Light Chain 3 Research Articles

Semaphorin‑3A alleviates cardiac hypertrophy by regulating autophagy.

Cardiac hypertrophy, characterized by cardiomyocyte enlargement, is an adaptive response of the heart to certain hypertrophic stimuli; however, prolonged hypertrophy results in cardiac dysfunction and can ultimately cause heart failure. The present study evaluated the role of semaphorin-3A (Sema3A), a neurochemical inhibitor, in cardiac hypertrophy, utilizing an isoproterenol (ISO) induced H9c2 cell model. Cells were stained with rhodamine-phalloidin to assess the cell surface area and reverse transcription-quantitative PCR was performed to quantify mRNA expression levels of Sema3A, brain natriuretic factor (BNF) and β-myosin heavy chain (β-MHC). The protein expression levels of the autophagy-related proteins light chain 3 (LC3), p62 and Beclin-1, and the Akt/mTOR signaling pathway associated proteins Akt, phosphorylated (p)-Akt, mTOR, p-mTOR, 4E-binding protein 1 (4EBP1) and p-4EBP1 were semi-quantified using western blotting. Rapamycin, a canonical autophagy inducer, was administered to H9c2 cells to elucidate the regulatory mechanism of Sema3A. The results indicated significantly increased cell surface area and elevated BNF and β-MHC mRNA expression levels, increased LC3II/I ratio and Beclin-1 protein expression levels and significantly decreased p62 protein expression levels after treatment of H9c2 cardiomyocytes with ISO for 24 h. Sema3A overexpression improved ISO-induced hypertrophy in H9c2 cells, indicated by decreased cell surface area and reduced BNF and β-MHC mRNA expression levels. Moreover, Sema3A overexpression inhibited ISO-induced autophagy in H9c2 cells, indicated by decreased LC3II/I ratio and Beclin-1 protein expression levels and increased p62 protein expression levels. The autophagy activator rapamycin partially inhibited the protective effect of Sema3A on ISO-induced hypertrophy. Sema3A overexpression suppressed the decrease of the protein expression levels of p-Akt, mTOR and their downstream target 4EBP1, which is induced by ISO. Collectively, these results suggested Sema3A prevented ISO-induced cardiac hypertrophy by inhibiting autophagy via the Akt/mTOR signaling pathway.

Stabilization of mitochondrial function by chlorogenic acid protects against kainic acid-induced seizures and neuronal cell death in rats

The current study investigated the effect of chlorogenic acid, a polyphenolic compound found in numerous plant products, on a kainic acid-induced seizure rat model and its potential mechanism. Rats were administered chlorogenic acid (10 and 50 mg/kg) intraperitoneally for 30 min before kainic acid (15 mg/kg) intraperitoneal administration. Pretreatment with chlorogenic acid decreased the seizure score, increased the latency to onset of the first seizure, and decreased the mortality rate. Chlorogenic acid pretreatment also resulted in a significant reduction in glutamate elevation and neuronal death in the hippocampus of kainic acid-treated rats. In addition, electron microscopy revealed that kainic acid-induced changes in hippocampal mitochondrial structure were prevented by chlorogenic acid pretreatment. Additionally, the levels of mitochondrial function-related proteins, including sirtuin 3, Complex I, glutamate dehydrogenase 1 and ATP synthase, were increased, and the level of the mitochondrial damage marker cytochrome C was decreased in the hippocampus of chlorogenic acid/kainic acid rats. Furthermore, the expression of mitochondrial biogenesis-related proteins [AMP-activated protein kinase (AMPK), sirtuin1, and peroxisome proliferator-activated receptor γ-coactivator-1α (PGC-1α)] and mitophagy-related proteins [phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1), Parkin, and microtubule-associated protein 1 light chain 3 (LC3)] was decreased in the hippocampus of kainic acid-treated rats, which was reversed by chlorogenic acid pretreatment. These observations reveal the marked neuroprotective potential of chlorogenic acid against kainic acid-induced neurotoxicity and seizures through prevention of glutamate increase and preservation of AMPK/sirtuin 1/PGC-1α-mediated mitochondrial biogenesis and PINK1/Parkin-induced mitophagy to maintain adequate mitochondrial homeostasis and function.

