ISO Treatment Research Articles

Abstract Tu074: A Potential Link Between Stress Kinase JNK2 and AKAP-1 in Catecholamine-Induced Acute Heart Failure

Takotsuba Syndrome (TTS) is a type of emotional/physical stress-induced heart failure (HF). Stress-elevated catecholamines activate adrenergic signaling. Cardiac A-Kinase Anchoring Protein-1 (AKAP1) is known to regulate the adrenergic signaling via protein complex formation and phosphorylation. We recently discovered that activation of the stress response kinase JNK2 is critical in HF onset. Whether AKAP1 is involved with stress-activated JNK2 and HF onset remains completely unknown. Isoproterenol (Iso; synthetic catecholamine) effects on JNK2 activation were assessed in left ventricles (LV) from Iso-treated wildtype (WT) mice (67 mg/kg i.p., 2 days) and in heterologous HEK-RyR2 cells (inducible cardiac ryanodine receptor-2; myocyte mimic; 5 mM Iso, 24 h). A JNK2 effect on cardiac function was reflected by cardiac output (CO) using echocardiography in cardiac-specific MKK7D (inducible overexpression of active MKK7D; a JNK upstream activator) and MKK7D-JNK2KD (50% JNK2 knockdown in MKK7D) mice. The AKAP1-JNK2 link was assessed using immunoprecipitation (IP) and immunoblotting in tGFP -JNK2 and/or FLAG -AKAP1 transfected HEK293 cells. Iso-WT LV had increased JNK phosphorylation (JNK-p, activated) and reduced SERCA2 (a hallmark of HF) vs controls. Likely, MKK7-induction in MKK7D LV increased JNK2 and JNK-p (vs WT-littermates; p<0.05, n=5,4), which led to reduced CO by 68% (n=5,6, p<0.01) and downregulated SERCA2 by 79% (p<0.01, n=4,8), also evidenced by preserved CO and SERCA2 in MKK7D-JNK2KD mice (50% ablated JNK2; n=9). Moreover, Iso treatment in HEK-RyR2 cells activated JNK2, but not JNK1, compared to sham controls. Intriguingly, we found that JNK2 was associated with AKAP1, given the co-IP of tGFP -JNK2 with anti- FLAG antibody pulldown of FLAG -AKAP1 co-transfected HEK293 cells (n=3). Further, overexpressed FLAG -AKAP1 increased the phosphorylation of co-transfected tGFP -JNK2 as well as endogenous JNK (p<0.01 & p<0.001, n=3,3), while the notable JNK downstream target c-Jun also showed increased phosphorylation (c-Jun-p; p<0.05, n=3,3), supporting a previously unrecognized role for AKAP1 in JNK2 activation. In total, our findings suggest that stress activates JNK2 via an adrenergic-AKAP1 activation pathway to drive HF/TTS onset.

Isolinderalactone Ameliorates the Pathology of Alzheimer's Disease by Inhibiting the JNK Signaling Pathway.

Neuronal cell damage is a major cause of cognitive impairment in Alzheimer's disease (AD). Multiple factors, such as amyloid deposition, tau hyperphosphorylation, and neuroinflammation, can lead to neuronal cell damage. Therefore, the development of multi-target drugs with broad neuroprotective effects may be an effective strategy for the treatment of AD. Natural products have become an important source of drug discovery because of their good pharmacological activity, multiple targets, and low toxicity. In this study, we screened a natural compound library and found that the fat-soluble sesquiterpene natural compound isolinderalactone (Iso) extracted from the dried root pieces of Lindera aggregata had the ability to alleviate cellular damage induced by β-amyloid-1-42 (Aβ1-42). The role and mechanism of Iso in AD have not yet been reported. Herein, we demonstrated that Iso significantly reduced the level of apoptosis in PC12 cells. Besides, Iso treatment reduced amyloid deposition, neuron apoptosis, and neuroinflammation, ultimately improving the cognitive dysfunction of APP/PS1 (APPswe/PSEN 1dE9) mice. Notably, Iso-10 mg/kg showed superior improved effects in APP/PS1 mice compared with the positive control drug donepezil-5 mg/kg. Mechanistically, the results of RNA sequencing combined with Western blots showed that Iso exerted its therapeutic effect by inhibiting the c-Jun N-terminal kinase (JNK) signaling pathway. Taken together, our findings suggest that Iso is a potential drug candidate for the treatment of AD.

