Autophagic Pathway Research Articles

Autophagic Mechanisms in Longevity Intervention: Role of Natural Active Compounds.

The term 'autophagy' literally translates to 'self-eating' and alterations to autophagy have been identified as one of the several molecular changes that occur with aging in a variety of species. Autophagy and aging, have a complicated and multifaceted relationship that has recently come to light thanks to breakthroughs in our understanding of the various substrates of autophagy on tissue homoeostasis. Several studies have been conducted to reveal the relationship between autophagy and age-related diseases. The present review looks at a few new aspects of autophagy and speculates on how they might be connected to both aging and the onset and progression of disease. Additionally, we go over the most recent preclinical data supporting the use of autophagy modulators as age-related illnesses including cancer, cardiovascular and neurodegenerative diseases, and metabolic dysfunction. It is crucial to discover important targets in the autophagy pathway in order to create innovative therapies that effectively target autophagy. Natural products have pharmacological properties that can be therapeutically advantageous for the treatment of several diseases and they also serve as valuable sources of inspiration for the development of possible new small-molecule drugs. Indeed, recent scientific studies have shown that several natural products including alkaloids, terpenoids, steroids, and phenolics, have the ability to alter a number of important autophagic signalling pathways and exert therapeutic effects, thus, a wide range of potential targets in various stages of autophagy have been discovered. In this review, we summarised the naturally occurring active compounds that may control the autophagic signalling pathways.

Autophagy and anti-inflammation ameliorate diabetic neuropathy with Rilmenidine.

To evaluate the neuroprotective effects of Rilmenidine on diabetic peripheral neuropathy (DPN) in a rat model of diabetes induced by streptozotocin (STZ). STZ (60 mg/kg) was administered to adult Sprague-Dawley rats to induce diabetes. On the 30th day after STZ administration, electromyography (EMG) and motor function tests confirmed the presence of DPN. Group 1: Control (n = 10), Group 2: DM + 0.1 mg/kg Rilmenidine (n = 10), and Group 3: DM + 0.2 mg/kg Rilmenidine (n = 10) were administered via oral lavage for four weeks. EMG, motor function test, biochemical analysis, and histological and immunohistochemical analysis of sciatic nerves were then performed. The administration of Rilmenidine to diabetic rats substantially reduced sciatic nerve inflammation and fibrosis and prevented electrophysiological alterations. Immunohistochemistry of sciatic nerves from saline-treated rats revealed increased perineural thickness, HMGB-1, tumor necrosis factor-α, and a decrease in nerve growth factor (NGF), LC-3. In contrast, Rilmendine significantly inhibited inflammation markers and prevented the reduction in NGF expression. In addition, Rilmenidine significantly decreased malondialdehyde and increased diabetic rats' total antioxidative capacity. The findings of this study suggest that Rilmenidine may have therapeutic effects on DNP by modulating antioxidant and autophagic pathways.

Selectively attacking tumor cells of Ru/Ir-arene complexes based on meclofenamic acid via cyclooxygenase-2 inhibition.

Breast cancer (BC) is one of the most common malignant tumors and often accompanied by inflammatory processes. Inflammation is an essential component of the tumor microenvironment, which might influence tumor proliferation and metastasis. Herein, three metal-arene complexes MA-bip-Ru, MA-bpy-Ir, and MA-bpy-Ru were prepared by tethering the non-steroidal anti-inflammatory drug meclofenamic acid (MA). Among them, MA-bip-Ru and MA-bpy-Ir showed lower cytotoxicity towards cancer cells, but MA-bpy-Ru showed significantly high selectivity and cytotoxicity towards MCF-7 cells through the autophagic pathway and exhibited no toxicity against normal HLF cells, showing potential for selective treatment of tumor cells. MA-bpy-Ru could also effectively destroy the 3D multicellular tumor spheroids, demonstrating its potential for clinical application. Besides, MA-bip-Ru, MA-bpy-Ir, and MA-bpy-Ru exhibited anti-inflammatory properties superior to MA, notably downregulating the expression of cyclooxygenase-2 (COX-2) and inhibiting the secretion of prostaglandin E2 in vitro. These findings demonstrated that MA-bpy-Ru was capable of intervening in inflammatory processes and showed the potential of MA-bpy-Ru to act as a selective anticancer agent, thus presenting a new mechanism of action for metal-arene complexes.

