PURPOSE:Ultraviolet B (UVB) irradiation is closely associated with human skin damage. Collagen I is found to be protective against UVB-caused apoptosis in human keratinocytes HaCaT, and in this study, we further investigated the potential underlying mechanisms. MATERIALS AND METHODS:Oxidative DNA damage was evaluated by assessing the DNA damage marker γ-H2AX, 8-OHdG, and reactive oxygen species (ROS). Relative cell numbers were determined by MTT assays, while apoptosis was evaluated by Hoechst 33,342 and Annexin V/PI double staining. Western blotting and RT-PCR were used to examine protein and mRNA expressions, respectively. Immunofluorescence confocal microscopy was used to visualize targeted proteins. RESULTS:Collagen I inhibits DNA damage and oxidative stress in HaCaT cells exposed to UVB irradiation. The YAP-p73 complex responds to DNA damages caused by UVB irradiation, and leads to the transcription of pro-apoptotic genes, including p21, PML and PUMA. DNA damage enhances ROS production, which further oxidatively damages DNA, forming a positive feedback loop. The protective effects of collagen I depend on the ROS restriction. Mitophagy, the process specifically eliminates the damaged mitochondria which are the main sources of cellular ROS, is restored by collagen I, and this contributes to the decreased ROS production, and the subsequent attenuation of cell apoptosis after UVB irradiation. CONCLUSIONS:Collagen I-coating protects UVB-irradiated cells from YAP-p73 mediated apoptosis through restricting ROS production and thus alleviating DNA damages, owing to the restored mitophagy. Our study supports the clinical application of collagen products as anti-UVB agents.

Microglia are resident immune cells in the central nervous system mediating brain inflammatory responses. The flavonoid silibinin has been found to restrict the neuronal inflammatory conditions in vivo. To fully reveal the underlying mechanisms, effects of silibinin on neuroinflammation was evaluated in lipopolysaccharides (LPS)-stimulated murine microglia BV2. The increased NO level, and the up-regulated pro-inflammatory proteins including iNOS and COX-2 in LPS-treated cells were all restricted by the treatment with silibinin. Further investigation showed that, mitochondrial disorders caused by LPS, including the excessive fission, loss of mitochondrial membrane potentials and intracellular ATP levels, augmented ROS and oxidative damages of mitochondrial DNA (mtDNA), were all attenuated by the treatment with silibinin. The protective effect of silibinin against the STING and NLRP3 inflammasome pathways is attributed to its ability to restore mitochondrial quality control. Of note, triggering receptors expressed on myeloid cells 2 (TREM2), a transmembrane receptor important for modulating microglia-associated inflammation, was low in LPS-treated cells but largely preserved in cells co-treated with silibinin. Molecular docking results show that silibinin has a binding potential with TREM2, which has also been confirmed in CETSA assay. TREM2 knockdown in microglia promotes a proinflammatory phenotype and mitochondrial damage which was reversed with silibinin treatment by increasing the stability of TREM2. Our data show that silibinin reduces mitochondrial damage and proinflammatory activation in microglia through the stabilization of TREM2. These results highlight the potential of silibinin as a treatment in neuroinflammatory diseases.

PURPOSE:Neuronal senescence contributes to several age-related neurodegenerative diseases occurring with aging. Meanwhile, aging of our bodies is accompanied by the systemic dysregulation of type I collagen, the major component in the extracellular matrix. This study investigates whether type I collagen plays a protective role in neuronal senescence. METHODS:Cellular senescence was assessed by evaluating senescence-associated β-galactosidase staining, cell area, senescence-associated proteins, and senescence-associated secretory phenotypes (SASPs); ferroptosis was evaluated using C11-BODIPY 581/591 fluorescent probe, GSH, MDA, and total iron assay kits; fluorescently labeled phalloidin staining was employed to examine the changes in F-actin levels. RESULTS:Coating the cell plates with type I collagen attenuates the senescence of murine hippocampal neuronal HT22 cells and alleviates ferroptosis induced by tert-butyl hydroperoxide (tBHP), a donor of reactive oxygen species. Enhancing ferroptosis-associated signaling by inhibiting GPX4 or FSP1 pathways promotes senescence of HT22 cells, suggesting that the ferroptosis signaling accelerates senescence. Integrin β1, a major component of receptors for collagen, is downregulated in tBHP-treated cells but rescued by collagen. F-actin was markedly disrupted in tBHP-treated cells, while restored by collagen, it protects cells against tBHP-induced ferroptosis and senescence. CONCLUSIONS:Our study reveals an F-actin/ferroptosis axis for regulating senescence of HT22 neuronal cells by extracellular type I collagen. The findings here suggest new anti-senescent strategies targeting the F-actin and ferroptosis pathways.