DJ1 Ameliorates AD-like Pathology in the Hippocampus of APP/PS1 Mice

ObjectiveTo explore whether the protein Deglycase protein 1 (DJ1) can ameliorate Alzheimer's disease (AD)-like pathology in Amyloid Precursor Protein/Presenilin 1 (APP/PS1) double transgenic mice and its possible mechanism to provide a theoretical basis for exploring the pathogenesis of AD. MethodsAdeno-associated viral vectors (AAV) of DJ1-overexpression or DJ1-knockdown were injected into the hippocampus of 7-month-old APP/PS1 mice to construct models of overexpression or knockdown. Mice were divided into the AD model control group (MC), AAV vector control group (NC), DJ1-overexpression group (DJ1+), and DJ1-knockdown group (DJ1–). After 21 days, the Morris water maze test, immunohistochemistry, immunofluorescence, and western blotting were used to evaluate the effects of DJ1 on mice. ResultsDJ1+ overexpression decreased the latency and increased the number of platform traversals in the water maze test. DJ1– cells were cured and atrophied, and the intercellular structure was relaxed; the number of age spots and the expression of AD-related proteins were significantly increased. DJ1+ increased the protein expression of Nuclear factor erythroid 2-related factor 2 (NRF2), heme oxygenase-1 (HO-1), light chain 3 (LC3), phosphorylated AMPK (p-AMPK), and B cell lymphoma-2 (BCL-2), as well as the antioxidant levels of total superoxide dismutase (T-SOD), total antioxidant capacity (T-AOC), and Glutathione peroxidase (GSH-PX), while decreasing the levels of Kelch-like hydrates-associated protein 1 (Keap1), mammalian target of rapamycin (mTOR), p62/sequestosome1 (p62/SQSTM1), Caspase3, and malondialdehyde (MDA). ConclusionsDJ1-overexpression can ameliorate learning, memory, and AD-like pathology in APP/PS1 mice, which may be related to the activation of the NRF2/HO-1 and AMPK/mTOR pathways by DJ1.

Canagliflozin ameliorates high glucose-induced apoptosis in NRK-52E cells via inhibiting oxidative stress and activating AMPK/mTOR-mediated autophagy.

Sodium-glucose cotransporter-2 (SGLT-2) inhibitors, as a new type of hypoglycemic drug, can prevent proximal renal tubule injury related to glucose toxicity and play a renoprotective role. Canagliflozin, a recognized SGLT-2 inhibitor, has been proved to have potential protection in diabetic nephropathy (DN), but its mechanism has not been fully elucidated. In this study, the protective effect of canagliflozin against high glucose (HG)-induced renal tubular epithelial cell (NRK-52E) injury in vitro was assessed. The viability and apoptosis of NRK-52E cells were detected using cell counting kit-8 (CCK-8) assay and flow cytometry analysis, respectively. The expression levels of cleaved caspase-3, oxidative stress-related proteins (NOX4 and Nrf2), autophagy marker light chain 3 (LC3) I/II, and adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway-related proteins were evaluated by Western blot. Reactive oxygen species (ROS) level was evaluated by dihydroethidium (DHE) reactive oxygen species assay, the activities of SOD, CAT, GSH-Px and MDA were analyzed using kits. The changes of morphology and red fluorescent protein (RFP)-LC3 fluorescence were observed under microscopy. Canagliflozin significantly ameliorated HG-induced NRK-52E cell apoptosis and caspase-3 cleavage. Furthermore, canagliflozin markedly ameliorated HG-induced NRK-52E cell oxidative stress. Moreover, canagliflozin significantly increased LC3-II levels and induced RFP-LC3-containing punctate structures in NRK-52E cells. Finally, canagliflozin increased the phosphorylation of AMPK and suppressed the phosphorylation of mTOR. The AMPK inhibitor compound C abolished canagliflozin-induced autophagy activation, as well as the anti-apoptotic effect of canagliflozin. Canagliflozin effectively ameliorate HG-induced apoptosis of NRK-52E cells in vitro that involved its antioxidant effect and induction of autophagy through the AMPK/mTOR pathway.

