Combination treatment of recombinant growth differentiation factor-9 and Cetrorelix improves gestational origin of the polycystic ovarian syndrome in female rats.

AIMS: Polycystic ovary syndrome (PCOS) is a hyper-androgenic endocrinopathy prevalent in premenopausal women with no cure available. The current study aimed to investigate the therapeutic effect of recombinant GDF-9 and Cetrorelix on the gestational origin of dehydroepiandrosterone (DHEA) induced PCOS in postnatal pups' delivered to rat dams. MAIN METHODS: The body weight measurement, blood and serum analysis for glucose tolerance, lipid profile, liver enzymes, sex hormones (Testosterone, Estradiol, and Progesterone), estrus cyclicity assessment, histological staining of ovary and liver, molecular markers expressions of pro-inflammatory by qRT-PCR and immuno-histochemistry technique for folliculogenesis genes and histological staining studies of liver and ovary were done. KEY FINDINGS: The combinational treatment was found to normalize the biochemical parameters and reduction in the estrus irregularity by altering the sex hormones as well as the glucose metabolism and insulin resistance via HOMA-IR value. Further, molecular markers expression confirmed the pro-inflammatory (IL-1ß, TNF-α, and IL-6) and folliculogenesis (GDF-9, BMPR2, and TGF-ßR1) genes associated with PCOS were improved by combinational therapy. SIGNIFICANCE: In conclusion, rGDF-9 could be a potential therapeutic agent in combination with Cetrorelix as a better treatment regime for metabolic and reproductive phenotypes in PCOS. However, the effect of rGDF-9 on infertility-associated phenotypes in PCOS needs further evaluation.

Sinigrin Attenuates the Dextran Sulfate Sodium-induced Colitis in Mice by Modulating the MAPK Pathway.

Ulcerative colitis (UC) is an intestinal inflammatory disease characterised by the loss of intestinal crypts, edema, mucosal ulceration, and infiltration of inflammatory cells in the mucosa. The current study aimed to investigate the protective and therapeutic effects of sinigrin and underlying mechanisms in a dextran sulfate sodium (DSS)-induced mouse model of ulcerative colitis. DSS-induced colitis models were used to demonstrate sinigrin's therapeutic/protective action. Mice were orally administered with sinigrin (15 mg/kg or 30 mg/kg) for a period of 12 days in both prophylactic and therapeutic models. Animal weights, stool consistency, and bleeding parameters were measured throughout the experimental period. After the experimental period, colon lengths were measured, and colon tissues were harvested to determine the levels of oxidative stress-inducing factors (nitrates and MDA levels) and anti-oxidant components (GSH, SOD, and catalase). Furthermore, gene expression analysis, IL-17 levels, and inflammatory marker expressions were measured using RT-qPCR, ELISA, and immunohistochemical methods respectively. Furthermore, histopathological observations and elucidation of the mechanism of action were determined using H&E analysis and Western blot analysis. Sinigrin treatment (in both prophylactic and therapeutic models) significantly mitigated the DSS-induced body weight loss, attenuated the colon length shrinkage, and improved the disease index score (p < 0.001). Further results revealed that sinigrin's protective/therapeutic effect is associated with a significant attenuation of pro­inflammatory cytokine production (p < 0.001), reversing the anti-oxidant enzyme levels (p < 0.001) and substantial improvement (2 folds) of the disruption of the colonic morphology in colon tissues compared to DSS control. Immunohistochemical analysis showed that sinigrin treatment remarkably reduced the DSS-induced myeloperoxidase, neutrophil elastase, and CD68 expression in colon tissues. Additionally, sinigrin successfully abrogated the DSS-induced IL-17 levels (p < 0.001) and improved the colonic barrier in colon tissues. Overall, these results demonstrated that sinigrin exerts protective and therapeutic effects on DSS­induced colitis, by enhancing the anti-oxidant enzymes and suppressing the intestinal inflammatory cascade of markers by regulating the MAPK pathway.

Long-term and non-invasive in vivo tracking of DiD dye-labeled human hepatic progenitors in chronic liver disease models.