Cardiomyocyte NOX4 regulates resident macrophage-mediated inflammation and diastolic dysfunction in stress cardiomyopathy

In acute sympathetic stress, catecholamine overload can lead to stress cardiomyopathy. We tested the hypothesis that cardiomyocyte NOX4 (NADPH oxidase 4)-dependent mitochondrial oxidative stress mediates inflammation and diastolic dysfunction in stress cardiomyopathy. Isoproterenol (ISO; 5 mg/kg) injection induced sympathetic stress in wild-type and cardiomyocyte (CM)-specific Nox4 knockout (Nox4CM−/−) mice. Wild-type mice treated with ISO showed higher CM NOX4 expression, H2O2 levels, inflammasome activation, and IL18, IL6, CCL2, and TNFα levels than Nox4CM−/− mice. Spectral flow cytometry and t-SNE analysis of cardiac cell suspensions showed significant increases in pro-inflammatory and pro-fibrotic embryonic-derived resident (CCR2−MHCIIhiCX3CR1hi) macrophages in wild-type mice 3 days after ISO treatment, whereas Nox4CM−/− mice had a higher proportion of embryonic-derived resident tissue-repair (CCR2−MHCIIloCX3CR1lo) macrophages. A significant increase in cardiac fibroblast activation and interstitial collagen deposition and a restrictive pattern of diastolic dysfunction with increased filling pressure was observed in wild-type hearts compared with Nox4CM−/− 7 days post-ISO. A selective NOX4 inhibitor, GKT137831, reduced myocardial mitochondrial ROS, macrophage infiltration, and fibrosis in ISO-injected wild-type mice, and preserved diastolic function. Our data suggest sympathetic overstimulation induces resident macrophage (CCR2−MHCII+) activation and myocardial inflammation, resulting in fibrosis and impaired diastolic function mediated by CM NOX4-dependent ROS.

Abstract P2007: Novel Regulatory Axis In Heart-adrenal Cross Talk Mediated By Mtor-eprs

Adrenal catecholamines, including norepinephrine and epinephrine play an important role in cardiovascular physiology. Serum catecholamine level is tightly regulated in response to metabolic and cardiac stress. The biosynthesis of catecholamine is driven by a series of enzymatic reaction carried out by the key rate-limiting enzymes, including tyrosine hydroxylase (TH), DOPA decarboxylase (DDC) and phenylethanolamine-N-methyltransferase (PNMT). Although the transcription regulation of these enzymes has been described before, the regulatory mechanism at post-transcription or protein translation level has never been explored. Using a system genetics approach, we explored genes associated with the adrenal gland traits in HMDP at basal and post isoproterenol treatment. We have identified EPRS (Glutamyl-Prolyl-tRNA synthetase) to be significantly associated with adrenal gland mass post ISO treatment. EPRS is a tRNA synthetase responsible for charging tRNA during protein translation, EPRS also regulates mRNA translation as an RNA binding protein. To explore the functional impact of EPRS in adrenal gland physiology, we generated an EPRS adrenal gland specific knockout mouse model (EPRS-aKO). Inactivation of EPRS in adrenal gland led to a decrease of adrenal gland mass associated with reduced systemic catecholamine level. Interestingly, we further observed a moderate yet significantly decreased heart rate and ejection fraction in EPRS-aKO mice. Through both in vivo and in vitro analysis, we confirmed inactivation of EPRS led to a reduced protein, but not mRNA expression for both TH and DDC. In adipose tissue, activation of EPRS through mTOR signaling pathway is critical for its function as RNA binding protein. To further explore the mechanism through EPRS mediated catecholamine synthesis, we treated PC12 cells with rapamycin to inhibit mTOR activity. Treatment with Rapamycin diminished EPRS activity and expression, reduced TH protein expression along with a decrease of catecholamine level. Our results identified a novel regulatory scheme of catecholamine synthesis through mTOR-EPRS axis, adding a novel layer of gene regulation in catecholamine production and providing new mechanistic insights for heart-adrenal cross talk.

Benzylisoquinoline alkaloids inhibit lung fibroblast activation mainly via inhibiting TGF-β1/Smads and ERK1/2 pathway proteins

BackgroundsLiensinine (Lien), Neferine (Nef), Isoliensinine (Iso) and Tetrandrine (Tet), benzylisoquinoline alkaloids (BIAs), have been shown inhibitory effects on pulmonary fibrosis (PF) through anti-inflammatory, anti-oxidative activities, inhibition of cytokines and NF-κB. Effects of other similar BIAs, Dauricine (Dau), Papaverine (Pap) and lotusine (Lot), on PF remain unclear. Here, we explored the effects of five bisbenzylisoquinoline (Lien, Nef, Iso, Tet and Dau) and two monobenzylisoquinoline (Pap, Lot) alkaloids on normal and PF fibroblasts. MethodsPrimary normal and PF lung fibroblasts were cultured and treated with these alkaloids. Proliferation, activation, migration and apoptosis changes were detected by MTT, wound healing assay, flow cytometry. Protein level was analyzed by Western blot. ResultsAll BIAs inhibited proliferation of normal and PF lung fibroblasts induced by TGF-β. α-SMA protein level in normal and PF lung fibroblasts decreased after Lien, Nef, Iso, Tet and Dau treatment. Pap and Lot had no influence on α-SMA expression. Dau showed the strongest inhibitory effects on proliferation and activation among alkaloids. The migration rates of normal and PF lung fibroblasts were inhibited by Lien, Nef, Iso, and Dau. Lien, Nef, Iso and Dau significantly promoted apoptosis, while Tet had no effect on apoptosis. Pap and Lot had no influence on activation, migration and apoptosis. Dau significantly inhibited Smad3/4 and p-ERK1/2 protein overexpression induced by TGF-β1. ConclusionsBisbenzylisoquinoline alkaloids had stronger effects on inhibiting lung fibroblasts than monobenzylisoquinoline alkaloids. Dau expressed the strongest inhibitory effects, which may be related to its inhibition of TGF-β1/Smad3/4 and p-ERK1/2 pathway proteins.

