Gastrodin alleviates mitochondrial dysfunction by regulating SIRT3-mediated TFAM acetylation in vascular dementia.

BACKGROUND: Mitochondrial dysfunction is key to the pathogenesis of vascular dementia (VaD). Sirtuin-3 (SIRT3), an essential member of the sirtuins family, has been proven to be a critical sirtuin in regulating mitochondrial function. The phenolic glucoside gastrodin (GAS), a bioactive ingredient from Gastrodiae Rhizome (known in Chinese as Tian ma) demonstrates significant neuroprotective properties against central nervous system disorders; however, the precise mechanisms through which GAS modulates VaD remain elusive. PURPOSE: This study aims to investigate whether GAS confers a protective role against VaD, and to figure out the underlying molecular mechanisms. METHODS: A bilateral common carotid artery occlusion (BCCAO)-mediated chronic cerebral hypoperfusion (CCH) VaD rat model and a hypoxia model using HT22 cells were employed to investigate pharmacological properties of GAS in mitigating mitochondrial dysfunction. A SIRT3 agonist resveratrol (RES), a SIRT3 inhibitor 3-TYP and SIRT3-knockdown in vitro were used to explore the mechanism of GAS in association with SIRT3. The ability of SIRT3 to bind and deacetylate mitochondrial transcription factor A (TFAM) was detected by immunoprecipitation assay, and TFAM acetylation sites were further validated using mass spectrometry. RESULTS: GAS increased SIRT3 expression and ameliorated mitochondrial structure, mitochondrial respiration, mitochondrial dynamics along with upregulated TFAM, mitigating oxidative stress and senescence. Comparable results were noted with the SIRT3 agonist RES, indicating an impactful neuroprotection played by SIRT3. Specifically, the attenuation of SIRT3 expression through knockdown techniques or exposure to the SIRT3 inhibitor 3-TYP in HT22 cells markedly abrogated GAS-mediated mitochondrial rescuing function. Furthermore, our findings elucidate a novel facet: SIRT3 interacted with and deacetylated TFAM at the K5, K7, and K8 sites. Decreased SIRT3 is accompanied by hyper-acetylated TFAM. CONCLUSION: The present results were the first to demonstrate that the SIRT3/TFAM pathway is a protective target for reversing mitochondrial dysfunction in VaD. The findings suggest that GAS-mediated modulation of the SIRT3/TFAM pathway, a novel mechanism, could ameliorate CCH-induced VaD, offering a potentially beneficial therapeutic strategy for VaD.

Essential oil from Fructus Alpinia zerumbet ameliorates atherosclerosis by activating PPARγ-LXRα-ABCA1/G1 signaling pathway.

BACKGROUND: Atherosclerosis (AS) is a progressive chronic disease. Currently, cardiovascular diseases (CVDs) caused by AS is responsible for the global increased mortality. Yanshanjiang as miao herb in Guizhou of China is the dried and ripe fruit of Fructus Alpinia zerumbet. Accumulated evidences have confirmed that Yanshanjiang could ameliorate CVDs, including AS. Nevertheless, its effect and mechanism on AS are still largely unknown. PURPOSE: To investigate the role of essential oil from Fructus Alpinia zerumbet (EOFAZ) on AS, and the potential mechanism. METHODS: A high-fat diet (HFD) ApoE-/- mice model of AS and a oxLDL-induced model of macrophage-derived foam cells (MFCs) were reproduced to investigate the pharmacological properties of EOFAZ on AS in vivo and foam cell formation in vitro, respectively. The underlying mechanisms of EOFAZ were investigated using Network pharmacology and molecular docking. EOFAZ effect on PPARγ protein stability was measured using a cellular thermal shift assay (CETSA). Pharmacological agonists and inhibitors and gene interventions were employed for clarifying EOFAZ's potential mechanism. RESULTS: EOFAZ attenuated AS progression in HFD ApoE-/- mice. This attenuation was manifested by the reduced aortic intima plaque development, increased collagen content in aortic plaques, notable improvement in lipid profiles, and decreased levels of inflammatory factors. Moreover, EOFAZ inhibited the formation of MFCs by enhancing cholesterol efflux through activiting the PPARγ-LXRα-ABCA1/G1 pathway. Interestingly, the pharmacological knockdown of PPARγ impaired the beneficial effects of EOFAZ on MFCs. Additionally, our results indicated that EOFAZ reduced the ubiquitination degradation of PPARγ, and the chemical composition of EOFAZ directly bound to the PPARγ protein, thereby increasing its stability. Finally, PPARγ knockdown mitigated the protective effects of EOFAZ on AS in HFD ApoE-/- mice. CONCLUSION: These findings represent the first confirmation of EOFAZ's in vivo anti-atherosclerotic effects in ApoE-/- mice. Mechanistically, its chemical constituents can directly bind to PPARγ protein, enhancing its stability, while reducing PPARγ ubiquitination degradation, thereby inhibiting foam cell formation via activation of the PPARγ-LXRα-ABCA1/G1 pathway. Simultaneously, EOFAZ could ameliorates blood lipid metabolism and inflammatory microenvironment, thus synergistically exerting its anti-atherosclerotic effects.