BACKGROUND: Chronic liver diseases (CLD) are the major public health burden due to the continuous increasing rate of global morbidity and mortality. The inherent limitations of organ transplantation have led to the development of stem cell-based therapy as a supportive and promising therapeutic option. However, identifying the fate of transplanted cells in vivo represents a crucial obstacle. AIM: To evaluate the potential applicability of DiD dye as a cell labeling agent for long-term, and non-invasive in vivo tracking of transplanted cells in the liver. METHODS: Magnetically sorted, epithelial cell adhesion molecule positive (1 × 106 cells/mL) fetal hepatic progenitor cells were labeled with DiD dye and transplanted into the livers of CLD-severe combined immunodeficiency (SCID) mice. Near-infrared (NIR) imaging was performed for in vivo tracking of the DiD-labeled transplanted cells along with colocalization of hepatic markers for up to 80 d. The existence of human cells within mouse livers was identified using Alu polymerase chain reaction and sequencing. RESULTS: NIR fluorescence imaging of CLD-SCID mice showed a positive fluorescence signal of DiD at days 7, 15, 30, 45, 60, and 80 post-transplantation. Furthermore, positive staining of cytokeratin, c-Met, and albumin colocalizing with DiD fluorescence clearly demonstrated that the fluorescent signal of hepatic markers emerged from the DiD-labeled transplanted cells. Recovery of liver function was also observed with serum levels of glutamic-oxaloacetic transaminase, glutamate-pyruvate transaminase, and bilirubin. The detection of human-specific Alu sequence from the transplanted mouse livers provided evidence for the survival of transplanted cells at day 80. CONCLUSION: DiD-labeling is promising for long-term and non-invasive in vivo cell tracking, and understanding the regenerative mechanisms incurred by the transplanted cells.

Mitochondria-targeted esculetin mitigates atherosclerosis in the setting of aging via the modulation of SIRT1-mediated vascular cell senescence and mitochondrial function in Apoe-/- mice.

BACKGROUND AND AIMS: Age is a dominant and independent risk factor for the development of atherosclerosis, a major cardiovascular disease, and if left untreated leads to myocardial infarction and death. Mitochondria-targeted anti-oxidants are evolving as a new class of compounds that can alter the pathophysiology of age-related diseases, including atherosclerosis, where mitochondrial dysfunction plays a critical role in disease progression. METHODS: We recently synthesized an alkyl TPP + -tagged esculetin (mitochondria-targeted esculetin or Mito-Esc). Apoe-/- mice were chronically (14 months) administered with Mito-Esc to investigate its efficacy in the mitigation of atherosclerosis in the setting of aging. We monitored BP, and performed various biochemical assays, histopathology, immunohistochemistry, inflammatory factors, qPCR, and Western blotting. Simultaneously, human aortic endothelial cells (HAECs) were used as a model system to study the mechanistic aspects. RESULTS: A chronic low-dose administration of Mito-Esc to Apoe-/- mice greatly prevented alterations in lipid profile, blood pressure, and atherosclerotic plaque formation in the setting of aging. Mito-Esc administration significantly reduced vascular senescence and pro-inflammatory cytokines levels and prevented dysregulation of mitochondrial biogenesis markers in aortic tissue. Further, Mito-Esc treatment prevented replicative and stress-induced premature senescence (SIPS) in HAEC. Importantly, Mito-Esc treatment delayed endothelial cell senescence by increasing human telomerase reverse transcriptase (hTERT) levels via SIRT1 activation. Moreover, Mito-Esc treatment by altering miR-19b and miR-30c via a SIRT1 activation significantly inhibited the increase in PAI-1 levels in HAEC as well as in the serum of Apoe-/- mice. In addition, Mito-Esc treatment improved mitochondrial function in late passage (aged) HAECs by enhancing the oxygen consumption rate (OCR). Furthermore, Mito-Esc administration counteracted the decline in GSH and nitrite levels in Apoe-/- mice and in HAECs. CONCLUSIONS: Overall, Mito-Esc alleviates atherosclerosis in the setting of aging by delaying vascular senescence and pro-inflammatory processes, and by improving mitochondrial biogenesis and function.