Exposure of the Gestating Mother to Sympathetic Stress Modifies the Cardiovascular Function of the Progeny in Male Rats

Sympathetic stress stimulates norepinephrine (NE) release from sympathetic nerves. During pregnancy, it modifies the fetal environment, increases NE to the fetus through the placental NE transporter, and affects adult physiological functions. Gestating rats were exposed to stress, and then the heart function and sensitivity to in vivo adrenergic stimulation were studied in male progeny. Pregnant Sprague-Dawley rats were exposed to cold stress (4 °C/3 h/day); rats' male progeny were euthanized at 20 and 60 days old, and their hearts were used to determine the β-adrenergic receptor (βAR) (radioligand binding) and NE concentration. The in vivo arterial pressure response to isoproterenol (ISO, 1 mg/kg weight/day/10 days) was monitored in real time (microchip in the descending aorta). Stressed male progeny presented no differences in ventricular weight, the cardiac NE was lower, and high corticosterone plasma levels were recorded at 20 and 60 days old. The relative abundance of β1 adrenergic receptors decreased by 36% and 45%, respectively (p < 0.01), determined by Western blot analysis without changes in β2 adrenergic receptors. A decrease in the ratio between β1/β2 receptors was found. Displacement of 3H-dihydroalprenolol (DHA) from a membrane fraction with propranolol (β antagonist), atenolol (β1 antagonist), or zinterol (β2 agonist) shows decreased affinity but no changes in the β-adrenergic receptor number. In vivo exposure to ISO to induce a β-adrenergic overload provoked death in 50% of stressed males by day 3 of ISO treatment. These data suggest permanent changes to the heart's adrenergic response after rat progeny were stressed in the uterus.

Isoliquiritin modulates ferroptosis via NF-κB signaling inhibition and alleviates doxorubicin resistance in breast cancer

Context Breast cancer (BC) is the most prevalent diagnosed tumor and the major reason for tumor-related death in females around the world. Isoliquiritin, a type of plant extract, has exhibited a probable inhibitory effect in a variety of cancers. However, the anti-tumor effect on BC is still unclear. Objective To reveal the effect and potential mechanism of Isoliquiritin on BC. Materials and methods The cell viabilities were detected by CCK-8 assay. The levels of indicators of ferroptosis, oxidative stress, glycolysis, and inflammation were evaluated by commercial kits, flow cytometry, western blot, spectrophotometry, and ELISA assays. Mechanically, the expressions expression of the NF-κB pathway was determined by western blot. In vivo assay was also yielded on the BALB/c nude mice. Results Iso induced a concentration and time-dependent decrease of viability in both MDA-MB-231 and MCF-7 cells. Iso treatment significantly increased the levels of Fe2+, ROS, and MDA, and decreased the GSH level, and the relative protein expressions of GPX4 and xCT. Furthermore, Iso modulated oxidative stress, glycolysis, and inflammation through ferroptosis. In addition, Iso induced a concentration-dependent decrease in cell viability and a concentration-dependent increase in apoptosis rate in both MDA-MB-231/Dox and MCF-7/Dox cells. Iso notably counteracted the LPS-induced relative protein levels of p-p50/p50, p-p65/p65, and IκB, and the levels of ferroptosis, oxidative stress, glycolysis, and inflammation. The same results were also verified in vivo. Conclusion Iso inhibited the NF-κB signaling to regulate ferroptosis and improved Dox-resistance in breast cancer.

Suppression of RBFox2 by Multiple MiRNAs in Pressure Overload-Induced Heart Failure.