The recruitment of macrophages to a pathological site is accompanied by the change in surrounding extracellular matrix. The pathological foci in a highly inflammatory status contain certain amounts of gelatin, the denatured form of collagen. We previously revealed that precoating the cell dishes with gelatin, but not type I collagen, enhances bacteria-phagocytosis capacity of phorbol 12-myristate 13-acetate (PMA)-treated macrophage-like human histiocytic lymphoma U937 cells. The present study further reveals that gelatin-precoating increases the amount of reactive oxygen species (ROS) in PMA-treated U937 cells, which contributes to the enhanced phagocytosis of bacteria, including both Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. ROS in cells on gelatin-precoated culture plates cause impairments on mitochondria, as shown by the reduced mitochondrial membrane potential and ATP levels, as well as the increase in oxidative lesions in mitochondrial DNA. These mitochondrial damages lead to the activation of stimulator of interferon genes (STING) pathway, which enhances the bacteria-phagocytosis in PMA-treated U937 cells. Simultaneously, mitophagy-related proteins, such as PINK1, parkin and LC3 II, all increase following the elevation of ROS levels. Of note, mitophagy restricts the mitochondrial disorders, forming a feedback negative regulation for the effects of ROS, and works against bacteria-phagocytosis. This study reveals a core function of ROS-mitochondria-STING axis during gelatin-enhanced bacteria-phagocytosis in PMA-stimulated macrophage-like U937 cells, and provides possibility for clinically applying gelatin as a protectant for bacterial infection in some lesions.

Breast cancer as a multi-factorial disease has been widely concerned due to its high incidence. It is urgent to find new targets to treat breast cancers. Our previous research found silibinin induced apoptosis in both the hormone-sensitive breast cancer cells MCF-7 and the triple-negative breast cancer (TNBC) cells MDA-MB-231, through inhibiting the YAP pathway. Besides apoptosis, we here discover silibinin induces G2/M cell cycle arrest in both cells, which are also dependent on the inhibition of YAP. Interestingly, the F-actin assembly is markedly reduced by Silibinin. F-actin is found to be positively regulated by YAP, due to its transcriptional regulations of factors for polymerization. Meanwhile, disturbance of F-actin assembly by using Cytochalasin D contributes to cell cycle arrest, suggesting that F-actin disassembly is not just a consequence following YAP inhibition, but also plays a critical role in modulating cell cycle arrest. Further study on the interaction of silibinin and F-actin reveals that silibinin directly targets Capza1, which causes F-actin disassembly. Moreover, promoting F-actin assembly by si-Capza1 transfection restores YAP's activity, suggesting a positive interaction between YAP and F-actin. Of note, by simultaneous transfection of si-YAP/TAZ and si-Capza1, we find that although YAP has a regulatory effect on F-actin assembly, Capza1-mediated F-actin disassembly is decisive for silibinin-induced cell cycle arrest. Our results reveal the F-actin assembly is inhibited by silibinin, and this results in G2/M cell cycle arrest in human breast cancer cells, providing new ideas for anti-cancer therapies including TNBCs. Abbreviations: ABPs, actin binding proteins; ARP2, actin-related protein2; Capza1, capping actin protein of muscle Z-line subunit alpha 1; CDC2, Cell Division Cycle protein 2/CDK1, Cyclin-Dependent Kinase 1; CDKi, cyclin-dependent kinase inhibitors; CDKs, cyclin-dependent kinases; CETSA, cellular thermal shift assay; CFL1, cofilin 1; Cyto D, Cytochalasin D; DARTS, drug affinity responsive target stability; DIAPH3, diaphanous related formin 3; DMEM, Dulbecco's Modified Eagle medium; ER, estrogen receptor; F-actin, filamentous actin; FBS, fetal bovine serum; G-actin, globular actin; GSN, gelsolin; HER2, human epidermal growth factor receptor 2; LAMP1, lysosomal associated membrane protein 1; NLS, nuclear localization signal; PDB, protein data bank; PFN1, profilin 1; PR, progesterone receptor; qRT-PCR, quantitative real-time polymerase chain reaction; RT, room temperature; Sili, silibinin; si-RNAs, small interfering RNAs; TNBC, triple-negative breast cancer; VP, verteporfin; YAP, Yes-associated protein.