A Unique G-Quadruplex Aptamer: A Novel Approach for Cancer Cell Recognition, Cell Membrane Visualization, and RSV Infection Detection.

Surface staining has emerged as a rapid technique for applying external stains to trace cellular identities in diverse populations. In this study, we developed a distinctive aptamer with selective binding to cell surface nucleolin (NCL), bypassing cytoplasmic internalization. Conjugation of the aptamer with a FAM group facilitated NCL visualization on live cell surfaces with laser confocal microscopy. To validate the aptamer-NCL interaction, we employed various methods, including the surface plasmon resonance, IHC-based flow cytometry, and electrophoretic mobility shift assay. The G-quadruplex formations created by aptamers were confirmed with a nuclear magnetic resonance and an electrophoretic mobility shift assay utilizing BG4, a G-quadruplex-specific antibody. Furthermore, the aptamer exhibited discriminatory potential in distinguishing between cancerous and normal cells using flow cytometry. Notably, it functioned as a dynamic probe, allowing real-time monitoring of heightened NCL expression triggered by a respiratory syncytial virus (RSV) on normal cell surfaces. This effect was subsequently counteracted with dsRNA transfection and suppressed the NCL expression; thus, emphasizing the dynamic attributes of the probe. These collective findings highlight the robust versatility of our aptamer as a powerful tool for imaging cell surfaces, holding promising implications for cancer cell identification and the detection of RSV infections.

Unveiling the role of G-quadruplex structure in promoter region: Regulation of ABCA1 expression in macrophages possibly via NONO protein recruitment.

ABCA1 has been found to be critical for cholesterol efflux in macrophages. Understanding the mechanism regulating ABCA1 expression is important for the prevention and treatment of atherosclerosis. In the present study, a G-quadruplex (G4) structure was identified in the ABCA1 promoter region. This G4 was shown to be essential for ABCA1 transcription. Stabilizing the G4 by ligands surprisingly upregulated ABCA1 expression in macrophages. Knocking out the G4 remarkably reduced ABCA1 expression, and abolished the increase of ABCA1 expression induced by the G4 ligand. By pull-down assays, the protein NONO was identified as an ABCA1 G4 binder. Overexpression or repression of NONO significantly induced upregulation and downregulation of ABCA1 expression, respectively. ChIP and EMSA experiments showed that the G4 ligand promoted the binding between the ABCA1 G4 and NONO, which led to more recruitment of NONO to the promoter region and enhanced ABCA1 transcription. Finally, the G4 ligand was shown to significantly reduce the accumulation of cholesterol in macrophages. This study showed a new insight into the regulation of gene expression by G4, and provided a new molecular mechanism regulating ABCA1 expression in macrophages. Furthermore, the study showed a possible novel application of the G4 ligand: preventing and treating atherosclerosis.

Gastrodin relieves cognitive impairment by regulating autophagy via PI3K/AKT signaling pathway in vascular dementia.