Heart failure is the final stage of various cardiovascular diseases and seriously threatens human health. Increasing mediators have been found to be involved in the pathogenesis of heart failure, including the RNA binding protein RBFox2. It participates in multiple aspects of the regulation of cardiac function and plays a critical role in the process of heart failure. However, how RBFox2 itself is regulated remains unclear. Here, we dissected transcriptomic signatures, including mRNAs and miRNAs, in a mouse model of heart failure after TAC surgery. A global analysis showed that an asymmetric alternation in gene expression and a large-scale upregulation of miRNAs occurred in heart failure. An association analysis revealed that the latter not only contributed to the degradation of numerous mRNA transcripts, but also suppressed the translation of key proteins such as RBFox2. With the aid of Ago2 CLIP-seq data, luciferase assays verified that RBFox2 was targeted by multiple miRNAs, including Let-7, miR-16, and miR-200b, which were significantly upregulated in heart failure. The overexpression of these miRNAs suppressed the RBFox2 protein and its downstream effects in cardiomyocytes, which was evidenced by the suppressed alternative splicing of the Enah gene and impaired E-C coupling via the repression of the Jph2 protein. The inhibition of Let-7, the most abundant miRNA family targeting RBFox2, could restore the RBFox2 protein as well as its downstream effects in dysfunctional cardiomyocytes induced by ISO treatment. In all, these findings revealed the molecular mechanism leading to RBFox2 depression in heart failure, and provided an approach to rescue RBFox2 through miRNA inhibition for the treatment of heart failure.

Isoproterenol Alters Metabolism, Promotes Survival and Migration in 5-Fluorouracil-Treated SW480 Cells with and without Beta-hydroxybutyrate.

People with cancer often experience long-term physical and psychological stress, which can have a significant impact on tumor metabolism and treatment. The effects of adrenergic signaling on metabolic pathways are well known, but only a few studies have looked into the connection between this signaling and tumor metabolism. This study examined the effects of treatment with isoproterenol (Iso) alone and in combination with β-hydroxybutyrate (βHB), a mitochondrial fuel, on the metabolism, survival, and migration of SW480 colon cancer cells treated with 5-fluorouracil (5FU). The researchers measured the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) to determine the metabolic profile of these cells. They also analyzed the gene expression of PGC-1α, c-MYC, and NANOG to investigate the relationship between metabolic phenotype and stemness status. Scratch assays were used to assess cell migration. The results showed that Iso treatment increased cell viability in both SW480 and 5FU-treated SW480 cells. There was a significant decrease in ECAR and an increase in OCR after Iso treatment in both cell types. The expression of c-MYC and NANOG, genes associated with stemness, increased, while the expression of PGC-1α, a gene related to oxidative phosphorylation, decreased following Iso treatment. Iso treatment also increased the migration potential of both SW480 and 5FU-treated SW480 cells. These findings suggest that under stressful conditions, 5FU-treated colon cancer cells can utilize the oxidative phosphorylation pathway for growth and migration.

Role of cardiac β1-adrenergic and A1-adenosine receptors in severe arrhythmias related to Parkinson's disease

AimsAlthough reduced life expectancy in Parkinson's Disease (PD) patients has been related to severe cardiac arrhythmias due to autonomic dysfunctions, its molecular mechanisms remain unclear. To investigate the role of cardiac β1-Adrenergic (β1AR) and A1-Adenosine (A1R) receptors in these dysfunctions, the pharmacological effects of stimulation of cardiac β1AR (isoproterenol, ISO), in the absence and presence of cardiac β1AR (atenolol, AT) or A1R (1,3-dipropyl-8-cyclopentyl xanthine, DPCPX) blockade, on the arrhythmias induced by Ischemia/Reperfusion (CIR) in an animal PD model were studied. MethodsPD was produced by dopaminergic lesions (confirmed by immunohistochemistry analysis) caused by the injection of 6-hydroxydopamine (6-OHDA, 6 μg) in rat striatum. CIR was produced by a surgical interruption for 10 min followed by reestablishment of blood circulation in the descendent left coronary artery. On the incidence of CIR-Induced Ventricular Arrhythmias (VA), Atrioventricular Block (AVB), and Lethality (LET), evaluated by Electrocardiogram (ECG) analysis, the effects of intravenous treatment with ISO, AT and DPCPX (before CIR) were studied. ResultsVA, AVB and LET incidences were significantly higher in 6-OHDA (83%, 92%, 100%, respectively) than in control rats (58%, 67% and 67%, respectively). ISO treatment significantly reduced these incidences in 6-OHDA (33%, 33% and 42%, respectively) and control rats (25%, 25%, 33%, respectively), indicating that stimulation of cardiac β1AR induced cardioprotection. This response was prevented by pretreatment with AT and DPCPX, confirming the involvement of cardiac β1AR and A1R. ConclusionPharmacological modulation of cardiac β1AR and A1R could be a potential therapeutic strategy to reduce severe arrhythmias and increase life expectancy in PD patients.