In scenarios like burns or local infections, monocytes migrate from the circulatory system to the injured sites, which are rich in extracellular matrix (ECM) components, collagen and its derivative gelatin, to differentiate into macrophages. The roles of ECM components in the phagocytosis of macrophages raised our interest. We previously found that precoating the culture dishes with gelatin markedly enhances phagocytosis of bacteria in PMA-stimulated human lymphoma U937 cells which have macrophage-like properties. But type I collagen has no such effects. Here we reveal that AMPK pathway is impaired in gelatin-treated U937 cells, and this subsequently induces mitochondrial fission. Mitochondrial fission further leads to mitochondrial dysfunction and leakage of mitochondrial DNA into the cytoplasm, activating the STING pathway which is responsible for the enhanced phagocytosis. Additionally, mitochondrial dysfunction impairs aerobic respiration and this compensatively elevates glycolytic levels, contributing to the enhanced phagocytosis. In summary, our study comprehensively unveils that gelatin promotes bacteria phagocytosis via modulating an AMPK-mitochondrial fission axis, which leads to the STING activation and glycolytic reprogramming. Our findings offer new insights for the influence of gelatin on immune cells.

Collagen I is one of the major components of the extracellular matrix in human skin, and is frequently used in skin cares and medications. Previously, we revealed that human keratinocytes HaCaT cells grown on collagen I (Col)-coated dishes gain resistance against UVB damages owing to the restored mitophagy. In this study, we further investigate the mechanisms by which collagen I modulates mitophagy. UVB irradiation causes loss of integrin β1 and collapse of F-actin cytoskeleton. Considering the requirement of actin skeleton in various cellular processes, we are curious about the participation of F-actin collapse in UVB damage. Integrin β1, whose activation enhances F-actin assembly, is a potential target for Col in UVB-treated cells. Notably, inhibiting integrin by adding an inhibitor RGDS or siRNA attenuates the effect of Col against UVB damages, confirming the participation of integrin in cell protection. The collapse of F-actin is rescued by Col, accompanying increases in the mRNA of F-actin polymerization-associated proteins and decreases in the mRNA of depolymerization-associated proteins. Inhibiting actin polymerization by using cytochalasin D represses the protective effect of Col, confirming the cytoprotective role of F-actin in UVB-treated cells. Remarkably, mitophagy in UVB-treated cells restored by Col-coating is inhibited by adding cytochalasin D or RGDS, as shown by the decreases of lysosomes, mitochondrial ubiquitin proteins, and co-localization of autophagosomes and mitochondria, resulting in accumulation of damaged mitochondria, which stresses the importance of F-actin and integrin in mitophagy. In summary, integrins and F-actin are required for mitophagy in UVB-irradiated HaCaT cells, and their enhancements by Col-coating facilitate timely elimination of damaged mitochondria caused by UVB, finally contributing to cell survival.