Targeting Annexin A1 as a Druggable Player to Enhance the Anti-Tumor Role of Honokiol in Colon Cancer through Autophagic Pathway

Colon cancer is one of the most common digestive tract malignancies, having the second highest mortality rate among all tumors, with a five-year survival of advanced patients of only 10%. Efficient, targeted drugs are still lacking in treating colon cancer, so it is urgent to explore novel druggable targets. Here, we demonstrated that annexin A1 (ANXA1) was overexpressed in tumors of 50% of colon cancer patients, and ANXA1 overexpression was significantly negatively correlated with the poor prognosis of colon cancer. ANXA1 promoted the abnormal proliferation of colon cancer cells in vitro and in vivo by regulating the cell cycle, while the knockdown of ANXA1 almost totally inhibited the growth of colon cancer cells in vivo. Furthermore, ANXA1 antagonized the autophagic death of honokiol in colon cancer cells via stabilizing mitochondrial reactive oxygen species. Based on these results, we speculated that ANXA1 might be a druggable target to control colon cancer and overcome drug resistance.

Physiology of autophagy

The process of intracellular components being broken down by lysosomes is known as autophagy. By destroying intracellular components and giving cells breakdown products, autophagy plays a crucial part in preserving and regulating cell homeostasis [1]. The most recent studies have demonstrated that autophagy serves a variety of physiological and pathological functions, some of which are intricate. There are fewer autophagic vacuoles and poorer lysosome fusion as autophagy decreases with age. The innate immune system is strengthened by activating the autophagic pathway, which has also been shown to be helpful in avoiding different foreign infections. Tau and synuclein protein accumulation result from a decrease in autophagic protein clearance in neurodegenerative diseases. Similar to this, lysosomal lipid accumulation results from autophagic failure. Tumors are first suppressed by autophagy, but they are subsequently immune system-protected. Muscle and cardiac issues are favourably and negatively regulated by autophagy, respectively.

Effect on hypoxia/reoxygenation-induced cardiomyocyte injury and Pink1/Parkin pathway.

Our study aimed to detect the effects of proprotein convertase subtilisin/kexin type 9 (PCSK9) on exacerbating cardiomyocyte hypoxia/reoxygenation (H/R) injury and the possible mechanism. A cell model of H/R was constructed. PCSK9 mRNA and protein levels were significantly upregulated during AC16 cardiomyocyte H/R. Flowmetry detection of apoptosis, as well as JC-1, confirmed that PCSK9 upregulation of autophagy levels was accompanied by apoptosis. Furthermore, in the H/R+si-PCSK9 group, the expression of autophagy-related protein LC3 decreased and P62 increased. At the same time, the presentation of the autophagic pathway Pink1/Parkin was also downregulated. In conclusion, in AC16 cardiomyocytes treated with H/R, PCSK9 expression and autophagy levels were increased; a possible molecular mechanism was the activation of the Pink1/Parkin pathway.

An aqueous olive leaf extract (OLE) ameliorates parameters of oxidative stress associated with lipid accumulation and induces lipophagy in human hepatic cells.

Fatty liver is a disease characterized by a buildup of lipids in the liver, often resulting from excessive consumption of high-fat-containing foods. Fatty liver can degenerate, over time, into more severe forms of liver diseases, especially when oxidative stress occurs. Olive leaf extract (OLE) is a reliable source of polyphenols with antioxidant and hypolipidemic properties that have been successfully used in medicine, cosmetics, and pharmaceutical products. Using "green" solvents with minimal impact on the environment and human health, which simultaneously preserves the extract's beneficial properties, represents one of the major challenges of biomedical research. In the present study, we assayed the potential antioxidant and lipid-lowering effect of a "green" OLE obtained by a water ultrasound-assisted extraction procedure, on the human hepatic HuH7 cell line, treated with a high concentration of free fatty acids (FFA). We found that high FFA concentration induced lipid accumulation and oxidative stress, as measured by increased hydrogen peroxide levels. Moreover, the activity of antioxidant enzymes, catalase, superoxide dismutase, and glutathione peroxidase, was reduced upon FFA treatment. Coincubation of high FFA with OLE reduced lipid and H2O2 accumulation and increased the activity of peroxide-detoxifying enzymes. OLE ameliorated mitochondrial membrane potential, and hepatic parameters by restoring the expression of enzymes involved in insulin signaling and lipid metabolism. Electron microscopy revealed an increased autophagosome formation in both FFA- and FFA + OLE-treated cells. The study of the autophagic pathway indicated OLE's probable role in activating lipophagy.