Sub-chronic oral toxicity evaluation of herbo-metallic formulation Arshakuthar rasa in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Arshakuthar rasa (AR) is a mercury based Ayurvedic herbo-metallic formulation. The concerns are being raised about the probable toxicity of mercury after prolonged use of AR. Hence, there is need for a long-term repeated in vivo toxicity study. The study will provide data with scientific evidence to enable safe use of the drug. Moreover, lack of toxicity study with AR incited us to perform sub-chronic study on rats. AIM OF THE STUDY: The aim of the study is to generate data by performing a sub-chronic study to assess the toxicity of AR after its prolonged oral intake. MATERIALS AND METHODS: The female and male rats were administered with 30 (low), 300 (medium) and 600 mg/kg BW/day (high) dose of AR for 90 consecutive days. The body weight, feed consumption and water intake were monitored weekly. On 91st day, blood was collected from retro-orbital plexus of rats and then sacrificed to harvest the vital organs for biochemical, haematological, histopathological, genotoxicity along with the expression study of oxidative stress related genes and the biodistribution of elements in the blood. RESULTS: Significant alterations in serum biochemical parameters were observed at the medium and high doses. The histopathological changes were in corroboration with biochemical changes at high dose in liver. There was no detectable level of mercury in blood, less to moderate biochemical changes, no haematological changes, moderate regulation of stress-related genes, and low genotoxicity. These results indicated that AR can be considered as moderately toxic above 600 mg/kg BW and mildly toxic at 300 mg/kg BW. CONCLUSIONS: It may be interpreted that AR may not induce grave toxic response in human after long-duration of oral administration at therapeutic doses.

Dehydrozingerone ameliorates Lipopolysaccharide induced acute respiratory distress syndrome by inhibiting cytokine storm, oxidative stress via modulating the MAPK/NF-κB pathway.

BACKGROUND: Inflammation-mediated lung injury is a major cause of health problems in many countries and has been the leading cause of morbidity/mortality in intensive care units. In the current COVID-19 pandemic, the majority of the patients experienced serious pneumonia resulting from inflammation (Acute respiratory distress syndrome/ARDS). Pathogenic infections cause cytokine release syndrome (CRS) by hyperactivation of immune cells, which in turn release excessive cytokines causing ARDS. Currently, there are no standard therapies for viral, bacterial or pathogen-mediated CRS. PURPOSE: This study aimed to investigate and validate the protective effects of Dehydrozingerone (DHZ) against LPS induced lung cell injury by in-vitro and in-vivo models and to gain insights into the molecular mechanisms that mediate these therapeutic effects. METHODS: The therapeutic activity of DHZ was determined in in-vitro models by pre-treating the cells with DHZ and exposed to LPS to stimulate the inflammatory cascade of events. We analysed the effect of DHZ on LPS induced inflammatory cytokines, chemokines and cell damage markers expression/levels using various cell lines. We performed gene expression, ELISA, and western blot analysis to elucidate the effect of DHZ on inflammation and its modulation of MAPK and NF-κB pathways. Further, the prophylactic and therapeutic effect of DHZ was evaluated against the LPS induced ARDS model in rats. RESULTS: DHZ significantly (p < 0.01) attenuated the LPS induced ROS, inflammatory cytokine, chemokine gene expression and protein release in macrophages. Similarly, DHZ treatment protected the lung epithelial and endothelial cells by mitigating the LPS induced inflammatory events in a dose-dependent manner. In vivo analysis showed that DHZ treatment significantly (p < 0.001) mitigated the LPS induced ARDS pathophysiology of increase in the inflammatory cells in BALF, inflammatory cytokine and chemokines in lung tissues. LPS stimulated neutrophil-mediated events, apoptosis, alveolar wall thickening and alveolar inflammation were profoundly reduced by DHZ treatment in a rat model. CONCLUSION: This study demonstrates for the first time that DHZ has the potential to ameliorate LPS induced ARDS by inhibiting cytokine storm and oxidative through modulating the MAPK and NF-κB pathways. This data provides pre-clinical support to develop DHZ as a potential therapeutic agent against ARDS.