Perimenopause raises the risk and incidence of depression, whereas the underlying molecular mechanism remains unclear. Disturbed glucose regulation has been widely documented in depressive disorders, which renders the brain susceptible to various stresses such as estrogen depletion. However, whether and how glucose dysfunction regulates depression-like behaviors and neuronal damage in perimenopausal transition remains unexplored. Here, a prominent depressive phenotype was found in perimenopausal mice induced by the ovarian toxin 4-vinylcyclohexene diepoxide (VCD). The VCD depression susceptible group (VCDSS) and the VCD depression resilient group (VCDRES) were determined using a ROC-based behavioral screening approach. We found that the hippocampus, a crucial region linked to depression, had hyperglycemia and mitochondrial abnormalities. Interestingly, oral administration of the SGLT2 inhibitor empagliflozin (EMPA) and intrahippocampal glucose infusion suggest a close relationship between hyperglycemia in the hippocampus and the susceptibility to depression. We verified that cytochrome c oxidase 7c (COX7C) downregulation is a potential cause of the high glucose-induced neuronal injury using proteomic screening and biochemical validations. High glucose causes COX7C to be ubiquitinated in a S-phase kinase associated protein 1 (SKP1)-dependent manner. According to these results, SKP1/COX7C represents a unique therapeutic target and a novel molecular route for treating perimenopausal depression.

Adipocyte hyperplasia refers to the increase in the number of adipocytes, whereas adipocyte hypertrophy pertains to the enlargement of individual adipocytes resulting from the accumulation of lipid droplets. In this study, we found that activation of the STING signalling pathway occurs during adipogenic differentiation of 3 T3-L1 preadipocytes. Interestingly, inhibiting the STING pathway by using STING antagonist H151 or siRNA targeting STING promotes adipocyte differentiation and increases adipocyte numbers, while activation of STING inhibits adipogenic differentiation. Silencing the STING canonical downstream IRF3, or inhibiting the proton channel activity of STING enhances adipogenic differentiation, confirming the negative modulation of adipogenic differentiation by STING. In vivo, intraperitoneal injection of H151 into mice with a high-fat diet further enhances the adipocyte hyperplasia, as shown by the increased volume of adipose tissues, but consistent sizes of adipocytes. During the adipogenic differentiation of 3 T3-L1 cells, DRP1-mediated mitochondrial fission is enhanced, and causes mitochondrial DNA leakage, which in turn activates the STING pathway. However, inhibition of mitochondrial fission represses adipogenic differentiation of 3 T3-L1 cells in spite of the down-regulation of STING pathway. Therefore, our results indicate that adipogenic differentiation is associated with DRP1-induced mitochondrial fission. However, the leakage of mitochondrial DNA caused by DRP1-induced mitochondrial fission activates the STING signalling pathway, which negatively regulates adipogenic differentiation. Tissue specific reduction of DRP1-associated mitochondrial fission or STING enhancement might be new strategies for the therapy of obesity-associated diseases.

Effective neovascularization is critical for tissue repair and the enhancement of cardiac function following myocardial infarction (MI). However, the hypoxic microenvironment post-MI significantly impedes neovascular formation. Although ATF4 has been implicated in heart failure and myocardial cell regeneration and repair, its role in angiogenesis remains unclear. This study utilized both in vitro and in vivo models to investigate the role of ATF4 in neovascularization after MI. In hypoxia-cultured murine endothelial cells (ECs), hypoxia was observed to inhibit EC proliferation, migration, and tube formation. In contrast, overexpression of ATF4 ameliorated these hypoxia-induced impairments. Conversely, inhibition of ATF4 further exacerbated the reduction in EC proliferation, migration, and tube formation induced by hypoxia. Notably, the beneficial effects of ATF4 were reversed by the PI3K/AKT inhibitor LY294002. Under hypoxic conditions, ATF4 overexpression significantly upregulated phosphorylated (p)-PI3K, p-AKT (T308), and p-AKT (S473) in ECs. LY294002, however, markedly reduced the expression of p-PI3K, p-AKT (T308), and p-AKT (S473) in hypoxic ECs overexpressing ATF4. In a murine MI model, ATF4 overexpression partially mitigated cardiac dysfunction and promoted neovascularization, effects that were significantly attenuated by LY294002. These findings suggest that ATF4 plays a crucial role in endothelial cell-mediated neovascularization under post-MI hypoxia by modulating the PI3K/AKT signaling pathway.