Autophagy in Atherosclerotic Plaque Cells: Targeting NLRP3 Inflammasome for Self-Rescue.

Atherosclerosis (AS) is a lipid-driven disorder of the artery intima characterized by the equilibrium between inflammatory and regressive processes. A protein complex called NLRP3 inflammasome is involved in the release of mature interleukin-1β (IL-1β), which is connected to the initiation and progression of atherosclerosis. Autophagy, which includes macroautophagy, chaperone-mediated autophagy (CMA), and microautophagy, is generally recognized as the process by which cells transfer their constituents to lysosomes for digestion. Recent studies have suggested a connection between vascular inflammation and autophagy. This review summarizes the most recent studies and the underlying mechanisms associated with different autophagic pathways and NLRP3 inflammasomes in vascular inflammation, aiming to provide additional evidence for atherosclerosis research.

The Anticancer Effect of Phytochemicals and Potential of Breynia cernua: An overview

Cancer treatment still has challenges from its expense, side effect, and survival rate. One of the actions to improve this is searching for new anticancer agents. Medicinal plants are a candidate source since they have traditionally been used to treat illness. Phytochemicals of medicinal plants play a significant role in exhibiting anticancer effects. Literature studies of the phytochemicals of existing medicinal plants can be a clue to finding out the potential other plants whose studies are still limited, such as Breynia cernua, a plant with anticancer effects used traditionally. This study will provide information on the phytochemicals effect of medicinal plants or other compounds against cancer and their anticancer mechanisms. The agents are collected based on their compound's group, and each group's anticancer mechanism is resumed. The results showed that phytochemicals (flavonoids, alkaloids, saponins, quinone, tannins, and terpenoids) affect cancer cell through variant mechanism; induction of apoptosis, inhibition of cell growth, inhibition of cell migration, and induction of autophagic pathway. Most of the studies used methanol extracts, and most showed very strong toxicity to cancer cells. For further study, we suggest using isolated compounds from methanol, ethanol, or N-hexane extracts of Breynia cernua to get better anticancer activity, especially compounds belonging to the flavonoid or quinone group.

Ursolic Acid Impairs Cellular Lipid Homeostasis and Lysosomal Membrane Integrity in Breast Carcinoma Cells.

Cancer is one of the leading causes of death worldwide, thus the search for new cancer therapies is of utmost importance. Ursolic acid is a naturally occurring pentacyclic triterpene with a wide range of pharmacological activities including anti-inflammatory and anti-neoplastic effects. The latter has been assigned to its ability to promote apoptosis and inhibit cancer cell proliferation by poorly defined mechanisms. In this report, we identify lysosomes as the essential targets of the anti-cancer activity of ursolic acid. The treatment of MCF7 breast cancer cells with ursolic acid elevates lysosomal pH, alters the cellular lipid profile, and causes lysosomal membrane permeabilization and leakage of lysosomal enzymes into the cytosol. Lysosomal membrane permeabilization precedes the essential hallmarks of apoptosis placing it as an initial event in the cascade of effects induced by ursolic acid. The disruption of the lysosomal function impairs the autophagic pathway and likely partakes in the mechanism by which ursolic acid kills cancer cells. Furthermore, we find that combining treatment with ursolic acid and cationic amphiphilic drugs can significantly enhance the degree of lysosomal membrane permeabilization and cell death in breast cancer cells.