Galangin ameliorates Imiquimod-Induced psoriasis-like skin inflammation in BALB/c mice via down regulating NF-κB and activation of Nrf2 signaling pathways.

Psoriasis is a most common chronic autoimmune-arbitrated cutaneous inflammatory skin disorder by unclear pathogenesis. In this current study we demonstrated the effect of galangin (GAL) on imiquimod (IMQ)-induced psoriasis-like skin inflammation and decipher its possible protective mechanism which has not been investigated. The in vivo results revealed that GAL at 1% w/w and 2% w/w for six consecutive days markedly reduced IMQ-induced PASI scoring, skin, ear thickness, hematological markers, levels of nitrites, TBARS, MPO, histopathological, as well modulated the protein levels of pro-inflammatory mediators of COX-2, iNOS, NF-κB pathway and pro-inflammatory cytokines IL-17, IL-23, IL-1ß in the skin and also IL-6, TNF-α in both skin and serum. Besides, GAL restored the levels of antioxidants markers such as SOD, CAT, GST, GSH, GR and Vit-C, anti-inflammatory cytokine of IL-10, and the protein levels of Nrf2/HO-1 in the skin compared to the IMQ group. Finally, our study demonstrates that GAL exerted its protective effect by up-regulating the anti-inflammatory and the antioxidant markers against psoriasis pre-clinical models indicating its potency for treating psoriasis in humans.

Fluorinated thiazolidinol drives autophagic cell death in pancreatic cancer cells via AMPK activation and perturbation of critical sentinels of oncogenic signaling.

Pancreatic cancer is one of the most malignant cancers around the world. The co-occurrence of mutation in KRAS and p53 makes it highly aggressive, proliferative, metastatic, and resistant to apoptotic cell death. Therefore, there is a need to trigger an alternate mechanism of cancer cell death in apoptosis-resistant pancreatic cancer. Autophagic cell death could be an alternate viable option for treatment in such cases. Thus, the identification of small molecules as autophagy modulators with potent anticancer efficacy would be of great importance in pancreatic cancer. The present study investigates fluorinated thiazolidionol (FTZ) driven autophagy modulation, underlying mechanism, and regulation of critical sentinels of oncogenic signaling in pancreatic cancer cells. We identified that FTZ triggered autophagic cell death in pancreatic cancer cells, independent of apoptosis evidenced by an increase in cytoplasmic vacuoles formation, autophagy flux, LC3-II expression, and p62 degradation. Further, the crucial events of apoptosis i.e., Caspase-3 activation and PARP cleavage, were not observed, indicating the non-occurrence of apoptotic cell death. Moreover, FTZ was able to activate AMPK and suppress PI3k/Akt/mTOR as well as MEK/ERK, the key oncogenic signaling pathways in cancer cells. Furthermore, treatment with FTZ suppressed migration, invasion, and angiogenesis in pancreatic cancer cells. Studies in vivo revealed significant regression of tumors by FTZ in nude mice model. Overall, our study demonstrates that FTZ induces autophagic cell death in pancreatic cancer cells independent of apoptosis, which is accompanied by AMPK activation and suppression of critical sentinels of oncogenic signaling in pancreatic cancer cells.

Anti-inflammatory effect of stevioside abates Freund's complete adjuvant (FCA)-induced adjuvant arthritis in rats.