694 DRUG INDUCED KIDNEY INFARCTION: A CASE REPORT

Abstract Isotretinoin (ISO) is a synthetic analog of vitamin A. Notwithstanding the well-known long-term efficacy in the treatment of severe resistant cystic acne, ISO induces several side effects too. Dyslipidemia, increased liver enzymes, as well as reduction of biotidinase have been already reported [1]. Homocysteine (HCY), a sulfur-containing amino acid, is recycled into methionine by a transmethylation reaction requiring folate and vitamin B12. Given that during ISO treatment elevated liver enzymes (SGOT, SGPT) are often observed, it has been hypothesized that iatrogenic liver dysfunction could affect cystathionine-b-synthase, an enzyme involved in HCY metabolism, [2]. Several studies have highlighted that elevated plasma HCY concentrations are associated with an increased risk of premature occlusive vascular disease. We hereby report a case of drug-induced kidney infarction after ISO therapy. A previously healthy 18-year-old male, on therapy with 40 mg/die isotretinoin for acne, was admitted to the Emergency Unit for abdominal pain, nausea and vomiting and dyspnea. A trans-thoracic echocardiogram showed dilated and hypocontractile left ventricle. During the hospitalization he had a total body CT that showed the presence of an ischemic lesion on left kidney without other pathological findings. He started therapy with 12 IU/kg UFH as soon as a surgical emergency was excluded. Subsequently, he underwent a coronary angiogram that resulted unremarkable. Cardiac MRI showed extensive delayed gadolinium late enhancement with intramural linear pattern. In the hypothesis of drug-induced vasculitis, an extensive serological testing was performed. Elevation of serum liver enzymes (SGOT, SGPT), CRP, LDH was found. Mutations on MTHFR gene and Leiden Factor were not detected, neither alteration in coagulation parameters. Finally the dosage of plasmatic HCY resulted over the range of normality and ISO was discontinued. The patient was discharged on optimized medical therapy for heart failure. He is currently managed as outpatient. HCY levels could be found elevated in patients with ischemic events during ISO treatment. Given the widespread use of this drug for acne, it could be reasonable to dose HCY in patients with suspected liver dysfunction. Physicians should be aware of this scenario and should act swiftly to avoid uneventful outcomes. [1] Schulpis KH et al. Elevated plasma homocysteine levels in patients on isotretinoin therapy for cystic acne. Int J Dermatol. 2001 Jan;40(1):33-6. [2] Polat M, et al. Plasma homocysteine level is elevated in patients on isotretinoin therapy for cystic acne: a prospective controlled study. J Dermatolog Treat. 2008;19(4):229-32.

Akne hastalarında sistemik izotretinoin kullanımının nötrofil/lenfosit oranı üzerine etkisi: Retrospektif bir çalışma

Objective: Acne vulgaris (AV) describes a chronic inflammatory condition that occurs in the pilosebaceous unit, which is common in the community. Increase in sebum production, abnormal follicular keratinization, Propionibacterium acnes (P.acnes) colonization and inflammation play a role in acne development. ISO, comedolytic, is the only agent that has an effect on all four conditions that determine the pathogenesis of acne, such as a large reduction in sebaceous gland size, suppression of sebum production and reduction in inflammation. In this study, it was aimed to compare the N/L ratio values at the beginning of the treatment and at the 4th month of the treatment in acne vulgaris patients receiving systemic ISO treatment. Material and Method 50 patients with acne vulgaris who applied to the dermatology outpatient clinics of Karaman Training and Research Hospital between October 2020 and February 2021, had moderate and severe acne vulgaris and were started on oral isotretinoin were included in the study. The medical records of the patients were reviewed retrospectively. The neutrophil/lymphocyte (N/L) ratio values of the patients at the beginning of Isotretinoin treatment and after four months were recorded. Results The mean age of the patients was 23.1±6.23, 52.0% (26 persons) were male. There was a significant decrease in the N/L ratio in total and in both genders with isotretinoin use (p

Isoginkgetin—A Natural Compound to Control U87MG Glioblastoma Cell Growth and Migration Activating Apoptosis and Autophagy

Isoginkgetin (Iso) is a natural bioflavonoid isolated from the leaves of Ginkgo biloba, this natural substance exhibits many healing properties, among which the antitumor effect stands out. Here we tested the effect of Iso on the growth of U87MG glioblastoma cells. Growth curves and MTT toxicity assays showed time and dose-dependent growth inhibition of U87MG after treatment with Iso (15/25 µM) for 1, 2, and 3 days. The cell growth block of U87MG was further investigated with the colony formation test, which showed that iso treatment for 24 h reduced colony formation. The present study also aimed to evaluate the effect of Iso on U87MG glioblastoma cell migration. The FACS analysis, on the other hand, showed that treatment with Iso 15 µM determines a blockage of the cell cycle in the S1 phase. Further investigation shows that Iso treatment of U87MG altered the protein pathways of homeostasis including autophagy and apoptosis. The present study demonstrated, for the first time, that Iso could represent an excellent adjuvant drug for the treatment of glioblastoma by simultaneously activating multiple mechanisms that control the growth and migration of neoplastic cells.