Mechanism involved in cell death induced by DEHP (Di-ethylhexyl phthalate) on primary brain cells from Zebrafish (Danio rerio)

Human and ecological exposure to chemical contaminants continue to increase given anthropogenic activities and sources. Phthalates are among the most persistent toxic chemicals found in aquatic systems. Phthalates belong to a family of chemical compounds that are heavily used in global manufacturing, distinguished for being able to provide durability and elasticity to plastic products, with poor water solubility and low evaporation. In terms of health impact, these compounds have been shown to breach the placental barrier having effect on human development and neurodegeneration. The main purpose of our study has been to assess the neurotoxicity of phthalates implementing the zebrafish (Danio rerio) model to determine the cell death induction of DEHP on brain cells. For many years, the zebrafish embryonic model has been used to study vertebrate development. Our study, in contrast, used adult zebrafish to resemble mature species that can be continuously exposed to low concentrations of DEHP. To assess the neurotoxicity, brain tissue from zebrafish was extracted via dissection, and primary neural cells dissociated to obtain primary cell subcultures. The neural cells where subsequently exposed to DEHP (Di-ethylhexyl phthalate) at concentrations from 10µM to 100µM for 48 hours to observe cell growth inhibition (GI50). The results indicate neural cell death at a GI50 of 62.9 µM. Apoptosis induction, caspase activation, mitochondrial membrane damage as well as autophagy cell death were analyzed to identify DEHP cell death mechanism at the GI50 concentration. In conclusion, this study clearly demonstrated that DEHP can induce apoptosis cell death on zebrafish neural brain cells in culture through the activation of an intrinsic apoptotic pathways with caspases 3 and 7 activation as well as autophagic pathway and mitochondrial damage. The presence of phthalates as well as other chemical contaminants on aquatic environments needs to be earnestly controlled in order to reduce neurotoxicity to aquatic species as well as human.

Catalase-deficient mice induce aging faster through lysosomal dysfunction

BackgroundLysosomes are a central hub for cellular metabolism and are involved in the regulation of cell homeostasis through the degradation or recycling of unwanted or dysfunctional organelles through the autophagy pathway. Catalase, a peroxisomal enzyme, plays an important role in cellular antioxidant defense by decomposing hydrogen peroxide into water and oxygen. In accordance with pleiotropic significance, both impaired lysosomes and catalase have been linked to many age-related pathologies with a decline in lifespan. Aging is characterized by progressive accumulation of macromolecular damage and the production of high levels of reactive oxygen species. Although lysosomes degrade the most long-lived proteins and organelles via the autophagic pathway, the role of lysosomes and their effect on catalase during aging is not known. The present study investigated the role of catalase and lysosomal function in catalase-knockout (KO) mice.MethodsWe performed experiments on WT and catalase KO younger (9 weeks) and mature adult (53 weeks) male mice and Mouse embryonic fibroblasts isolated from WT and KO mice from E13.5 embryos as in vivo and in ex-vivo respectively. Mouse phenotyping studies were performed with controls, and a minimum of two independent experiments were performed with more than five mice in each group.ResultsWe found that at the age of 53 weeks (mature adult), catalase-KO mice exhibited an aging phenotype faster than wild-type (WT) mice. We also found that mature adult catalase-KO mice induced leaky lysosome by progressive accumulation of lysosomal content, such as cathespin D, into the cytosol. Leaky lysosomes inhibited autophagosome formation and triggered impaired autophagy. The dysregulation of autophagy triggered mTORC1 (mechanistic target of rapamycin complex 1) activation. However, the antioxidant N-acetyl-L-cysteine and mTORC1 inhibitor rapamycin rescued leaky lysosomes and aging phenotypes in catalase-deficient mature adult mice.ConclusionsThis study unveils the new role of catalase and its role in lysosomal function during aging.D3gySv46Ggy6LMJqdgNDW3Video abstract

The role of autophagy in disease

AbstractDefective proteostasis is one of the hallmarks neurodegenerative diseases. Of the different components of the proteostasis network, we are interested in autophagy and its changes with age. We have previously shown that, out of the different autophagic pathways that co‐exist in all mammalian cells, macroautophagy is the first that declines with age in the retina, A crosstalk between macroautophagy and chaperone mediated autophagy (CMA) has been described whereby, in aged mice retinas, the decrease in macroautophagy correlates with increased CMA. Our working hypothesis is that in aged and degenerated retinal cells increase CMA to compensate for macroautophagy loss, in an attempt to maintain cell viability. Here, I will present our latest data showing how CMA upregulation can protect photoreceptor cells in retinitis pigmentosa models.

Cardioprotective effects of preconditioning exercise in the female tumor bearing mouse.