Vascular dementia (VaD), the second most common type of dementia, is attributed to lower cerebral blood flow. To date, there is still no available clinical treatment for VaD. The phenolic glucoside gastrodin (GAS) is known for its neuroprotective effects, but the role and mechanisms of action on VD remains unclear. In this study, we aim to investigate the neuroprotective role and underlying mechanisms of GAS on chronic cerebral hypoperfusion (CCH)-mediated VaD rats and hypoxia-induced injury of HT22 cells. The study showed that GAS relieved learning and memory deficits, ameliorated hippocampus histological lesions in VaD rats. Additionally, GAS down-regulated LC3II/I, Beclin-1 levels and up-regulated P62 level in VaD rats and hypoxia-injured HT22 cells. Notably, GAS rescued the phosphorylation of PI3K/AKT pathway-related proteins expression, which regulates autophagy. Mechanistic studies verify that YP-740, a PI3K agonist, significantly resulted in inhibition of excessive autophagy and apoptosis with no significant differences were observed in the YP-740 and GAS co-treatment. Meantime, we found that LY294002, a PI3K inhibitor, substantially abolished GAS-mediated neuroprotection. These results revealed that the effects of GAS on VaD are related to stimulating PI3K/AKT pathway-mediated autophagy, suggesting a potentially beneficial therapeutic strategy for VaD.

Leocarpinolide B Attenuates Collagen Type II-Induced Arthritis by Inhibiting DNA Binding Activity of NF-κB.

Rheumatoid arthritis (RA) is a chronic autoimmune disease triggered by a cascading inflammatory response. Sigesbeckia Herba (SH) has long been utilized as a traditional remedy to alleviate symptoms associated with rheumatism. Our previous study found that leocarpinolide B (LB), a sesquiterpene lactone isolated from the whole plant of SH, possesses potent a anti-inflammatory effect on macrophages. This study was designed to evaluate the therapeutic effects of LB on RA, and further investigate the underlying mechanisms. In collagen type II-induced arthritic mice, LB was demonstrated to decrease the production of autoimmune antibodies in serum and inflammatory cytokines in the joint muscles and recover the decreased regulatory T lymphocytes in spleen. Moreover, LB significantly suppressed the inflammatory infiltration, formation of pannus and bone erosion in the paw joints. In vitro testing showed that LB inhibited the proliferation, migration, invasion, and secretion of inflammatory cytokines in IL-1ß-induced human synovial SW982 cells. Network pharmacology and molecular docking suggested NF-κB p65 could be the potential target of LB on RA treatment, subsequent experimental investigation confirmed that LB directly interacted with NF-κB p65 and reduced the DNA binding activity of NF-κB in synovial cells. In conclusion, LB significantly attenuated the collagen type II-induced arthritis, which was at least involved in the inhibition of DNA binding activity of NF-κB through a direct binding to NF-κB p65. These findings suggest that LB could be a valuable lead compound for developing anti-RA drugs.

Diversity, morphology, and molecular phylogeny of Diatrypaceae from southern China.

During an investigation of Diatrypaceae from southern China, 10 xylariales-like taxa have been collected. Morphological and multi-gene analyses confirmed that these taxa reside in Diatrypaceae and represent eight novel taxa and two new records belonging to six genera (viz., Allocryptovalsa, Diatrype, Diatrypella, Paraeutypella, Peroneutypa, and Vasilyeva gen. nov.). Vasilyeva gen. nov. was proposed to accommodate Vasilyeva cinnamomi sp. nov. Among the other collections, seven new species were introduced (viz., Diatrype camelliae-japonicae sp. nov., Diatrype rubi sp. nov., Diatrypella guiyangensis sp. nov., Diatrypella fatsiae-japonicae sp. nov., Paraeutypella subguizhouensis sp. nov., Peroneutypa hainanensis sp. nov., and Peroneutypa qianensis sp. nov.), while two were reported as new records from China (Allocryptovalsa rabenhorstii and Diatrype enteroxantha). For Diatrypaceae, the traditional taxonomic approach based on morphology may not be applicable.

Lignans and Flavonoids from Cajanus cajan (L.) Millsp. and Their α-Glucosidase Inhibitory Activities.