Galectin-3-centered paracrine network mediates cardiac inflammation and fibrosis upon β-adrenergic insult.

Rapid over-activation of β-adrenergic receptors (β-AR) following acute stress initiates cardiac inflammation and injury by activating interleukin-18 (IL-18), however, the process of inflammation cascades has not been fully illustrated. The present study aimed to determine the mechanisms of cardiac inflammatory amplification following acute sympathetic activation. With bioinformatics analysis, galectin-3 was identified as a potential key downstream effector of β-AR and IL-18 activation. The serum level of galectin-3 was positively correlated with norepinephrine or IL-18 in patients with chest pain. In the heart of mice treated with β-AR agonist isoproterenol (ISO, 5 mg kg-1), galectin-3 expression was upregulated markedly later than IL-18 activation, and Nlrp3-/- and Il18-/- mice did not show ISO-induced galectin-3 upregulation. It was further revealed that cardiomyocyte-derived IL-18 induced galectin-3 expression in macrophages following ISO treatment. Moreover, galectin-3 deficiency suppressed ISO-induced cardiac inflammation and fibrosis without blocking ISO-induced IL-18 increase. Treatment with a galectin-3 inhibitor, but not a β-blocker, one day after ISO treatment effectively attenuated cardiac inflammation and injury. In conclusion, galectin-3 is upregulated to exaggerate cardiac inflammation and injury following acute β-AR activation, a galectin-3 inhibitor effectively blocks cardiac injury one day after β-AR insult.

Beta-Site Amyloid Precursor Protein-Cleaving Enzyme Inhibition Partly Restores Sevoflurane-Induced Deficits on Synaptic Plasticity and Spine Loss.

Evidence indicates that inhalative anesthetics enhance the β-site amyloid precursor protein (APP)-cleaving enzyme (BACE) activity, increase amyloid beta 1-42 (Aβ1–42) aggregation, and modulate dendritic spine dynamics. However, the mechanisms of inhalative anesthetics on hippocampal dendritic spine plasticity and BACE-dependent APP processing remain unclear. In this study, hippocampal slices were incubated with equipotent isoflurane (iso), sevoflurane (sevo), or xenon (Xe) with/without pretreatment of the BACE inhibitor LY2886721 (LY). Thereafter, CA1 dendritic spine density, APP processing-related molecule expressions, nectin-3 levels, and long-term potentiation (LTP) were tested. The nectin-3 downregulation on LTP and dendritic spines were evaluated. Sevo treatment increased hippocampal mouse Aβ1–42 (mAβ1–42), abolished CA1-LTP, and decreased spine density and nectin-3 expressions in the CA1 region. Furthermore, CA1-nectin-3 knockdown blocked LTP and reduced spine density. Iso treatment decreased spine density and attenuated LTP. Although Xe blocked LTP, it did not affect spine density, mAβ1–42, or nectin-3. Finally, antagonizing BACE activity partly restored sevo-induced deficits. Taken together, our study suggests that sevo partly elevates BACE activity and interferes with synaptic remodeling, whereas iso mildly modulates synaptic changes in the CA1 region of the hippocampus. On the other hand, Xe does not alternate dendritic spine remodeling.

Activated autophagy-lysosomal pathway in dairy cows with hyperketonemia is associated with lipolysis of adipose tissues