Cancer cachexia, a metabolic wasting syndrome, affects up to 80% of cancer patients and leads to the death in up to 20% of cancer patients. While research is growing in the field, there are still no clear diagnostic criteria and cancer cachexia remains an untreated condition. Aerobic exercise has been shown to positively impact cachexia by slowing its development and attenuating muscle loss. The most effective timing, duration, and intensity of exercise as a preventative and protective measure against cancer cachexia remains questionable. Therefore, the purpose of this study was to examine the effects of preconditioning exercise as a protective measure for tumor-mediated muscle wasting. Female LC3 Tg+ and wildtype mice were randomly separated into four groups, sedentary non-tumor bearing (SED + NT), sedentary tumor bearing (SED + T), treadmill exercise non-tumor bearing (TM + NT), and treadmill exercise tumor bearing (TM + T). Mice underwent an 8-week treadmill exercise training protocol (TM) or remained sedentary (SED). Next, mice were implanted with tumor cells (T group; 5 × 105 Lewis Lung Carcinoma cells in flank) or remained non-tumor (NT) for 4 weeks. Tumor bearing resulted in a significant decline in cardiac function. SED + T showed a significant decrease in fractional shortening (p < 0.05) when compared to the other groups. This coincided with an increase in beclin-1 and MyD88 protein expression and decrease in p-FOXO1 (inactivated) protein expression in SED + T mice. Interestingly, preconditioning exercise (exercise prior to tumor bearing) appeared to preserve cardiac function (TM + T not significantly different than SED + NT). Exercise-mediated cardioprotection also coincided with abolished beclin-1 and MyD88 signaling that was not significantly elevated in TM + T mice. Additionally, TM resulted in a 22-fold decrease in estimated tumor volume (p < 0.05) and a 45% decrease in tumor mass (p < 0.05) compared to SED tumors. The data indicate potential cardioprotective effects of preconditioning exercise on preserving cardiac structure and function, as well as regulating autophagic (beclin-1), inflammatory (TGF-β and MyD88), and atrophy (p-FOXO1) pathways during tumor bearing. Preconditioning exercise may be an effective and accessible treatment intervention for early-stage cancer survivors. This data is crucial in identifying the significance of exercise and the timing of exercise as a protective measure against the detrimental effects of cancer cachexia.

Design, synthesis and pharmacological evaluation of β-carboline derivatives as potential antitumor agent via targeting autophagy

A series of novel β-carboline derivatives was designed, synthesized and evaluated as potential anticancer agents. Among them, compound 6g showed the most potent antiproliferative activity against the 786–0, HT-29 and 22RV1 cell lines with IC50 values of 2.71, 2.02, and 3.86 μM, respectively. The antitumor efficiency of compound 6gin vivo was also evaluated, and the results revealed that compound 6g significantly suppressed tumor development and reduced tumor weight in a mouse colorectal cancer homograft model. Further investigation on mechanisms of action demonstrated that compound 6g inhibited HCT116 cell growth by stimulating the ATG5/ATG7-dependent autophagic pathway. These molecules might be served as candidates for further development of colorectal cancer therapy agent.

Advances in vesicle trafficking of membrane proteins and their regulatory mechanisms