Melatonin-mediated mitophagy protects against long-term impairments after repeated neonatal sevoflurane exposures

BackgroundMelatonin is known to have protective effects in aging, neurodegenerative disorders and mitochondria-related diseases, while there is a poor understanding of the effects of melatonin treatment on mitophagy in neonatal cognitive dysfunction after repeated sevoflurane exposures. This study explores the protective effects of melatonin on mitophagy and cognition in developing rats exposed to sevoflurane. MethodsPostnatal day six (P6) neonatal rats were exposed to 3 % sevoflurane for 2 h daily from P6 to P8. In the intervention groups, rats received 3-Methyladenine (3-MA) intracerebroventricularly from P6 to P8 and melatonin intraperitoneally from P6 to P8 following water drinking once daily from P21 to P41, respectively. Behavioral tests, including open field (OF), novel object recognition (NOR), and fear conditioning (FC) tests, were performed to assess cognitive function during young adulthood. In another experiment, rat brains were harvested for biochemical, histopathological, and electron microscopy studies. ResultsRats exposed to sevoflurane showed disordered mitophagy and mitochondrial dysfunction as revealed by increased mitophagy marker proteins (microtubule-associated protein 1 light chain 3 (LC3) II/I, and parkin), decreased autophagy marker protein (sequestosome 1 (P62/SQSTM1)), electron transport chain (ETC) proteins and ATP levels. Immunofluorescent staining of LC3 was co-localized mostly with a neuronal marker and microglial marker but was not co-localized with a marker for astrocytes in rats exposed to sevoflurane. These rats had poorer performance in the NOR and FC tests than control rats during young adulthood. Melatonin treatment reversed the abnormal expression of mitophagy proteins, mitochondrial energy metabolism, the activity of microglia, and impaired cognition. These ameliorations were blocked by an autophagy inhibitor, 3-MA, except for the activation of microglia. ConclusionWe have demonstrated that melatonin inhibits microglial activation by enhancing mitophagy and finally significantly reduces sevoflurane-induced deficits in cognition in neonatal rats. These results suggest that melatonin might be beneficial if considered when the anesthesia must be administered at a very young age.

Epithelial Cell NOD1/IRGM Recruits STX17 to Neisseria gonorrhoeae-Containing Endosomes to Initiate Lysosomal Degradation.

Neisseria gonorrhoeae establishes tight interactions with mucosal epithelia through activity of its type IV pilus, while pilus retraction forces activate autophagic responses toward invading gonococci. Here we studied pilus-independent epithelial cell responses and showed that pilus-negative gonococci residing in early and late endosomes are detected and targeted by nucleotide-binding oligomerization domain 1 (NOD1). NOD1 subsequently forms a complex with immunity-related guanosine triphosphatase M (IRGM) and autophagy-related 16-like 1 (ATG16L1) to activate autophagy and recruit microtubule-associated protein light chain 3 (LC3) to the intracellular bacteria. IRGM furthermore directly recruits syntaxin 17 (STX17), which is able to form tethering complexes with the lysosome. Importantly, IRGM-STX17 interactions are enhanced by LC3 but were still observed at lower levels in an LC3 knockout cell line. These findings demonstrate key roles for NOD1 and IRGM in the sensing of intracellular N gonorrhoeae and subsequent directing of the bacterium to the lysosome for degradation.