A pair of new lignan conformers (1-2), one new flavonoid glycoside (3), as well as nineteen known compounds were purified from the twigs and leaves of Cajanus cajan (L.) Millsp.. The planar structures of the unknown compounds were determined via NMR and high-resolution mass spectrometry, while their absolute configurations were elucidated via comparison between their experimental and calculated electronic circular dichroism (ECD) values. All the isolated compounds were assayed for their α-glucosidase inhibitory activities. The results demonstrated that compounds 8-12, 15-16, 18-19, 21-22 had strong inhibition activities, with compound 10 (IC50 =0.4±0.21 µM) most active. The structure-activity relationships were preliminarily summarized. Enzyme kinetics showed that compounds 8, 9, 15-16, 18-19, 21-22 were non-competitive inhibitors and compounds 10-12 were anti-competitive ones.

Tetraphenylethene derivative that discriminates parallel G-quadruplexes.

G-Quadruplex (G4), as a non-canonical nucleic acid secondary structure, has been proved to be prevalent in genomes and plays important roles in many biological processes. Ligands targeting G4, especially small-molecular fluorescent light-up probes with selectivity for special conformations, are essential for studying the relationship between G4 folding and the cellular response. However, their development still remains challenging but is attracting massive attention. Here, we synthesized a new tetraphenylethene derivative, namely TPE-B, as a parallel G4 probe. Fluorescence experiments showed that TPE-B could give out a strong fluorescence response to the G4 structure. Moreover, it gave a much higher fluorescence intensity response to parallel G4s than anti-parallel ones, which indicated that TPE-B could serve as a special tool for probing parallel G4s. The circular dichroism (CD) spectra and melting curves showed that TPE-B could selectively bind and stabilize parallel G4s without changing their topology. ESI-MS studies showed that TPE-B could bind to parallel G4 with a 1 : 1 stoichiometry. The gel staining results showed that TPE-B was a good candidate for probing parallel G4s. Altogether, the TPE-B molecule may serve as a promising new probe that can discriminate parallel G4s.

Morpho-molecular characterisation of Arecophila, with A.australis and A.clypeata sp. nov. and A.miscanthi comb. nov.

Three arecophila-like fungal samples were collected on dead culms of gramineous plants in China. Morphological studies of our new collections and the herbarium specimen of Arecophilagulubiicola (generic type) were conducted and the morphological affinity of our new collections with Arecophila was confirmed. Maximum likelihood and Bayesian analyses using combined ITS, LSU, rpb2 and ß-tubulin data from our collections revealed the phylogeny of Cainiaceae. The monospecific genus Alishanica (type species Al.miscanthi), which had been accepted in Cainiaceae, is revisited and synonymised under Arecophila. Based on morphology and phylogeny, Arecophilaaustralis sp. nov. and A.clypeata sp. nov. are introduced as new species, while A.miscanthi is a new record for China. All the new collections are illustrated and described.

Spectrum-Efficacy Relationships between GC-MS Fingerprints of Essential Oil from Valerianae Jatamansi Rhizoma et Radix and the Efficacy of Inhibiting Microglial Activation.

The bioactive ingredients of essential oil from Valerianae Jatamansi Rhizoma et Radix (the Rhizome et Radix from Valerianae Jatamansi Jones) (EOVJRR) on the efficacy of inhibiting microglial activation were investigated with the approach of spectrum-efficacy relationship. Fourteen batches of Valerianae Jatamansi Rhizoma et Radix were extracted and analyzed by gas chromatography-mass spectrometry (GC-MS), and their activities in the efficacy of inhibiting microglial activation were assayed by measuring the inflammatory responses induced by lipopolysaccharide (LPS) in microglia cells from mice. The spectrum-efficacy relationships between fingerprints and the efficacy of inhibiting microglial activation of EOVJRR were established by grey relational analysis (GRA). Twenty common peaks were obtained from the GC-MS fingerprints of EOVJRR. P12 (vetivenol), P1 (bornyl acetate), P5 (seychellene), and P3 (ß-elemene) indicated inhibition on microglia activation together, according to the spectrum-efficacy relationships. The current results established a general model for the spectrum-efficacy relationships of EOVJRR by GC-MS and the efficacy of inhibiting microglial activation, which could be applied to identify the bioactive ingredient and control the quality of herbs.