Adjuvant arthritis is a chronic, autoimmune and inflammatory disorder of the joints. The occurrence of disorder causes a severe damage to the connective tissue eventually leading to progressive physical disability and eventual death. The recent years of evidence suggests the anti-inflammatory properties of stevioside, a diterpene glycoside. However, the effect of stevioside against adjuvant arthritis, a chronic inflammatory disorder is not known. Hence, the present study was designed to study the effect of stevioside against Freund's complete adjuvant induced arthritis model in rats. The acute anti-inflammatory effect of stevioside also studied by employing carrageenan-induced paw oedema model in rats. The biochemical markers, haematological parameters, lipid peroxidation, myeloperoxidase activity, lipoxygenase activity, the levels of PGE2 and pro-inflammatory (TNF-α, IL-6 & IL-1ß) and anti-inflammatory cytokine (IL-10) were analysed. The protein expression of NF-κB (p65) COX-2 and iNOS in paw tissues were estimated by western blotting. Stevioside treatment significantly ameliorates the adjuvant induced arthritic scoring, histological alterations, paw volume, elevation of biochemical (AST, ALT, ALP and glucose levels) and haematological (haemoglobin, differential and platelet count) parameters and restored the endogenous anti-oxidant (SOD, CAT, GSH and GST) activities. Treatment with stevioside also significantly prevented the adjuvant induced elevation of pro-inflammatory cytokines (TNF-α, IL-6 and IL-1ß), pro-inflammatory protein expressions (iNOS, COX-2, NF-κB (p65) and pIκB/IκB ratio), prevented the increase in myeloperoxidase activity and significantly restored the anti-inflammatory (IL-10) cytokine level in paw tissues. Collectively, our findings suggest that stevioside may serve as anti-inflammatory agent and could serve as a potential adjunct therapeutic option in treating adjuvant arthritis.

Arbutin Attenuates Isoproterenol-Induced Cardiac Hypertrophy by Inhibiting TLR-4/NF-κB Pathway in Mice.

Arbutin is a glycoside reported for its anti-oxidant, anti-inflammatory and anti-tumor properties. However, the cardioprotective effect of Arbutin is not well established. The study aims to understand the effect of arbutin on isoproterenol (ISO)-induced cardiac hypertrophy in mice. The animals were pretreated with Arbutin for a week and ISO was administered for 10 days and then sacrificed. Cardiac injury markers such as creatinine kinase and lactate dehydrogenase concentrations were measured in the serum. The mRNA expression of cardiac hypertrophy markers namely atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) were measured using qRT-PCR. The levels of pro-inflammatory cytokines TNF-α and IL-6 were quantified by ELISA in isolated tissues and serum. Other tissue anti-oxidant parameters such as GST, GSH, SOD and TBARS were also measured. TUNEL assay was performed to detect apoptosis. Histology studies were performed using H & E and Masson trichome staining. Immunoblot analysis was used to quantify the protein expression of TLR-4 and NF-κB. ISO-alone-treated group showed significant increase in CK-MB, LDH along with increase in hypertrophic markers ANP and BNP, TNF-α and IL-6 levels in serum and tissues and increased cardiomyocyte apoptosis. Anti-oxidant parameters were significantly decreased and TLR-4 and NF-κB protein expression was found to be upregulated in comparison to the control group. Pretreatment with Arbutin-exhibited significant inhibition of TLR-4/NF-κB pathway with decreased levels of pro-inflammatory cytokines and enhanced myocardial anti-oxidant status. Our study demonstrated that pretreatment with Arbutin exhibits marked protective effects on ISO-induced cardiac hypertrophy in mice. Thus, Arbutin may be used as potential pharmacological interventions in the management of cardiac hypertrophy.

Protective effect of galangin against dextran sulfate sodium (DSS)-induced ulcerative colitis in Balb/c mice.

OBJECTIVE AND DESIGN: Inflammatory bowel disease (IBD) is known to cause chronic inflammation in the digestive tract by the immune malfunction. Herein, we demonstrate the protective effect of galangin (GAL), a phytochemical, on LPS-induced inflammation in cultured mouse macrophages (RAW 264.7) and the treatment of DSS-induced ulcerative colitis in Balb/c mice. However, the anti-inflammatory effect of GAL in DSS-exposed experimental colitis has not been investigated. MATERIALS AND METHODS: We determined the levels of proinflammatory cytokines by ELISA, biochemical analysis using standard protocols and protein expression level of NF-κB signaling pathway and activation of Nrf2 gene pathway were analyzed by western blot analysis in colitis-induced mice. RESULTS: Our in vitro studies showed that LPS-stimulated RAW 264.7 cells treated with GAL reduced the levels of nitrites, IL-6, and TNF-α in a concentration-dependent manner. The results demonstrated that oral administration of GAL at 20 mg/kg (lower dose) and 40 mg/kg (higher dose) significantly reduced the severity of colitis and mitigated the clinical signs of both macroscopic and microscopic of the disease. The levels of proinflammatory cytokines (TNF-α and IL-6) in colonic tissue and serum were reduced significantly and in GAL + DSS-treated group relative to DSS alone treated group.  Increased levels of anti-inflammatory cytokine (IL-10) was detected in colon tissues in GAL + DSS-treated groups relative to DSS alone treated group. We also observed decreased levels of myeloperoxidase (MPO), nitrites and TBARS with increased SOD in colonic tissue of GAL + DSS group. Besides, GAL + DSS-treated animals significantly suppressed protein expressions of p-NF-κB and p-Ikk-ßα, COX-2, iNOS, Nrf2 and increased HO-1 levels in colon tissues by inhibiting inflammation and oxidative stress. CONCLUSION: Our study highlights the protective effect of galangin as an anti-inflammatory agent against the severe form of colitis in pre-clinical models suggesting its potency for the treatment of IBD in humans.