Alcohol induces hepatocytes necroptosis through the LC3/RIPK1/RIPK3 pathway

Excessive alcohol consumption leads to serious liver injury. Necroptosis is a programmed cell death form, which has been confirmed to be involved in alcoholic liver injury. However, the exact mechanism remains still unclear. In this study, we found that ethanol caused hepatocytes necroptosis by activating receptor-interacting serine/threonine-protein kinase 1 and 3 (RIPK1 and RIPK3). Meanwhile, autophagy was activated in ethanol-treated hepatocytes. Accumulative studies have demonstrated a possible link between autophagy and necroptosis. Microtubule-associated protein 1 light chain 3 (LC3), an autophagy marker protein, is essential for autophagosome biogenesis/maturation. But little attention has been paid to its functional role. In this study, we explored whether LC3 was involved in ethanol-induced necroptosis. The data showed that LC3 interacted with RIPK1 and RIPK3 in ethanol-treated AML12 cells and mice liver by co-immunoprecipitation (co-IP) and colocalization assay. Ethanol-induced necrosome formation and subsequent necroptosis were alleviated in hepatocytes by knockdown of LC3 or autophagy inhibitor 3-methyladenine (3-MA). These results demonstrated that LC3 accumulation facilitated the formation of necrosome by LC3-RIPK1 and LC3-RIPK3 interactions, eventually caused hepatocytes necroptosis after acute ethanol exposure. Our current research could potentially offer a new understanding of the intricate mechanisms involved in the development of acute alcoholic liver injury.

The possible effects of the aqueous extract of Stevia Rebaudiana on autophagic clearance and sympathetic nerve density in pancreatic β cells of type 2 diabetic rats

BackgroundDiabetes mellitus (DM) is a common metabolic disorder affecting more than 400 million people worldwide. Pancreatic islet cell failure due to partial loss or dysfunction of islet cells is a common feature in type 2 DM. The aim of this study to assess the possible protective role of aqueous extract of stevia on pancreatic β cells of type 2 diabetic rats. MethodsThirty-two male Sprague Dawley rats were divided into four groups; control, control received stevia, type 2 DM and type 2 DM treated with stevia. For all groups; fasting blood glucose, fasting insulin, pancreatic Malondialdehyde (MDA) and Catalase (CAT) were measured. Western blot analysis for measuring LC3 (microtubule associated protein-1 light chain-3) and LAMP2 (lysosomal associated membrane protein 2) was done. Also, histopathological, histomorphometric studies and tyrosine hydroxylase (TH) and norepinephrine (NE) expression by immunostaing in pancreatic tissues were detected. ResultsType 2 DM resulted in a significant increase in fasting blood glucose, pancreatic MDA, LC3 and LAMP2 expression, TH and NE density with a significant decrease in fasting insulin, pancreatic CAT, pancreatic islet area, the number of islets per unit area and the number of β cell per islet. Also, treatment with stevia extract significantly improved the laboratory and histopathological features of pancreas ConclusionStevia extract has protective effects on type 2 DM pancreas by its hypoglycemic and antioxidant actions in addition to its ability to attenuate hyperactive autophagy and sympathetic nerve density.

Effect of electroacupuncture on enteric neuronal autophagy in functional constipation mice.