<p indent="0mm">Membrane proteins are essential components of cell membrane structures and can be transported through vesicles as signaling molecules. The vesicle trafficking of membrane proteins is crucial for maintaining biological processes such as plant growth and development, substance exchange, cell recognition, immune response, and signal transduction. Both vesicle exocytosis and endocytosis play integral roles in regulating the activities and turnover of plasma membrane proteins required for signal triggering or attenuation at the cell surface. Exocytosis involves the transport of newly synthesized or recycled material to the plasma membrane (PM) in vesicles where they are released into the extracellular space. Recent investigations have shown that changing the balance between conventional and unconventional protein secretion can be an efficient strategy to respond to stressful conditions in eukaryotes. With the aid of an N-terminal located signal peptide (SP), conventional protein secretion delivers newly synthesized proteins from the endoplasmic reticulum (ER) to the Golgi apparatus and the <italic>trans</italic>-Golgi network (TGN) and finally to the PM or extracellular space via secretory vesicles or secretory granules. Unconventional protein secretion is responsible for the secretion of such SP-lacking protein. Notably, several unconventional protein secretion pathways mediated by distinct mechanisms have been reported in plants, most of which are related to stress responses. Conversely to exocytosis, endocytosis is the process by which material associated with the cellular surface is uptaken by invagination of the plasma membrane and internalized as an endocytic vesicle. In recent years, substantial progress has been made in the characterization of the plant components involved in clathrin-mediated and membrane microdomain-associated endocytic pathways. Moreover, the coordination of exocytic and endocytic trafficking has been shown to be important for various plant cell functions. An increasing pool of evidence has indicated that multiple cellular processes require tightly regulated exocytosis-endocytosis coupling, including nutrient uptake, signaling transduction, and plant–microbe interactions. However, compared to animal systems in which multiple mechanisms have been described, the functions and regulation of coupled trafficking processes in plants remain poorly understood. All proteins in the cells of an organism and most extracellular proteins are continually being degraded and replaced. The autophagy pathway is a conserved protein auto degradation pathway that delivers dysfunctional organelles or other cytoplasmic components to vacuoles for degradation and recycling in plants. In addition, autophagy is reported to be involved in plant development as part of processes such as root tip cell growth and differentiation. Recent observations have revealed possible interplay between vesicle trafficking and autophagic pathways in regulating vacuolar degradation in plants. In recent years, with constant advances in super-resolution microscopy and labeling methods, much progress has been made in mechanistic studies of membrane protein transportation, and a diversity of exocytic and endocytic trafficking pathways have been uncovered in plants. Although a variety of methods and techniques have been used to study plant vesicle trafficking, a systematic summary of vesicle trafficking pathways of plant membrane proteins has not been published. This review first introduces the relevant organelles involved in membrane protein vesicle trafficking, and then summarizes the different plant vesicle trafficking pathways for membrane proteins. Based on the review, we systematically consolidated the chemical treatments and mutants used in the study of plant vesicle trafficking. Finally, future directions for research in vesicle trafficking pathways of plant membrane proteins are discussed. With recent advancements, we expect major breakthroughs in our understanding of the regulatory mechanisms that determine the perception and adaptability of plants to their environments in the coming years.

Melatonin Attenuates Ischemic-like Cell Injury by Promoting Autophagosome Maturation via the Sirt1/FoxO1/Rab7 Axis in Hippocampal HT22 Cells and in Organotypic Cultures.

Dysfunctional autophagy is linked to neuronal damage in ischemia/reperfusion injury. The Ras-related protein 7 (Rab7), a member of the Rab family of small GTPases, appears crucial for the progression of the autophagic flux, and its activity is strictly interconnected with the histone deacetylase Silent information regulator 1 (Sirt1) and transcription factor Forkhead box class O1 (FoxO1). The present study assessed the neuroprotective role of melatonin in the modulation of the Sirt1/FoxO1/Rab7 axis in HT22 cells and organotypic hippocampal cultures exposed to oxygen-glucose deprivation followed by reoxygenation (OGD/R). The results showed that melatonin re-established physiological levels of autophagy and reduced propidium iodide-positive cells, speeding up autophagosome (AP) maturation and increasing lysosomal activity. Our study revealed that melatonin modulates autophagic pathways, increasing the expression of both Rab7 and FoxO1 and restoring the Sirt1 expression affected by OGD/R. In addition, the Sirt1 inhibitor EX-527 significantly reduced Rab7, Sirt1, and FoxO1 expression, as well as autolysosomes formation, and blocked the neuroprotective effect of melatonin. Overall, our findings provide, for the first time, new insights into the neuroprotective role of melatonin against ischemic injury through the activation of the Sirt1/FoxO1/Rab7 axis.

Emerging Drug Targets for Endometriosis.

Endometriosis is a chronic inflammatory disease causing distressing symptoms and requiring a life-long management strategy. The objective of this review is to evaluate endometriosis-related pathways and identify novel therapies to treat it. We focused on the crucial role of inflammation and inflammatory molecules in order to define new perspectives for non-hormonal treatment of the disease by targeting inflammation, nuclear factor kappa B and cytokines, or reactive oxygen species, apoptotic and autophagic pathways, regulators of epithelial-mesenchymal transition, and angiogenesis and neuroangiogenesis. Novel non-steroidal therapies targeting these pathways for endometriosis were explored, but multiple challenges remain. While numerous agents have been investigated in preclinical trials, few have reached the clinical testing stage because of use of inappropriate animal models, with no proper study design or reporting of preclinical strategies. Targeting estrogens is still the best way to control endometriosis progression and inflammation.

