Methionine redox regulation of actin-interacting proteins primarily governs antioxidative signaling and response to the salvianolic acid B treatment in EA.hy926 cells.

Actin-interacting proteins are important molecules for filament assembly and cytoskeletal signaling within vascular endothelium. Disruption in their interactions causes endothelial pathogenesis through redox imbalance. Actin filament redox regulation remains largely unexplored, in the context of pharmacological treatment. This work focused on the peptidyl methionine (M) redox regulation of actin-interacting proteins, aiming at elucidating its role on governing antioxidative signaling and response. Endothelial EA.hy926 cells were subjected to treatment with salvianolic acid B (Sal B) and tert-butyl-hydroperoxide (tBHP) stimulation. Mass spectrometry was employed to characterize redox status of proteins, including actin, myosin-9, kelch-like erythroid-derived cap-n-collar homology-associated protein 1 (Keap1), plastin-3, prelamin-A/C and vimentin. The protein redox landscape revealed distinct stoichiometric ratios or reaction site transitions mediated by M sulfoxide reductase and reactive oxygen species. In comparison with effects of tBHP stimulation, Sal B treatment prevented oxidation at actin M325, myosin-9 M1489/1565, Keap1 M120, plastin-3 M592, prelamin-A/C M187/371/540 and vimentin M344. For Keap1, reaction site was transitioned within its scaffolding region to the actin ring. These protein M oxidation regulations contributed to the Sal B cytoprotective effects on actin filament. Additionally, regarding the Keap1 homo-dimerization region, Sal B preventive roles against M120 oxidation acted as a primary signal driver to activate nuclear factor erythroid 2-related factor 2 (Nrf2). Transcriptional splicing of non-POU domain-containing octamer-binding protein was validated during the Sal B-mediated overexpression of NAD(P)H dehydrogenase [quinone] 1. This molecular redox regulation of actin-interacting proteins provided valuable insights into the phenolic structures of Sal B analogs, showing potential antioxidative effects on vascular endothelium.

Exosome lncRNA IFNG-AS1 derived from mesenchymal stem cells of human adipose ameliorates neurogenesis and ASD-like behavior in BTBR mice.

BACKGROUND: The transplantation of exosomes derived from human adipose-derived mesenchymal stem cells (hADSCs) has emerged as a prospective cellular-free therapeutic intervention for the treatment of neurodevelopmental disorders (NDDs), as well as autism spectrum disorder (ASD). Nevertheless, the efficacy of hADSC exosome transplantation for ASD treatment remains to be verified, and the underlying mechanism of action remains unclear. RESULTS: The exosomal long non-coding RNAs (lncRNAs) from hADSC and human umbilical cord mesenchymal stem cells (hUCMSC) were sequenced and 13,915 and 729 lncRNAs were obtained, respectively. The lncRNAs present in hADSC-Exos encompass those found in hUCMSC-Exos and are associated with neurogenesis. The biodistribution of hADSC-Exos in mouse brain ventricles and organoids was tracked, and the cellular uptake of hADSC-Exos was evaluated both in vivo and in vitro. hADSC-Exos promote neurogenesis in brain organoid and ameliorate social deficits in ASD mouse model BTBR T + tf/J (BTBR). Fluorescence in situ hybridization (FISH) confirmed lncRNA Ifngas1 significantly increased in the prefrontal cortex (PFC) of adult mice after hADSC-Exos intraventricular injection. The lncRNA Ifngas1 can act as a molecular sponge for miR-21a-3p to play a regulatory role and promote neurogenesis through the miR-21a-3p/PI3K/AKT axis. CONCLUSION: We demonstrated hADSC-Exos have the ability to confer neuroprotection through functional restoration, attenuation of neuroinflammation, inhibition of neuronal apoptosis, and promotion of neurogenesis both in vitro and in vivo. The hADSC-Exos-derived lncRNA IFNG-AS1 acts as a molecular sponge and facilitates neurogenesis via the miR-21a-3p/PI3K/AKT signaling pathway, thereby exerting a regulatory effect. Our findings suggest a potential therapeutic avenue for individuals with ASD.

Bidirectional crosstalk of the cAMP/ROS-dependent signaling pathways in inflammatory macrophage: An activation of formononetin.

