cerebral ischemia. PPI network and enrichment results showed that 8 targets, including AKT1, PTGS2 and TLR4, were core targets of HLJDD in CI. And 19 signaling pathways, including Rap1 signaling pathway, cAMP signaling pathway and arachidonic acid metabolism, were identified as key pathways to the therapeutic activity of HLJDD in CI. Combined with proteomics studies, we identified that Rap1 signaling pathway and upstream and downstream targets were the key mechanisms. Molecular biology experiments showed that RAP1A and AKT expression levels were significantly up-regulated in middle cerebral artery occlusion (MCAO) rats treated with HLJDD (P < 0.0001), GRIN1 expression level was significantly down-regulated (P < 0.0001). However, ACTB expression level was slightly down-regulated (P > 0.05), which may be related to the biological function. CONCLUSION: This study confirms the pharmacological effect of HLJDD on cerebral ischemia. These results indicate that HLJDD mediates various pathways such as inhibition of apoptosis, regulation of oxygen balance, inhibition of excitatory toxicity and maintenance of basic cell functions to improve CI by regulating Rap1 signaling pathway.

RIF1-ASF1-mediated high-order chromatin structure safeguards genome integrity.

The 53BP1-RIF1 pathway antagonizes resection of DNA broken ends and confers PARP inhibitor sensitivity on BRCA1-mutated tumors. However, it is unclear how this pathway suppresses initiation of resection. Here, we identify ASF1 as a partner of RIF1 via an interacting manner similar to its interactions with histone chaperones CAF-1 and HIRA. ASF1 is recruited to distal chromatin flanking DNA breaks by 53BP1-RIF1 and promotes non-homologous end joining (NHEJ) using its histone chaperone activity. Epistasis analysis shows that ASF1 acts in the same NHEJ pathway as RIF1, but via a parallel pathway with the shieldin complex, which suppresses resection after initiation. Moreover, defects in end resection and homologous recombination (HR) in BRCA1-deficient cells are largely suppressed by ASF1 deficiency. Mechanistically, ASF1 compacts adjacent chromatin by heterochromatinization to protect broken DNA ends from BRCA1-mediated resection. Taken together, our findings identify a RIF1-ASF1 histone chaperone complex that promotes changes in high-order chromatin structure to stimulate the NHEJ pathway for DSB repair.

An OB-fold complex controls the repair pathways for DNA double-strand breaks.

53BP1 with its downstream proteins, RIF1, PTIP and REV7, antagonizes BRCA1-dependent homologous recombination (HR) and promotes non-homologous end joining (NHEJ) in an unclear manner. Here we show that REV7 forms a complex with two proteins, FAM35A and C20ORF196. We demonstrate that FAM35A preferentially binds single-strand DNA (ssDNA) in vitro, and is recruited to DSBs as a complex with C20ORF196 and REV7 downstream of RIF1 in vivo. Epistasis analysis shows that both proteins act in the same pathway as RIF1 in NHEJ. The defects in HR pathway to repair DSBs and the reduction in resection of broken DNA ends in BRCA1-mutant cells can be largely suppressed by inactivating FAM35A or C20ORF196, indicating that FAM35A and C20ORF196 prevent end resection in these cells. Together, our data identified a REV7-FAM35A-C20ORF196 complex that binds and protects broken DNA ends to promote the NHEJ pathway for DSB repair.

Identification of a small molecule that downregulates MITF expression and mediates antimelanoma activity in vitro.

Melanoma is a type of cancer arising from the melanocytes, which are the cells that make up the pigment melanin and are derived from the neural crest. There is no particularly effective therapy once the disease is metastatic, highlighting the need for discovery of novel potent agents. In this investigation, we adopted a zebrafish embryonic pigmentation model to identify antimelanoma agents by screening an in-house small molecule library. With this assay, we found that a small molecule compound, SKLB226, blocked zebrafish pigmentation and pigment cell migration. Mechanism of action studies showed that SKLB226 downregulated MITF mRNA level in both zebrafish embryos and mammalian melanoma cells. Further studies showed that it could efficiently suppress the viability and migration of mammalian melanoma cells. In summary, SKLB226 can be used as a chemical tool to study melanocyte development as well as an antimelanoma lead compound that should be subjected to further structural optimization.

Thermosensitive hydrogel containing dexamethasone micelles for preventing postsurgical adhesion in a repeated-injury model.

Tissue adhesion is a common complication after surgery. In this work, a dexamethasone loaded polymeric micelles in thermosensitive hydrogel composite (Dex hydrogel) was prepared, which combined the anti-adhesion barrier with controlled release of anti-adhesion drug. Dexamethasone (Dex) was encapsulated in polymeric micelles (Dex micelles), and then the Dex micelles were loaded into biodegradable and thermosensitive hydrogel. The obtained Dex hydrogel showed a temperature-dependent sol-gel-sol phase transition behavior. The Dex hydrogel could form a non-flowing gel in situ upon subcutaneous injection and gradually degrade in about 20 days. In addition, Dex hydrogel was assigned for anti-adhesion studies in a more rigorous recurrent adhesion animal model. Compared with normal saline (NS) and Dex micelles group, tissue adhesions in hydrogel and Dex hydrogel group were significantly alleviated. In Dex hydrogel group, the media adhesion score is 0, which was dramatically lower than that in blank hydrogel group (2.50, P < 0.001). In histopathological examination and scanning electron microscopy (SEM) analysis, an integral neo-mesothelial cell layer with microvilli on their surface was observed, which revealed that the injured parietal and visceral peritoneum were fully recovered without the concerns of adhesion formation. Our results suggested that Dex hydrogel may serve as a potential anti-adhesion candidate.