Activated autophagy-lysosomal pathway (ALP) can degrade virtually all kinds of cellular components, including intracellular lipid droplets, especially during catabolic conditions. Sustained lipolysis and increased plasma fatty acids concentrations are characteristic of dairy cows with hyperketonemia. However, the status of ALP in adipose tissue during this physiological condition is not well known. The present study aimed to ascertain whether lipolysis is associated with activation of ALP in adipose tissues of dairy cows with hyperketonemia and in calf adipocytes. In vivo, blood and subcutaneous adipose tissue (SAT) biopsies were collected from nonhyperketonemic (nonHYK) cows [blood β-hydroxybutyrate (BHB) concentration <1.2 mM, n = 10] and hyperketonemic (HYK) cows (blood BHB concentration 1.2-3.0 mM, n = 10) with similar days in milk (range: 3-9) and parity (range: 2-4). In vitro, calf adipocytes isolated from 5 healthy Holstein calves (1 d old, female, 30-40 kg) were differentiated and used for (1) treatment with lipolysis inducer isoproterenol (ISO, 10 µM, 3 h) or mammalian target of rapamycin inhibitor Torin1 (250 nM, 3 h), and (2) pretreatment with or without the ALP inhibitor leupeptin (10 μg/mL, 4 h) followed by ISO (10 µM, 3 h) treatment. Compared with nonHYK cows, serum concentration of free fatty acids was greater and serum glucose concentration, DMI, and milk yield were lower in HYK cows. In SAT of HYK cows, ratio of phosphorylated hormone-sensitive lipase to hormone-sensitive lipase, and protein abundance of adipose triacylglycerol lipase were greater, but protein abundance of perilipin 1 (PLIN1) and cell death-inducing DNA fragmentation factor-α-like effector c (CIDEC) was lower. In addition, mRNA abundance of autophagy-related 5 (ATG5), autophagy-related 7 (ATG7), and microtubule-associated protein 1 light chain 3 beta (MAP1LC3B), protein abundance of lysosome-associated membrane protein 1, and cathepsin D, and activity of β-N-acetylglucosaminidase were greater, whereas protein abundance of sequestosome-1 (p62) was lower in SAT of HYK cows. In calf adipocytes, treatment with ISO or Torin1 decreased protein abundance of PLIN1, and CIDEC, and triacylglycerol content in calf adipocytes, but increased glycerol content in the supernatant of calf adipocytes. Moreover, the mRNA abundance of ATG5, ATG7, and MAP1LC3B was upregulated, the protein abundance of lysosome-associated membrane protein 1, cathepsin D, and activity of β-N-acetylglucosaminidase were increased, whereas the protein abundance of p62 was decreased in calf adipocytes treated with ISO or Torin1 compared with control group. Compared with treatment with ISO alone, the protein abundance of p62, PLIN1, and CIDEC, and triacylglycerol content in calf adipocytes were higher, but the glycerol content in the supernatant of calf adipocytes was lower in ISO and leupeptin co-treated group. Overall, these data indicated that activated ALP is associated with increased lipolysis in adipose tissues of dairy cows with hyperketonemia and in calf adipocytes.

Mitochondrial-Targeted Therapies Require Mitophagy to Prevent Oxidative Stress Induced by SOD2 Inactivation in Hypertrophied Cardiomyocytes.

Heart failure, mostly associated with cardiac hypertrophy, is a major cause of illness and death. Oxidative stress causes accumulation of reactive oxygen species (ROS), leading to mitochondrial dysfunction, suggesting that mitochondria-targeted therapies could be effective in this context. The purpose of this work was to determine whether mitochondria-targeted therapies could improve cardiac hypertrophy induced by mitochondrial ROS. We used neonatal (NCMs) and adult (ACMs) rat cardiomyocytes hypertrophied by isoproterenol (Iso) to induce mitochondrial ROS. A decreased interaction between sirtuin 3 and superoxide dismutase 2 (SOD2) induced SOD2 acetylation on lysine 68 and inactivation, leading to mitochondrial oxidative stress and dysfunction and hypertrophy after 24 h of Iso treatment. To counteract these mechanisms, we evaluated the impact of the mitochondria-targeted antioxidant mitoquinone (MitoQ). MitoQ decreased mitochondrial ROS and hypertrophy in Iso-treated NCMs and ACMs but altered mitochondrial structure and function by decreasing mitochondrial respiration and mitophagy. The same decrease in mitophagy was found in human cardiomyocytes but not in fibroblasts, suggesting a cardiomyocyte-specific deleterious effect of MitoQ. Our data showed the importance of mitochondrial oxidative stress in the development of cardiomyocyte hypertrophy. We observed that targeting mitochondria by MitoQ in cardiomyocytes impaired the metabolism through defective mitophagy, leading to accumulation of deficient mitochondria.

Responsiveness of domesticated goats towards various stressors following long-term cognitive test exposure.

Current evidence suggests that frequent exposure to situations in which captive animals can solve cognitive tasks may have positive effects on stress responsiveness and thus on welfare. However, confounding factors often hamper the interpretation of study results. In this study, we used human-presented object-choice tests (in form of visual discrimination and reversal learning tests and a cognitive test battery), to assess the effect of long-term cognitive stimulation (44 sessions over 4–5 months) on behavioural and cardiac responses of female domestic goats in subsequent stress tests. To disentangle whether cognitive stimulation per se or the reward associated with the human–animal interaction required for testing was affecting the stress responsiveness, we conditioned three treatment groups: goats that were isolated for participation in human-presented cognitive tests and rewarded with food (‘Cognitive’, COG treatment), goats that were isolated as for the test exposure and rewarded with food by the experimenter without being administered the object-choice tests (‘Positive’, POS treatment), and goats that were isolated in the same test room but neither received a reward nor were administered the tests (‘Isolation’, ISO treatment). All treatment groups were subsequently tested in four stress tests: a novel arena test, a novel object test, a novel human test, and a weighing test in which goats had to enter and exit a scale cage. All treatment groups weretested at the same two research sites, each using two selection lines, namely dwarf goats, not selected for production traits, and dairy goats, selected for high productivity. Analysing the data with principal component analysis and linear mixed-effects models, we did not find evidence that cognitive testing per se (COG–POS contrast) reduces stress responsiveness of goats in subsequent stress tests. However, for dwarf goats but not for dairy goats, we found support for an effect of reward-associated human–animal interactions (POS–ISO contrast) at least for some stress test measures. Our results highlight the need to consider ontogenetic and genetic variation when assessing stress responsiveness and when interacting with goats.