P2Y1R Ligation Suppresses Th17 Cell Differentiation and Alleviates Colonic Inflammation in an AMPK-Dependent Manner.

P2Y1 receptor is a G-protein-coupled receptor that plays a critical role in the immune response of inflammatory bowel diseases. However, its regulatory effects on CD4+ T cell response have not been fully elucidated. The study aimed to characterize the role of P2Y1R in Th17 cell differentiation and colonic inflammation. Our results demonstrated that P2Y1R was significantly increased in the splenocytes of colitic mice, which was positively associated with the expression of RORγt and IL-17A. P2Y1R deficiency significantly ameliorated DSS-induced colitis and its Th17 responses. In parallel, P2Y1R deficiency greatly impaired the differentiation of Th17 cell, down-regulated the mRNA expression of IL-17A and RORγt, and protein expression of RORγt in vitro. More importantly, it was found that P2Y1R deficiency markedly increased AMPK phosphorylation of Th17 polarized CD4+ T cells, and antagonist of AMPK significantly reversed the inhibitory effect of P2Y1R deficiency on Th17 cell generation in vivo and in vitro. Overall, these findings demonstrated that P2Y1R deficiency could suppress Th17 cell differentiation in an AMPK-dependent manner to ameliorate colitis, and P2Y1R can act as an important regulator of Th17 cell differentiation to control colonic inflammation.

New species and records of Neomassaria, Oxydothis and Roussoella (Pezizomycotina, Ascomycota) associated with palm and bamboo from China.

Several micro fungi were gathered from bamboo and palm in Guizhou Province, China. In morphology, these taxa resemble Neomassaria, Roussoella and Oxydothis. Multi-gene phylogenetic analyses based on combined ITS, LSU, SSU, rpb2 and tef1 loci confirmed that two are new geographical records for China, (viz. Roussoellasiamensis, Neomassariafabacearum), while two of them are new to science (viz. Oxydothisfortunei sp. nov. and Roussoellabambusarum sp. nov.). The stromata of Roussoellabambusarum are similar to those of R.thailandica, but its ascospores are larger. In addition, multi-gene phylogenetic analyses show that Oxydothisfortunei is closely related to O.inaequalis, but the J- ascus subapical ring as well as the ascospores of O.inaequalis are smaller. Morphological descriptions and illustrations of all species are provided.

Genetic screening for hypertension and COVID-19 reveals functional variation of SPEG potentially associated with severe COVID-19 in women.

The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to more than 6.4 million deaths worldwide. The prevalent comorbidity between hypertension and severe COVID-19 suggests common genetic factors may affect the outcome of both diseases. As both hypertension and severe COVID-19 demonstrate sex-biased prevalence, common genetic factors between the two diseases may display sex-biased differential associations. By evaluating COVID-19 association signals of 172-candidate hypertension single nucleotide polymorphisms (SNPs) derived from more than 1 million European individuals in two sex-stratified severe COVID-19 genome-wide association studies from UK BioBank with European ancestry, we revealed one functional cis expression quantitative trait locus of SPEG (rs12474050) showing sex-biased association with severe COVID-19 in women. The risk allele rs12474050*T associates with higher blood pressure. In our study, we found it is significantly correlated with lower SPEG expression in muscle-skeletal but with higher expression in both brain cerebellum and cerebellar hemisphere. Additionally, nominal significances were detected for the association between rs12474050*T and lower SPEG expression in both heart left ventricle and atrial appendage; among these tissues, the SPEG expression is nominally significantly higher in females than in males. Further analysis revealed SPEG is mainly expressed in cardiomyocytes in heart and is upregulated upon SARS-CoV-2 infection, with significantly higher upregulation of SPEG only observed in female but not in male COVID-19 patients compared to both normal female and male individuals, suggesting upregulation of SPEG is a female-specific protective mechanism against COVID-19 induced heart damage. Taken together, our analyses suggest the involvement of SPEG in both hypertension and severe COVID-19 in women, which provides new insights for sex-biased effect of severe COVID-19 in women.