Collagen I is a major component of extracellular matrix in human skin, and is also widely used in a variety of skin-care products. In this study, we investigated the modulatory roles of collagen I on human immortalized keratinocytes HaCaT, especially when cells were irradiated with UVB. Interestingly, the cells grown on plates coated by molecular collagen I, but not fibrillar collagen I, acquired certain resistance against UVB damages, as shown by increased survival and reduced apoptosis. The accumulation of dysfunctional mitochondria in UVB-treated cells was attenuated by molecular collagen I-coating. Interestingly, molecular collagen I rescued the loss of mitochondrial biogenesis in cells treated with UVB. Loss of PINK1/parkin-mediated mitophagy was dominant for the accumulation of dysfunctional mitochondria after UVB irradiation. Of note, cells cultured on molecular collagen I-precoated plates exhibited reserved mitophagy after UVB irradiation, as reflected by the enhanced protein level of PINK1/parkin, increased mitochondrial ubiquitin and the co-localization of lysosomes and mitochondria. Moreover, in UVB-treated cells, inhibiting mitophagy by Cyclosporin A, or by silencing PINK1 or parkin, disturbed the resolution of mitochondrial stress and reduced the protective effect of molecular collagen I, indicating that mitophagy is pivotal for the protection of collagen I against UVB damage in keratinocytes HaCaT. Collectively, this study reveals an unexpected protective role of collagen I, which facilitates mitophagy to rescue cells under UVB irradiation, providing a new direction for clinical application of collagen products.

PURPOSE:Besides comprising scaffolding, extracellular matrix components modulate many biological processes including inflammation and cell differentiation. We previously found precoating cell plates with extracellular matrix collagen I, or its denatured product gelatin, causes aggregation of macrophage-like human lymphoma U937 cells, which are induced to differentiation by phorbol myristate treatment. In the present study, we investigated the influence of gelatin or collagen I precoating on the bacteria phagocytosis in PMA-stimulated U937 cells.MATERIALS AND METHODS:Colony forming units of phagocytosed bacteria, Giemsa-staining of cells with phagocytosed bacteria, confocal microscopic and flow cytometric analysis of cells with phagocytosed FITC-labeled bacteria and non-bioactive latex beats were conducted.RESULTS:Gelatin precoating enhances the phagocytosis of both Gram-negative and positive bacteria, as shown by the increased colony forming units of bacteria phagocytosed by cells, and increased intracellular bacteria observed after Giemsa-staining. But collagen I has no marked influence. Confocal microscopy reveals that both live and dead FITC-bacteria were phagocytosed more in the cells with gelatin-coating but not collagen-coating. Of note, both gelatin and collagen I coating had no influence on the phagocytosis of non-bioactive latex beads. Since gelatin-coating increases autophagy but collagen I has no such impact, we are curious about the role of autophagy. Inhibiting autophagy reduced the phagocytosis of bacteria, in cells with gelatin-coating, while stimulating autophagy enhanced phagocytosis.CONCLUSION:This study finds the bacteria-phagocytosis stimulatory effect of gelatin in PMA-treated U937 cells and reveals the positive regulatory role of autophagy, predicting the potential use of gelatin products in anti-bacterial therapy.

Breast cancer is one of the most common cancers threatening women's health. Our previous study found that silibinin induced the death of MCF-7 and MDA-MB-231 human breast cancer cells. We noticed that silibinin-induced cell damage was accompanied by morphological changes, including the increased cell aspect ratio (cell length/width) and decreased cell area. Besides, the cytoskeleton is also destroyed in cells treated with silibinin. YAP/TAZ, a mechanical signal sensor interacted with extracellular pressure, cell adhesion area and cytoskeleton, is also closely associated with cell survival, proliferation and migration. Thus, the involvement of YAP/TAZ in the cytotoxicity of silibinin in breast cancer cells has attracted our interests. Excitingly, we find that silibinin inhibits the nuclear translocation of YAP/TAZ in MCF-7 and MDA-MB-231 cells, and reduces the mRNA expressions of YAP/TAZ target genes, ACVR1, MnSOD and ANKRD. More importantly, expression of YAP1 gene is negatively correlated with the survival of the patients with breast cancers. Molecular docking analysis reveals high probabilities for binding of silibinin to the proteins in the YAP pathways. DARTS and CETSA results confirm the binding abilities of silibinin to YAP and LATS. Inhibiting YAP pathway either by addition of verteporfin, an inhibitor of YAP/TAZ-TEAD, or by transfection of si-RNAs targeting YAP or TAZ further enhances silibinin-induced cell damage. While enhancing YAP activity by silencing LATS1/2 or overexpressing YAPS127/397A, an active form of YAP, attenuates silibinin-induced cell damage. These findings demonstrate that inhibition of the YAP/TAZ pathway contributes to cytotoxicity of silibinin in breast cancers, shedding lights on YAP/TAZ-targeted cancer therapies.