To explore the effect mechanism of electroacupuncture (EA) on functional constipation (FC) at the combined lower he-sea and front-mu points of large intestine based on enteric neuronal autophagy. A total of 40 SPF Kunming mice were randomly divided into 5 groups (n = 8), i.e. a control group, a model group, an acupuncture group, a 3-methyl adenine (3-MA) group, and a 3-MA + acupuncture group. Except the control group, the FC model was established by gavage with compound diphenoxylate suspension for 14 days in the other 4 groups. After successful modeling, the mice of the acupuncture group and the 3-MA + acupuncture group received EA at bilateral "Tianshu" (ST 25) and "Shangjuxu" (ST 37), stimulated for 30 min with disperse-dense wave, 2 Hz/15 Hz of frequency, 1 mA of intensity. EA was delivered once daily. One course of treatment was composed of 5 days and 2 courses were needed, with an interval of 2 days. An intraperitoneal injection of 3-MA (15 mg/kg) was administered 30 min before EA in the mice of the 3-MA group and the 3-MA + acupuncture group, once daily. Before and after intervention, the time of the first black stool defecation and defecation behaviors in 6 h were observed in each group. After intervention, in every group, the small intestine propulsion rate was calculated, the colon tissue morphology was observed using HE staining, the ultrastructure of enteric neuronal autophagy was observed under transmission electron microscope, and the expressions of microtubule-associated protein 1 light chain 3 (LC3), Beclin-1 and neuronal nuclear antigen protein (NeuN) in neurons of colonic muscularis were determined by immunohistochemistry. Before intervention, when compared with those in the control group, the time of the first black stool defecation was prolonged (P<0.01, P<0.05), and numbers (P<0.01), wet weight (P<0.01, P<0.05) and water content (P<0.05, P<0.01) of stool in 6 h were reduced in the model, acupuncture, 3-MA and 3-MA + acupuncture groups. After intervention, compared with those in the control group, the time of the first black stool defecation was longer (P<0.05), and numbers (P<0.01), wet weight (P<0.01) and water content (P<0.01) of stool in 6 h were decreased in the model group. The time of the first black stool defecation was shortened (P<0.01), and numbers (P<0.01), wet weight (P<0.01) and water content (P<0.01) of stool in 6 h were increased in the acupuncture group when compared with those in the model group. The time of the first black stool defecation was extended (P<0.01), and numbers (P<0.01), wet weight (P<0.01) and water content (P<0.01) of stool in 6 h were declined in the 3-MA + acupuncture group in comparison with those in the acupuncture group. All layers of colon tissue were normal and intact in each group. When compared with the control group, the small intestine propulsion rate and the average optical density (OD) values of LC3, Beclin-1 and NeuN in neurons of colonic muscularis were decreased (P<0.01), and autophagosomes were dropped in the model group. In the acupuncture group, the small intestine propulsion rate and the average OD values of NeuN, LC3 and Beclin-1 in neurons of colonic muscularis increased (P<0.01,P<0.05), and autophagosomes were elevated when compared with those in the model group. The small intestine propulsion rate and the average OD values of NeuN, LC3 and Beclin-1 in neurons of colonic muscularis were dropped (P<0.05,P<0.01) in the 3-MA + acupuncture group in comparison with those in the acupuncture group. Electroacupuncture may promote enteric neuronal autophagy and increase the number of neurons so that the intestinal motility can be improved and constipation symptoms can be relieved in FC mice.

Serum levels of tumor necrosis factor (TNF-α) and vaginal gene expression of toll-like receptor 2 (TLR2) & microtubule-associated protein 1 light chain 3 (LC3) after treatment of vulvovaginal candidiasis with sertaconazole nitrate spanlastics

The irritating infection known as vulvovaginal candidiasis (VVC) affects women all over the world. Around 75% of women will acquire at least one attack throughout their lives. In addition to its demonstrated antifungal properties, sertaconazole nitrate (SCN) also exhibits anti-inflammatory effects, which are of significant importance in the therapeutic management of (VVC). Despite its benefits, SCN suffers from poor solubility, which affects its biological activity. This project's objective is to construct SCN spanlstics in order to enhance its solubility as well as its antifungal and anti-inflammatory activities. SCN spanlastics were created utilizing the ethanol injection technique, after which they were characterized. Chitosan (2.5%) w/w gels containing 2 % w/w of SCN in the form of spanlastics were prepared, and then the in vitro antifungal activity of the prepared chitosan gel and commercial SCN cream (Dermofix) was performed. After a complete gynecological examination, vaginal swabs were obtained from patients suspected of VVC and then cultured on Sabouraud dextrose agar for phenotypic identification. VVC was then induced in nonpregnant ovariectomized female rats using isolated and identified Candida albicans (C. albicans) species. In order to detect the difference between chitosan gel containing SCN spanlastics and the commercial (Dermofix) cream, serum levels of pro-inflammatory cytokine Tumor necrosis factor (TNF- α) were detected. After sacrificing the rats, gene expression of toll-like receptor 2 (TLR2) & microtubule-associated protein 1 light chain 3 (LC3) in vaginal tissue and immunohistochemical expression of interleukin-6 (IL-6) as an inflammatory chemokine were performed in all groups. Results revealed that SCN was successfully entrapped into spanlastics, the particle size ranged from 1.6 ± 0.050 to 5.7 ± 0.900 μm in diameter; in-vitro dissolution of SCN was significantly enhanced from chitosan gel compared to that from Dermofix cream, the inhibition zone of prepared chitosan gel containing SCN spanlastics was significantly larger compared to that of Dermofix cream. Our results revealed that the anti-inflammatory effect of SCN in the form of spanlastics was significantly greater than that of commercial (Dermofix) cream. Finally, we recommend the use of SCN spanlastics as the best antifungal and anti-inflammatory formulation in cases of vaginal candidiasis.