Bacterial lipopolysaccharide (LPS) is a toxic stimulant to macrophage inflammation. Inflammation intersects cell metabolism and often directs host immunopathogenesis stress. We aim here at pharmacological discovering of formononetin (FMN) action, to which anti-inflammatory signaling spans across immune membrane receptors and second messenger metabolites. In ANA-1 macrophage stimulated by LPS, and simultaneous treatment with FMN, results show the Toll-like receptor 4 (TLR4) and estrogen receptor (ER) signals, in concert with reactive oxygen species (ROS) and cyclic adenosine monophosphate (cAMP), respectively. LPS stimulates inactivation of the ROS-dependent nuclear factor erythroid 2-related factor 2 (Nrf2) by upregulating TLR4, but it does not affect cAMP. However, FMN treatment not only activates Nrf2 signaling by TLR4 inhibition, but also it activates cAMP-dependent protein kinase activities by upregulating ER. The cAMP activity gives rise to phosphorylation (p-) of protein kinase A, liver kinase B1 and 5'-AMP activated protein kinase (AMPK). Moreover, bidirectional signal crosstalk is amplified between p-AMPK and ROS, as FMN combinational validation with AMPK activator/inhibitor/target small-interfering RNA or ROS scavenger. The signal crosstalk is well positioned serving as the 'plug-in' knot for rather long signaling axis, and the immune-to-metabolic circuit via ER/TLR4 signal transduction. Collectively, convergence of the FMN-activated signals drives significant reduction of cyclooxygenase-2, interleukin-6 and NLR family pyrin domain-containing protein 3, in LPS-stimulated cell. Although anti-inflammatory signaling is specifically related to the immune-type macrophage, the p-AMPK antagonizing effect arises from FMN combination with ROS scavenger H-bond donors. Information of our work assists in predictive traits against macrophage inflammatory challenges, using phytoestrogen discoveries.

Improving endothelial cell junction integrity by diphenylmethanone derivatives at oxidative stress: A dual-action directly targeting caveolar caveolin-1.

Directly targeting caveolar caveolin-1 is a potential mechanism to regulate endothelial permeability, especially during oxidative stress, but little evidence on the topic limits therapeutics discoveries. In this study, we investigated the pharmacological effect of an antioxidant LM49 (5,2'-dibromo-2,4',5'-trihydroxydiphenylmethanoe) and its five diphenylmethanone derivatives on endothelial permeability and establish two distinct mechanisms of action. Multiplex molecular assays with theoretical modeling indicate that diphenylmethanone molecules, including LM49, directly bind the caveolin-1 steric pocket of ASN53/ARG54, ILE49/ASP50, ILE18, LEU59, ASN60, GLU48 and ARG19 residues. They also indicated dynamic binding-affinity for diphenylmethanone derivatives. First, this molecular interaction at caveolin-1 pocket inhibits its phosphorylation at TYR14 residue in H2O2-injured endothelial cell. A positive correlation was established between diphenylmethanone derivative binding-affinity and caveolin-1 phosphorylation inhibition. Inhibition of caveolin-1 phosphorylation, however, was independent of the LM49-mediated variation of protein tyrosine kinase activity, suggesting a direct blockage of adenosine triphosphate substrate diffusion into cavelion-1 structure. Second, LM49 increases the expression of cellular adhesive and tight junction proteins, VE-cadherin and occludin, in H2O2-injured cell, in a dose dependent manner. A leakage assay of fluorescein isothiocyanate-labeled dextran 40 across cell monolayer suggested improvement in endothelial barrier integrity with diphenylmethanone treatments. Our results demonstrate a direct targeting effect of caveolin-1 on endothelial permeability, and should guide the diphenylmethanone therapy against oxidative stress-induced junction dysfunction, especially at caveolar membrane invagination.

5,2'-dibromo-2,4',5'-trihydroxydiphenylmethanone attenuates LPS-induced inflammation and ROS production in EA.hy926 cells via HMBOX1 induction.