BackgroundMyoprotein degradation accelerates in obese individuals, resulting in a decline in muscular mass. Atg7 plays a crucial role in regulating protein stability and function through both autophagy-dependent and independent pathways. As obesity progresses, the expression of Atg7 gradually rises in muscle tissue. Nonetheless, the precise impact and mechanism of Atg7 in promoting muscle mass decline in obesity remain uncertain. The study aimed to elucidate the role and underly mechanism of Atg7 action in the context of obesity-induced muscle mass decline.MethodsIn this study, we established a murine model of high-fat diet-induced obesity (DIO) and introduced adeno-associated virus delivery of short hairpin RNA to knock down Atg7 (shAtg7) into the gastrocnemius muscle. We then examined the expressions of Atg7 and myoprotein degradation markers in the gastrocnemius tissues of obese patients and mice using immunofluorescence and western blotting techniques. To further investigate the effects of Atg7, we assessed skeletal muscle cell diameter and the myoprotein degradation pathway in C2C12 and HSkMC cells in the presence or absence of Atg7. Immunofluorescence staining for MyHC and western blotting were utilized for this purpose. To understand the transcriptional regulation of Atg7 in response to myoprotein degradation, we conducted luciferase reporter assays and chromatin immunoprecipitation experiments to examine whether FoxO3a enhances the transcription of Atg7. Moreover, we explored the role of Akt in Atg7-mediated regulation and its relevance to obesity-induced muscle mass decline. This was accomplished by Akt knockdown, treatment with MK2206, and GST pulldown assays to assess the interaction between Atg7 and Akt.ResultsAfter 20 weeks of being on a high-fat diet, obesity was induced, leading to a significant decrease in the gastrocnemius muscle area and a decline in muscle performance. This was accompanied by a notable increase in Atg7 protein expression (p < 0.01). Similarly, in gastrocnemius tissues of obese patients when compared to nonobese individuals, there was a significant increase in both Atg7 (p < 0.01) and TRIM63 (p < 0.01) levels. When palmitic acid was administered to C2C12 cells, it resulted in increased Atg7 (p < 0.01), LC3Ⅱ/Ⅰ (p < 0.01), and p62 levels (p < 0.01). Additionally, it promoted FoxO3a-mediated transcription of Atg7. The knockdown of Atg7 in the gastrocnemius partially reversed DIO-induced muscle mass decline. Furthermore, when Atg7 was knocked down in C2C12 and HSkMC cells, it mitigated palmitic acid-induced insulin resistance, increased the p-Akt/Akt ratio (p < 0.01), and reduced TRIM63 (p < 0.01). Muscular atrophy mediated by Atg7 was reversed by genetic knockdown of Akt and treatment with the p-Akt inhibitor MK2206. Palmitic acid administration increased the binding between Atg7 and Akt (p < 0.01) while weakening the binding of PDK1 (p < 0.01) and PDK2 (p < 0.01) to Akt. GST pulldown assays demonstrated that Atg7 directly interacted with the C-terminal domain of Akt.ConclusionThe consumption of a high-fat diet, along with lipid-induced effects, led to the inhibition of Akt signaling, which, in turn, promoted FoxO3a-mediated transcription, increasing Atg7 levels in muscle cells. The excess Atg7 inhibited the phosphorylation of Akt, leading to a cyclic activation of FoxO3a and exacerbating the decline in muscle mass regulated by obesity. Consequently, Atg7 serves as a regulatory point in determining the decline in muscle mass induced by obesity.