Abstract 15215: Autophagy Inhibition Attenuate the Development and Progression of Pulmonary Hypertension

Introduction: Autophagy plays an important role in the pathogenesis of pulmonary hypertension. However, the potential therapeutic benefit of autophagy inhibition remains undetermined. Hypothesis: ROC-325 was developed as a novel small molecule lysosomal autophagy inhibitor that has demonstrated significantly more potent anticancer activity than the antimalarial drug hydroxychloroquine (HCQ). Methods: Rats with monocrotaline-induced PH (MCT-PH) and hypoxia/Sugen-induced severe PH were treated with ROC-325 and the related effects on echocardiography, hemodynamics, right ventricle hypertrophy, and pulmonary vascular remodeling were assessed. Pulmonary arterial pressure (PAP) and isometric tension were measured in isolated perfused/ventilated mouse lungs and rat PA rings. The in vitro effects on ROC-325 on autophagy, proliferation, viability, apoptosis and endothelial nitric oxide (NO) synthase (eNOS)-NO signaling were tested in human and rat pulmonary artery smooth muscle cells (PASMCs) as well as human pulmonary artery endothelial cells (PAECs) and rat pulmonary microvascular endothelial cells (PMVECs). Results and Conclusions: The therapeutic benefits of a novel lysosomal autophagy inhibitor, ROC-325, in experimental pulmonary hypertension (PH) models. Specifically, ROC-325 treatment prevents the development of monocrotaline (MCT)-induced PH, right ventricular dysfunction, and pulmonary vascular remodeling, and reverses the progression of established MCT-PH. Meanwhile, ROC-325 treatment attenuates high K + or alveolar hypoxia-induced pulmonary vasoconstriction. Mechanistically, in PASMC and PAEC, ROC-325 inhibits autophagic pathways, reduces hypoxia induced-hypoxia inducible factor (HIF) accumulation and blocks endothelial nitric oxide (NO) synthase (eNOS)-NO signaling activation. These studies illustrate the discovery of ROC-325 as a novel promising agent for PH therapy.

PCSK9 Knockdown Can Improve Myocardial Ischemia/Reperfusion Injury by Inhibiting Autophagy

This study investigates the effect and mechanism of proprotein convertase subtilisin/Kexin type 9 (PCSK9) on myocardial ischemia-reperfusion injury (MIRI) and provides a reference for clinical prevention and treatment of acute myocardial infarction (AMI). We established a rat model of myocardial ischemia/reperfusion (I/R) and AC16 hypoxia/reoxygenation (H/R) model. A total of 48 adult 7-week-old male Sprague-Dawley rats were randomly assigned to three groups (n = 16): control, I/R, and I/R + SiRNA. In I/R and I/R + siRNA groups, myocardial ischemia was induced via occlusion of the left anterior descending branch (LAD) of the coronary artery in rats in I/R group for 30min and reperfused for 3days. To assess the myocardial injury, the rats were subjected to an electrocardiogram (ECG), cardiac function tests, cardiac enzymes analysis, and 2,3,5-triphenyl tetrazolium chloride (TTC)/Evan Blue (EB) staining. Meanwhile, differences in the expression of autophagy-level proteins and Bcl-2/adenovirus E1B 19-kDa interacting protein (Bnip3) signaling-related proteins were determined by protein blotting. In vitro and in vivo experimental studies revealed that siRNA knockdown of PCSK9 reduced the expression of autophagic protein Beclin-1, light chain 3 (LC3) compared to normal control-treated cells and control-operated groups. Simultaneously, the expression of Bnip3 pathway protein was downregulated. Furthermore, the PCSK9-mediated small interfering RNA (siRNA) group injected into the left ventricular wall significantly improved cardiac function and myocardial infarct size. In ischemic/hypoxic circumstances, PCSK9 expression was dramatically increased. PCSK9 knockdown alleviated MIRI via Bnip3-mediated autophagic pathway, inhibited inflammatory response, reduced myocardial infarct size, and protected cardiac function.