Isorhamnetin Alleviates Airway Inflammation by Regulating the Nrf2/Keap1 Pathway in a Mouse Model of COPD

Chronic obstructive pulmonary disease (COPD) is a severely disabling chronic lung disease characterized by persistent airway inflammation, which leads to limited expiratory airflow that deteriorates over time. Isorhamnetin (Iso) is one of the most important active components in the fruit of Hippophae rhamnoides L. and leaves of Ginkgo biloba L, which is widely used in many pulmonary disease studies because of its anti-inflammatory effects. Here, we investigated the pharmacological action of Iso in CS-induced airway inflammation and dissected the anti-inflammation mechanisms of Iso in COPD mice. A mouse model of COPD was established by exposure to cigarette smoke (CS) and intratracheal inhalation of lipopolysaccharide (LPS). Our results illustrated that Iso treatment significantly reduced leukocyte recruitment and excessive secretion of interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and regulated upon activation, normal T-cell expressed and secreted (RANTES) in BALF of CS-induced COPD mice in a dose-dependent manner. This improved airway collagen deposition and emphysema, and further alleviated the decline in lung functions and systemic symptoms of hypoxia and weight loss. Additionally, Iso treatment obviously improves the T lymphocyte dysregualtion in peripheral blood of COPD mice. Mechanistically, Iso may degrade Keap1 through ubiquitination of p62, thereby activating the nuclear factor erythroid 2-related factor (Nrf2) pathway to increase the expression of protective factors, such as heme oxygenase-1 (HO-1), superoxide dismutase (SOD) 1, and SOD2, in lungs of CS-exposed mice, which plays an anti-inflammatory role in COPD. In conclusion, our study indicates that Iso significantly alleviates the inflammatory response in CS-induced COPD mice mainly by affecting the Nrf2/Keap1 pathway. More importantly, Iso exhibited anti-inflammatory effects comparable with Dex in COPD and we did not observe discernible side effects of Iso. The high safety profile of Iso may make it a potential drug candidate for COPD.

YQFM alleviated cardiac hypertrophy by apoptosis inhibition and autophagy regulation via PI3K/AKT/mTOR pathway

Ethnopharmacological relevanceAs a traditional compound preparation of Chinese medicine, Yiqi Fumai lyophilized injection (YQFM) has protective effects on various cardiac diseases including cardiac hypertrophy, which is the primary cause of arrhythmia. However, the involved mechanism remains unclear. Aim of the study: This study was projected to investigate whether YQFM could prevent cardiac hypertrophy and arrhythmia concurrence. Materials and Methods: The cardiac hypertrophy rats were established by transverse aortic ligation and the H9c2 hypertrophy cardiomyocyte was induced by angiotensin II (AngII). The electrocardiogram (ECG) was conducted to estimate the arrhythmia occurrence of cardiac hypertrophy rats under isoprenaline (iso) treatment. The cardiac related indicators and histopathology were also detected. The protective effects of YQFM on H9c2 hypertrophy cardiomyocyte were determined by the cell size measurement, apoptosis detection and mitochondrial membrane potential measurement. The cardiac hypertrophy relative proteins (ANP and BNP), autophagy related factors (LC3II, p62 and Beclin-1), apoptosis related markers (p53, caspase 3, Bax and Bcl-2) and the PI3K/AKT/mTOR pathway expressions were all measured by Western blot. Results: YQFM decreased the arrhythmia occurrence and improved cardiac function in cardiac hypertrophy rats. YQFM also reduced the H9c2 cardiomyocyte size and alleviated the cardiomyocyte apoptosis induced by AngII. In addition, YQFM inhibited cell apoptosis by increasing Bcl-2/Bax ratio and decreasing caspase 3 and p53 expressions in vitro and vivo. Meanwhile, YQFM regulated the autophagy pathway by down-regulating of LC3II and Beclin-1 expressions, as well as up-regulating of p62 expression. Finally, the results showed that YQFM could activate the PI3K/AKT/mTOR pathway by enhancing the p-AKT, p-PI3K and p-mTOR expressions. Conclusion: Our results displayed that YQFM attenuated the cardiac hypertrophy by apoptosis inhibition and autophagy regulation via PI3K/AKT/mTOR pathway.