A Small Ligand That Selectively Binds to the G-quadruplex at the Human Vascular Endothelial Growth Factor Internal Ribosomal Entry Site and Represses the Translation.

G-quadruplexes are believed to have important biological functions, so many small molecules have been screened or developed for targeting G-quadruplexes. However, it is still a major challenge to find molecules that recognize specific G-quadruplexes. Here, by using a combination of surface plasmon resonance, electrospray ionization mass spectrometry, circular dichroism, Western blot, luciferase assay, and reverse transcriptase stop assay, we observed a small molecule, namely, oxymatrine (OMT) that could selectively bind to the RNA G-quadruplex in 5'-untranslated regions (UTRs) of human vascular endothelial growth factor (hVEGF), but could not bind to other G-quadruplexes. OMT could selectively repress the translation of VEGF in cervical cancer cells. Furthermore, it could recognize VEGF RNA G-quadruplexes in special conformations. The results indicate that OMT may serve as a potentially special tool for studying the VEGF RNA G-quadruplex in cells and as a valuable scaffold for the design of ligands that recognize different G-quadruplexes.

Intraarterial Lidocaine Administration for Pain Control by Water-in-Oil Technique in Transarterial Chemoembolization: in vivo and Randomized Clinical Trial.

OBJECTIVE: To investigate the sustained release of lidocaine from a lidocaine-epirubicin-lipiodol emulsion created by water-in-oil (W/O) technique in vivo and evaluate the efficacy and safety of intraarterial lidocaine administration for intra- and postoperative pain control in transarterial chemoembolization (TACE) for hepatocellular carcinoma (HCC). METHODS: The in vivo concentrations of lidocaine were determined in tumor tissues after VX2 rabbit models for hepatic tumor were administered with intra-arterial lidocaine-epirubicin-lipiodol emulsion. A prospective randomized controlled clinical trial was performed, enrolling 70 consecutive patients who underwent TACE. Patients were randomized into two groups: Group A received an immediate bolus intraarterial lidocaine injection before TACE, and Group B received a lidocaine-epirubicin-lipiodol emulsion during TACE. Pain intensity was compared between the two groups using a visual analog scale (VAS) score before (Tbefore) and at 0 h (T0), 4 h (T4), 8 h (T8), 24 h (T24), 48 h (T48), and 72 h (T72) after the procedure. Adverse events and intake of analgesics were evaluated and compared between the two groups. RESULTS: The concentrations of lidocaine in tumor tissues were higher in experimental group than in control group at T0.5 (P=0.004), T1 (P=0.038), T4 (P=0.036), and T8 (P=0.029). In the clinical trial, VAS scores in Group B were significantly lower than in Group A at T0 (P=0.006), T4 (P=0.001), T8 (P=0.002), and T24 (P=0.005). The tramadol intake in Group B was significantly lower than in Group A (P=0.021). No significant difference was observed regarding the incidence of adverse events between the two groups. CONCLUSION: This study demonstrated the effectiveness and safety of intraarterial lidocaine administration using the W/O technique in controlling intra- and post-TACE pain.

A taxonomic study of Nemania from China, with six new species.

During an investigation of Xylariaceae from 2019 to 2020, isolates representing eight Nemania (Xylariacese) species were collected from Yunnan, Guizhou and Hainan Provinces in China. Morphological and multi-gene phylogenetic analyses, based on combined ITS, α-actin, rpb2 and ß-tubulin sequences, confirmed that six of them are new to science, viz. Nemaniacamelliae, N.changningensis, N.cyclobalanopsina, N.feicuiensis, N.lishuicola and N.rubi; one is a new record (N.caries) for China and one is a known species (N.diffusa). Morphological descriptions and illustrations of all species are detailed. In addition, the characteristics of Nemania are summarised and prevailing contradictions in generic concepts are discussed.