Type 2 diabetes (T2DM) is induced by the abundance of glucose and lipids, which causes glucolipotoxicity to the pancreatic β-cells. Silibinin is a natural flavonoid possessing the regulatory activity on insulin production and therapeutic activity in diabetic mice; however, its effect on glucolipotoxicity is not fully explained. This in vitro study investigates the effects of silibinin on palmitic acid (PA) and high glucose (HG)-induced cell loss and ferroptosis of rat insulinoma INS-1 cells. In the cells treated with PA and HG, expressions of glucose transporter 4 (Glut4) and carnitine acyltransferase I (CPT1) for β-oxidation of fatty acids are reduced. Mitochondria are the metabolic organelles for glucose and fatty acids. The mitochondrial membrane potential (MMP) and ATP production were decreased, while the ROS level was elevated in the cells treated with PA and HG, indicating an induction of mitochondrial disorder. Cell loss was partially rescued by ferroptosis inhibition, suggesting an involvement of ferroptosis in the cells treated with PA and HG. More importantly, the increases in total iron, lipid ROS, MDA and COX-2, and the decrease in ferroptosis inhibitory molecules GSH, GPX4 and FSP1 appeared in the cells treated with PA and HG, confirming the occurrence of ferroptosis. Moreover, PINK1/parkin-mediated mitophagy, a vital process for selective elimination of damaged mitochondria, was blocked. Interestingly, silibinin rescued the mitochondria, restricted the ferroptosis and restored the mitophagy. By using the pharmacological stimulator and inhibitor of mitophagy, and si-RNA transfection to silence PINK1 expression, silibinin's protective effect against ferroptosis caused by PA and HG treatment was found to depend on mitophagy. Collectively, our current study reveals the new mechanisms for the protection of silibinin against the injury of INS-1 cells treated with PA and HG, elucidates the participation of ferroptosis in glucolipotoxicity, highlighting the involvement of mitophagy in defense against ferroptotic cell death.

Background： The activated group 2 innate lymphocytes (ILC2s) play a crucial role in respiratory syncytial virus (RSV)-induced airway inflammation by secreting large amount of type 2 cytokines. Although the classical activator IL-33 is the key factor for ILC2 activation, a regulatory effect of neurotransmitter-neuromedin U (NMU) has also been reported. However, whether and how NMU can be elicited by RSV infection and regulate pulmonary ILC2 activation remains unclear. Methods： The regulatory effect and underlying mechanisms of NMU on ILC2 activation were determined by using RSV-infected wild-type and NMU-knockout mice. The expression of NMU in the lungs and NMUR1 on ILC2s were measured by Real-time PCR and Western blot. Flow cytometry and ELISA were used to detect the proliferation and activation of ILC2s. The type of neurons secreting NMU and its possible secretion mechanism during RSV infection were also analyzed. Results： Acute RSV infection induced the production of NMU in the lungs of mice and up-regulate the expression of NMUR1 on the pulmonary ILC2s. In vivo administration of NMU exacerbated RSV-induced airway inflammation by promoting the proliferation and activation of pulmonary ILC2s via NMUR1 pathway. In this process, PI3K, MEK and NFAT signal proteins might be involved. Furthermore, pulmonary neurons responded to the stimulation of RSV and secreted NMU in TLR4 and TLR7-dependent manners. Conclusion： Our data suggest that NMU is an ILC2 activator other than the classical activator, revealing a novel effect of neurotransmitter on RSV-induced airway inflammation.

Neurodegenerative diseases (NDs) affect 15% of the world's population and are becoming an increasingly common cause of morbidity and mortality worldwide. Circadian rhythm disorders (CRDs) have been reported to be involved in the pathogenic regulation of various neurologic diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis and amyotrophic lateral sclerosis. Proteomic technology is helpful to explore treatment targets for CRDs in patients with NDs. Here, we review the key differentially expressed (DE) proteins identified in previous proteomic studies investigating NDs, CRDs and associated models and the related pathways identified by enrichment analysis. Furthermore, we summarize the advantages and disadvantages of the above studies and propose new proteomic technologies for the precise study of circadian disorder-mediated regulation of ND pathology. This review provides a theoretical and technical reference for the precise study of circadian disorder-mediated regulation of ND pathology.