Inflammation and reactive oxygen species (ROS) are important factors in the pathogenesis of atherosclerosis (AS). 5,2'-dibromo-2,4',5'-trihydroxydiphenylmethanone (TDD), possess anti-atherogenic properties; however, its underlying mechanism of action remains unclear. Therefore, we sought to understand the therapeutic molecular mechanism of TDD in inflammatory response and oxidative stress in EA.hy926 cells. Microarray analysis revealed that the expression of homeobox containing 1 (HMBOX1) was dramatically upregulated in TDD-treated EA.hy926 cells. According to the gene ontology (GO) analysis of microarray data, TDD significantly influenced the response to lipopolysaccharide (LPS); it suppressed the LPS-induced adhesion of monocytes to EA.hy926 cells. Simultaneously, TDD dose-dependently inhibited the production or expression of IL-6, IL-1ß, MCP-1, TNF-α, VCAM-1, ICAM-1 and E-selectin as well as ROS in LPS-stimulated EA.hy926 cells. HMBOX1 knockdown using RNA interference attenuated the anti-inflammatory and anti-oxidative effects of TDD. Furthermore, TDD inhibited LPS-induced NF-κB and MAPK activation in EA.hy926 cells, but this effect was abolished by HMBOX1 knockdown. Overall, these results demonstrate that TDD activates HMBOX1, which is an inducible protective mechanism that inhibits LPS-induced inflammation and ROS production in EA.hy926 cells by the subsequent inhibition of redox-sensitive NF-κB and MAPK activation. Our study suggested that TDD may be a potential novel agent for treating endothelial cells dysfunction in AS.

PGC-1α/NRF1-dependent cardiac mitochondrial biogenesis: A druggable pathway of calycosin against triptolide cardiotoxicity.

Mitochondrion-related cardiotoxicity due to cardiotoxin stimuli is closely linked to abnormal activities of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), followed by co-inactivation of nuclear respiratory factor-1(NRF1). Pharmacological interventions targeting mitochondria may be effective for developing agents against cardiotoxicity. Herein, in triptolide-treated H9C2 cardiomyocytes, we observed defective mitochondrial biogenesis and respiration, characterized by depletion of mitochondrial mass and mitochondrial DNA copy number, downregulation of mitochondrial respiratory chain complexes subunits, and disorders of mitochondrial membrane potential and mitochondrial oxidative phosphorylation. Dysregulation of mitochondria led to cardiac pathological features, such as myocardial fiber fracture, intercellular space enlargement, and elevation of serum aspartate aminotransferase, creatine kinase isoenzyme, lactate dehydrogenase, and cardiac troponin I. However, following calycosin treatment, an active compound from Astragali Radix, the mitochondrion-related disorders at both cell and tissue levels were significantly ameliorated, which was facilitated by the activation of PGC-1α via deacetylation, followed by NRF1 co-activation. Calycosin-enhanced PGC-1α deacetylation is impelled by increasing sirtuin-1 expression and NAD+/NADH ratio. PGC-1α/NRF1 signaling in calycosin-mediated mitochondrial biogenesis protection was further confirmed by NRF1 knockdown and PGC-1α inhibition with SR18292. We conclude that calycosin ameliorated triptolide-induced cardiotoxicity by protecting PGC-1α/NRF1-dependent cardiac mitochondrial biogenesis and respiration, which is the druggable pathway for cardiotoxicity mitigation.

Loss of neurodevelopmental-associated miR-592 impairs neurogenesis and causes social interaction deficits.

microRNA-592 (miR-592) has been linked to neurogenesis, but the influence of miR-592 knockout in vivo remains unknown. Here, we report that miR-592 knockout represses IPC-to-mature neuron transition, impairs motor coordination and reduces social interaction. Combining the RNA-seq and tandem mass tagging-based quantitative proteomics analysis (TMT protein quantification) and luciferase reporter assays, we identified MeCP2 as the direct targetgene of miR-592 in the mouse cortex. In Tg(MECP2) mice, lipofection of miR-592 efficiently reduced MECP2 expression in the brains of Tg(MECP2) mice at E14.5. Furthermore, treatment with miR-592 partially ameliorated the autism-like phenotypes observed in adult Tg(MECP2) mice. The findings demonstrate that miR-592 might play a novel role in treating the neurodevelopmental-associated disorder.