Stevioside, a diterpenoid glycoside, shows anti-inflammatory property against Dextran Sulphate Sodium-induced ulcerative colitis in mice.

Inflammatory bowel disease is an umbrella-term used to describe a set of chronic inflammatory conditions that affect the gastro-intestinal tract. Since most of the inflammatory medications in current use have several undesirable side-effects, stevioside, a naturally occurring, high-intensity sweetener was assessed in our study for its anti-inflammatory properties by in-vitro and in-vivo experiments. Stevioside was observed to significantly inhibit the levels of LPS induced elevation of cytokines, TNF-α (P < 0.05) and IL-6 (P < 0.001) as well as the production of reactive oxygen species (P < 0.01) and nitrites (P < 0.001) in RAW264.7 cells. Stevioside has also been evaluated for its anti-inflammatory effect by using dextran sulfate sodium (DSS)-induced ulcerative colitis model in mice. Stevioside significantly reduced the disease activity index (DAI) score, ameliorated the inflammatory symptoms induced by DSS in mice and exhibited intact colon histo-architecture. Stevioside treatment significantly inhibited the levels of pro-inflammatory cytokines, TNF-α and IL-6, and the protein expressions of pro-inflammatory mediators, COX-2 (P < 0.01) and iNOS (P < 0.01) and restored the levels of endogenous anti-oxidants such as superoxide dismutase (P < 0.01), catalase (P < 0.001), glutathione s-transferase (P < 0.001) and reduced glutathione (P < 0.001) level in colon tissues. It was also observed that stevioside significantly suppressed NF-κB (p65) activation by abrogating IκB phosphorylation and attenuated the phosphorylation of p38, ERK and JNK proteins in colon tissues. The findings of the present study suggest that stevioside exhibits anti-inflammatory property by inhibiting NF-κB (p65) and MAPK pathways and can be employed as an adjunct in nutraceuticals to treat IBD.

Supplementation of oat (Avena sativa L.) extract abates alcohol-induced acute liver injury in a mouse model.

Dietary supplementation of oats has been associated with reduced risk of cardiovascular disease, diabetes, and gastrointestinal disorders. The role of oat extract as prophylactic in treating acute liver injury is not thoroughly established. We, therefore, hypothesized that oat extract would exert protective effect against alcohol-induced acute liver injury in a mouse model. To test this hypothesis, male C57BL/6 mice were pretreated with phenolic-enriched ethyl acetate (EA) fraction of oats (prepared by fractionating aqueous ethanolic extract with solvents of increasing polarity) at dosages of 125 and 250 mg kg-1 d-1 for 12 consecutive days. Acute liver injury was induced by administering 5 doses of 50% ethanol intragastrically (10 g/kg body weight) to mice at an interval of 12 hours. The alcohol-induced liver injury was evaluated by measuring serum levels of alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, antioxidant parameters, mitochondrial function, and histology of liver tissue. Our results demonstrated that pretreatment with EA fraction at 250 mg kg-1 d-1 significantly (P < .001 for aspartate aminotransferase, alanine aminotransferase, and thiobarbituric acid-reactive species and P < .01 for lactate dehydrogenase and nitrites) reduced the levels of liver injury markers and significantly (P < .001 for glutathione reductase and glutathione S-transferase; P < .01 for catalase, superoxide dismustase, and vitamin C; P < .05 for reduced glutathione and NAD(P)H quinone dehydrogenase 1) increased the levels of antioxidant defenses. Furthermore, EA-pretreated mice showed mechanistic inhibition of nuclear factor κB signaling pathway through decreased phosphorylation and degradation of IκBα. We conclude that phenolic-enriched EA fraction of oats has immense potential to serve as dietary intervention against alcohol-induced liver damage.