Ultraviolet B (UVB) irradiation causes skin inflammation and apoptosis. Mitochondria are highly dynamic and undergo constant fusion and fission that are essential for maintaining physiological functions of cells. Although dysfunction of mitochondria has been implicated in skin damages, little is known about the roles of mitochondrial dynamics in these processes. UVB irradiation increases abnormal mitochondrial content but decreases mitochondrial volume in immortalized human keratinocyte HaCaT cells. UVB irradiation resulted in marked upregulation of mitochondrial fission protein dynamin-related protein 1 (DRP1) and downregulation of mitochondrial outer membrane fusion proteins 1 and 2 (MFN1 and MFN2) in HaCaT cells. Mitochondrial dynamics was discovered to be crucial for NLRP3 inflammasome and cGAS-STING pathway activation, as well as the induction of apoptosis. Inhibition of mitochondrial fission by treatments with a DRP1 inhibitor, mdivi-1, or with DRP1-targeted siRNA, efficiently prevented UVB-induced NLRP3/cGAS-STING mediated pro-inflammatory pathways or apoptosis in the HaCaT cells, whereas inhibition of mitochondrial fusion with MFN1and 2 siRNA increased these pro-inflammatory pathways or apoptosis. The enhanced mitochondrial fission and reduced fusion caused the up-regulation of reactive oxygen species (ROS). Application of an antioxidant, N-acetyl-l-cysteine (NAC), which scavenges excessive ROS, attenuated inflammatory responses through suppressing NLRP3 inflammasome and cGAS-STING pathway activation, and rescued cells from apoptosis caused by UVB-irradiation. Together, our findings revealed the regulation of NLRP3/cGAS-STING inflammatory pathways and apoptosis by mitochondrial fission/fusion dynamics in UVB-irradiated HaCaT cells, providing a new strategy for the therapy of UVB skin injury.

Background: Over-activation of N-methyl-D-aspartate receptors (NMDARs) is involved in sporadic Alzheimer's disease. Silibinin, a natural flavonoid gained from the seeds of Silybum marianum, exerts neuroprotective effects on sporadic AD models, but its impacts on NMDARs remain unknown. Purpose: To study silibinin's regulatory effects on NMDARs pathway in sporadic AD models. Methods: MTT assay, western blotting, confocal microscopy, flow cytometry, RT-PCR, and siRNA transfection etc. were used for cellular and molecular studies. The direct interactions between silibinin and NMDAR subunits were evaluated by computational molecular docking, drug affinity responsive target stability (DARTS) assay and cellular thermal shift assay (CETSA). Y maze test, novel objects recognition test and Morris water maze test were conducted to examine the learning and memory ability of rats. Results: An in vitro AD model was established by treating HT22 murine hippocampal neurons with streptozotocin (STZ), as evidenced by the amyloid beta (A beta) deposition and hyperphosphorylation of tau proteins. Silibinin shows protection of neurons against STZ-induced cell damage. It is noteworthy that STZ-induced cellular calcium influx is inhibited by silibinin-treatment, indicating the possible modulation of calcium channels. Studies on NMDARs, the most widely distributed calcium channel, by using molecular docking, DARTS and CESTA, reveal that the GluN2B subunit, but not GluN2A, is the potential target of silibinin. Further studies using the pharmacological agonist (NMDA) and the GluN2B-specific inhibitor (Ifenprodil) or siRNA, indicate that the protection by silibinin treatment from STZ-induced cytotoxicity is medicated through interference with GluN2B-containing NMDARs, followed by the upregulation of CaMKII alpha/BDNF/TrkB signaling pathway and improved levels of synaptic proteins (SYP and PSD-95). The results in vivo using rats intracerebroventricularly injected with STZ (ICV-STZ), a well-established sporadic AD model, confirm that silibinin improves learning and memory ability in association with modulation of the GluN2B/CaMKII alpha/ BDNF/TrkB signaling pathway. Conclusion: Inhibiting over-activation of GluN2B-containing NMDARs is involved in the neuroprotective effect of silibinin on STZ-induced sporadic AD models.