5,2'-Dibromo-2,4,5-trihydroxydiphenylmethanone, a novel immunomodulator of T lymphocytes by regulating the CD4+ T cell subset balance via activating the mitogen-activated protein kinase pathway.

5,2'-Dibromo-2,4',5'-trihydroxydiphenylmethanone (LM49) exerted therapeutic effects against rat acute pyelonephritis by regulating immune responses, especially affecting T lymphocytes. However, its underlying action mechanism remains unclear. T lymphocytes play an irreplaceable role in immune responses. Therefore, we sought to understand whether LM49 is an immunomodulator of T lymphocytes. The results showed that LM49 promoted T lymphocyte proliferation, increased the number of CD4+ T cells, and increased the CD4+/CD8+ T cell ratio. LM49 regulated the CD4+ T cell subset balance by increasing the production of CD4+IL-2+, CD4+IL-4+, and CD4+IL-10+, and reducing the production of CD4+IL-17+, without changing the production of interferon-γ. LM49 had a significant effect on the mRNA expression of the transcription factors T-bet, GATA3, Foxp3, and RORγt. Furthermore, LM49 raised the phospho (p)-extracellular signal-regulated protein kinase 1/2, p-p38, and p-c-Jun N-terminal kinase expression levels. T cell proliferation, and the production of CD4+IL-2+, CD4+IL-4+, and CD4+IL-10+ induced by LM49, were decreased by inhibitors of mitogen-activated protein kinases (MAPKs). These results revealed that LM49 possesses immunomodulatory activity on T lymphocytes, in which the MAPK pathway plays an essential role.

Therapeutic effects of 5,2'-dibromo-2,4',5'-trihydroxydiphenylmethanone (LM49) in an experimental rat model of acute pyelonephritis by immunomodulation and anti-inflammation.

Antibiotics are still the primary therapy for acute pyelonephritis (APN); rarely, natural polyphenols are also used. LM49 is a novel marine bromophenol derivative displaying strong anti-inflammatory effects. We investigated the therapeutic efficacy of LM49 in an experimental rat model of APN. The model was established by injecting 0.5 mL Escherichia coli (ATCC 25922, 108 CFU/mL) into the urinary bladders of Sprague Dawley rats. This model showed increased kidney viscera indices and renal bacterial growth scores, as well as pathological changes in kidneys. We also performed a broth microdilution antimicrobial susceptibility test of the E. coli strain. Both norfloxacin and LM49 treatment reduced kidney viscera indices and decreased microbial counts in urine cultures and kidney homogenates in APN rats. However, in vitro experiments showed that LM49 did not directly inhibit bacteria. Rather, LM49 treatment inhibited inflammatory cell infiltration or abscess and improved tissue lesions in the renal medullary junction, renal pelvis, and calyx, and high-dose LM49 treatment inhibited the production of inflammatory interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) in serum. CD4+ T cells were higher in the LM49 groups treated with high, medium, and low doses than in the model group, whereas only high-dose LM49 treatment increased the number of CD8+ T cells, as compared with that in the model group. However, LM49 treatment did not influence hematological parameters. Our results show that LM49 therapeutic effects in an APN animal model may be achieved by regulating immune responses and inhibiting inflammatory mediators, suggesting it as a candidate APN treatment.

[Investigation of replantation of thumb rotation avulsion injury].

OBJECTIVE: To investigate replantation methods and clinical outcomes of thumb rotation avulsion injury,and to evaluate the advantages and disadvantages of each procedure. METHODS: From Feburary 2009 to March 2012, 21 thumbs suffered from rotation avulsion injuries and replanted by different methods, including 16 males and 5 females with an average age of 32 years old ranging from 16 to 45 years old. Diffierent methods were chosen according to the traumatic condition. And the survival condition and function of the thumbs after replantation were observed and evaluated. RESULTS: Among them, 19 replanted figers were survival, 2 cases failed. The mean follow-up period was 8 months (ranged from 3 to 14 months). According to the criteria for function assement of amputated finger issued by the Branch of Hand Surgery of Chinese Medicine Association, the results were excellent in 13 cases, good in 5, and poor in 1. CONCLUSION: According to the traumatic condition to choose various approach in replantation of thumb with rotation avulsion, leading to a higher success rate, and provide the patients with a more aesthetic appearance and satisfied function.