Metformin regulates mitochondrial biogenesis and senescence through AMPK mediated H3K79 methylation: Relevance in age-associated vascular dysfunction.

Endothelial senescence in conjunction with mitochondrial dysfunction orchestrates age-associated cardiovascular disorders. In this study we investigated the causal link between these two processes and studied the molecular mechanisms by which metformin acts to coordinate the delay of endothelial senescence via enhancing mitochondrial biogenesis/function. AMPK activators metformin and AICAR delayed endothelial senescence via SIRT1-mediated upregulation of DOT1L, leading to increased trimethylation of H3K79 (H3K79me3). Treatment of cells with either siAMPK or siSIRT1 repressed DOT1L-mediated enhancement of H3K79me3. Moreover, the increase in SIRT3 expression and mitochondrial biogenesis/function by AMPK activators was H3K79me-dependent as H3K79N mutant or siDOT1L abrogated these effects. This was confirmed by the enrichment of H3K79me3 in the SIRT3 promoter with AMPK activation. Intriguingly, enhanced PGC-1α expression by SIRT3 via AMPK activation was responsible for increased hTERT expression and delayed endothelial senescence. In contrast, SIRT3 knockdown caused increased oxidative stress and premature senescence, possibly by depleting hTERT expression. Furthermore, a chronic low dose administration of metformin significantly attenuated vascular aging and inhibited age-associated atherosclerotic plaque formation in ApoE-/- mice. Overall, the results of this study show a novel regulation of mitochondrial biogenesis/function, and cellular senescence by H3K79me acting through SIRT3, thus providing a molecular basis for metformin-mediated age-delaying effects.

Mitochondria-targeted esculetin inhibits PAI-1 levels by modulating STAT3 activation and miR-19b via SIRT3: Role in acute coronary artery syndrome.

In this study we explored the microRNAs responsible for the regulation of PAI-1 during LPS-stimulated inflammation in human aortic endothelial cells and subsequently studied the effect of a newly synthesized mitochondria-targeted esculetin (Mito-Esc) that was shown for its anti-atherosclerotic potential, in modulating PAI-1 levels and its targeted miRs during angiotensin-II-induced atherosclerosis in ApoE-/- mice. LPS-stimulated PAI-1 was accompanied with an upregulation of miR-19b and down-regulation of miR-30c. These effects of LPS on PAI-1 were reversed in the presence of both parent esculetin and Mito-Esc. However, the effect of Mito-Esc was more pronounced in the regulation of PAI-1. In addition, LPS-stimulated PAI-1 expression was significantly decreased in cells treated with Anti-miR-19b, thereby suggesting that miR-19b co-expression plays a key role in PAI-1 regulation. The results also show that incubation of cells with Stattic, an inhibitor of STAT-3, inhibited LPS-stimulated PAI-1 expression. Interestingly, knockdown of SIRT3, a mitochondrial biogenetic marker, enhanced PAI-1 levels via modulation of miR-19b and -30c. Mito-Esc treatment significantly inhibited Ang-II-induced PAI-1, possibly via altering miR-19b and 30c in ApoE-/- mice. The association between PAI-1, miR-19b and -30c were further confirmed in plasma and microparticles isolated from patients suffering from acute coronary syndrome of various degrees. Taken together, LPS-induced PAI-1 involves co-expression of miR-19b and down regulation of miR-30c, and Mito-Esc treatment by modulating miR-19b and miR-30c through SIRT3 activation, inhibits PAI-1 levels that, in part, contribute to its anti-atherosclerotic effects. Moreover, there exists a strong positive correlation between miR-19b and PAI-1 in patients suffering from ST-elevated myocardial infarction.