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.

[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.

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.