VEGF121 Mediates Post-Hypoxia Cardioprotective Effects Via CaSR and Mitochondria-Dependent Protease Pathway. / VEGF121 como Mediador de Efeitos Cardioprotetores Pós-Hipóxia via CaSR e via da Protease Dependente de Mitocôndria.

BACKGROUND: Cardiovascular disease is the major cause of death worldwide. Hypoxia-mediated apoptosis in cardiomyocytes is a major cause of cardiovascular disorders. Treatment with vascular endothelial growth factor (VEGF) protein has been tested but operational difficulties have limited its use. However, with the advancements of gene therapy, interest has risen in VEGF-based gene therapy in cardiovascular disorders. However, the precise mechanism by which VEGF replenishment rescues post-hypoxia damage in cardiomyocytes is not known. OBJECTIVES: To investigate the effect of post-hypoxia VEGF121 expression using neonatal rat cardiomyocytes. METHODS: Cardiomyocytes isolated from neonatal rats were used to establish an in vitro model of hypoxia-induced cardiac injury. The effect of VEGF overexpression, alone or in combination with small-molecule inhibitors targeting calcium channel, calcium sensitive receptors (CaSR), and calpain on cell growth and proliferation on hypoxia-induced cardiomyocyte injury were determined using an MTT assay, TUNEL staining, Annexin V/PI staining, lactate dehydrogenase and caspase activity. For statistical analysis, a value of P<0.05 was considered to be significant. RESULTS: The effect of VEGF121 was found to be mediated by CaSR and calpain but was not dependent on calcium channels. CONCLUSIONS: Our findings, even though using an in vitro setting, lay the foundation for future validation and pre-clinical testing of VEGF-based gene therapy in cardiovascular diseases.

FUNDAMENTO: A doença cardiovascular é a principal causa de morte em todo o mundo. A apoptose mediada por hipóxia em cardiomiócitos é uma das principais causas de distúrbios cardiovasculares. O tratamento com a proteína do fator de crescimento endotelial vascular (VEGF, do inglês vascular endothelial growth factor) foi testado, mas as dificuldades operacionais limitaram seu uso. Entretanto, com os avanços da terapia gênica, aumentou o interesse na terapia gênica baseada no VEGF em doenças cardiovasculares. No entanto, o mecanismo preciso pelo qual a reposição de VEGF resgata os danos pós-hipóxia em cardiomiócitos não é conhecido. OBJETIVOS: Investigar o efeito da expressão de VEGF121 pós-hipóxia utilizando cardiomiócitos de ratos neonatos. MÉTODOS: Cardiomiócitos isolados de ratos neonatos foram utilizados para estabelecer um modelo in vitro de lesão cardíaca induzida por hipóxia. O efeito da superexpressão de VEGF, isolado ou em conjunto com inibidores de moléculas pequenas que têm como alvo os canais de cálcio, receptores sensíveis ao cálcio (CaSR, do inglês calcium-sensitive receptors) e calpaína, no crescimento e proliferação celular em lesão de cardiomiócitos induzidos por hipóxia, foram determinados com ensaio de MTT, coloração TUNEL, coloração com Anexina V/PI, lactato desidrogenase e atividade da caspase. Para análise estatística, um valor de p<0,05 foi considerado significativo. RESULTADOS: Verificou-se que o efeito do VEGF121 foi mediado por CaSR e calpaína, mas não foi dependente dos canais de cálcio. CONCLUSÕES: Nossos resultados, mesmo em um ambiente in vitro, estabelecem as bases para uma validação futura e testes pré-clínicos da terapia gênica baseada em VEGF em doenças cardiovasculares.

VEGF121 como Mediador de Efeitos Cardioprotetores Pós-Hipóxia via CaSR e via da Protease Dependente de Mitocôndria / VEGF121 Mediates Post-Hypoxia Cardioprotective Effects Via CaSR and Mitochondria-Dependent Protease Pathway

Resumo Fundamento: A doença cardiovascular é a principal causa de morte em todo o mundo. A apoptose mediada por hipóxia em cardiomiócitos é uma das principais causas de distúrbios cardiovasculares. O tratamento com a proteína do fator de crescimento endotelial vascular (VEGF, do inglês vascular endothelial growth factor) foi testado, mas as dificuldades operacionais limitaram seu uso. Entretanto, com os avanços da terapia gênica, aumentou o interesse na terapia gênica baseada no VEGF em doenças cardiovasculares. No entanto, o mecanismo preciso pelo qual a reposição de VEGF resgata os danos pós-hipóxia em cardiomiócitos não é conhecido. Objetivos: Investigar o efeito da expressão de VEGF121 pós-hipóxia utilizando cardiomiócitos de ratos neonatos. Métodos: Cardiomiócitos isolados de ratos neonatos foram utilizados para estabelecer um modelo in vitro de lesão cardíaca induzida por hipóxia. O efeito da superexpressão de VEGF, isolado ou em conjunto com inibidores de moléculas pequenas que têm como alvo os canais de cálcio, receptores sensíveis ao cálcio (CaSR, do inglês calcium-sensitive receptors) e calpaína, no crescimento e proliferação celular em lesão de cardiomiócitos induzidos por hipóxia, foram determinados com ensaio de MTT, coloração TUNEL, coloração com Anexina V/PI, lactato desidrogenase e atividade da caspase. Para análise estatística, um valor de p<0,05 foi considerado significativo. Resultados: Verificou-se que o efeito do VEGF121 foi mediado por CaSR e calpaína, mas não foi dependente dos canais de cálcio. Conclusões: Nossos resultados, mesmo em um ambiente in vitro, estabelecem as bases para uma validação futura e testes pré-clínicos da terapia gênica baseada em VEGF em doenças cardiovasculares.

Abstract Background: Cardiovascular disease is the major cause of death worldwide. Hypoxia-mediated apoptosis in cardiomyocytes is a major cause of cardiovascular disorders. Treatment with vascular endothelial growth factor (VEGF) protein has been tested but operational difficulties have limited its use. However, with the advancements of gene therapy, interest has risen in VEGF-based gene therapy in cardiovascular disorders. However, the precise mechanism by which VEGF replenishment rescues post-hypoxia damage in cardiomyocytes is not known. Objectives: To investigate the effect of post-hypoxia VEGF121 expression using neonatal rat cardiomyocytes. Methods: Cardiomyocytes isolated from neonatal rats were used to establish an in vitro model of hypoxia-induced cardiac injury. The effect of VEGF overexpression, alone or in combination with small-molecule inhibitors targeting calcium channel, calcium sensitive receptors (CaSR), and calpain on cell growth and proliferation on hypoxia-induced cardiomyocyte injury were determined using an MTT assay, TUNEL staining, Annexin V/PI staining, lactate dehydrogenase and caspase activity. For statistical analysis, a value of P<0.05 was considered to be significant. Results: The effect of VEGF121 was found to be mediated by CaSR and calpain but was not dependent on calcium channels. Conclusions: Our findings, even though using an in vitro setting, lay the foundation for future validation and pre-clinical testing of VEGF-based gene therapy in cardiovascular diseases.

MicroRNAs Regulating Mitochondrial Function in Cardiac Diseases.

Mitochondria are the key organelles that supply cellular energy. As the most active organ in the body, the energy required to maintain the mechanical function of the heart requires a high quantity of high-quality mitochondria in cardiomyocytes. MicroRNAs (miRNAs) are single-stranded noncoding RNAs, approximately 22 nt in length, which play key roles in mediating post-transcriptional gene silencing. Numerous studies have confirmed that miRNAs can participate in the occurrence and development of cardiac diseases by regulating mitochondrial function-related genes and signaling pathways. Therefore, elucidating the crosstalk that occurs between miRNAs and mitochondria is important for the prevention and treatment of cardiac diseases. In this review, we discuss the biogenesis of miRNAs, the miRNA-mediated regulation of major genes involved in the maintenance of mitochondrial function, and the effects of miRNAs on mitochondrial function in cardiac diseases in order to provide a theoretical basis for the clinical prevention and treatment of cardiac disease and the development of new drugs.