The SGLT2 inhibitor empagliflozin attenuates atherosclerosis progression by inducing autophagy.

Cardiovascular disease due to atherosclerosis is one of the leading causes of death worldwide; however, the underlying mechanism has yet to be defined. The sodium-dependent glucose transporter 2 inhibitor (SGLT2i) empagliflozin is a new type of hypoglycemic drug. Recent studies have shown that empagliflozin not only reduces high glucose levels but also exerts cardiovascular-protective effects and slows the process of atherosclerosis. The purpose of this study was to elucidate the mechanism by which empagliflozin ameliorates atherosclerosis. Male apolipoprotein E-deficient (ApoE-/-) mice were fed a high-fat Western diet to establish an atherosclerosis model. The area and size of atherosclerotic lesions in ApoE-/- mice were then assessed by performing hematoxylin-eosin (HE) staining after empagliflozin treatment. Concurrently, oxidized low-density lipoprotein (oxLDL) was used to mimic atherosclerosis in three different types of cells. Then, following empagliflozin treatment of macrophage cells (RAW264.7), human aortic smooth muscle cells (HASMCs), and human umbilical vein endothelial cells (HUVECs), western blotting was applied to measure the levels of autophagy-related proteins and proinflammatory cytokines, and green fluorescent protein (GFP)-light chain 3 (LC3) puncta were detected using confocal microscopy to confirm autophagosome formation. Oil Red O staining was performed to detect the foaming of macrophages and HASMCs, and flow cytometry was used for the cell cycle analysis. 5-ethynyl-2'-deoxyuridine (EdU), cell counting kit-8 (CCK-8), and scratch assays were also performed to examine the proliferation and migration of HASMCs. Empagliflozin suppressed the progression of atherosclerotic lesions in ApoE-/- mice. Empagliflozin also induced autophagy in RAW246.7 cells, HASMCs, and HUVECs via the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway, and it significantly increased the levels of the Beclin1 protein, the LC3B-II/I ratio, and p-AMPK protein. In addition, empagliflozin decreased the expression of P62 and the protein levels of inflammatory cytokines, and it inhibited the foaming of RAW246.7 cells and HASMCs, as well as the expression of inflammatory factors by inducing autophagy. Empagliflozin activated autophagy through the AMPK signaling pathway to delay the progression of atherosclerosis. Furthermore, the results of flow cytometry, EdU assays, CCK-8 cell viability assays, and scratch assays indicated that empagliflozin blocked HASMCs proliferation and migration. Empagliflozin activates autophagy through the AMPK signaling pathway to delay the evolution of atherosclerosis, indicating that it may represent a new and effective drug for the clinical treatment of atherosclerosis.

Ferroptosis is involved in passive Heymann nephritis in rats

Ferroptosis is found to be involved in some experimental models of kidney diseases, but its role in membrane nephropathy (MN) is still unclear. The purpose of this study is to explore whether ferroptosis occurred in MN, and the role of ferritinophagy. In this study, passive Heymann nephritis (PHN) rats were induced by single tail vein injection of anti-Fx1A serum, and normal rats were used as control. The changes of 24 h urinary protein, serum biochemical parameters, renal pathological damage, iron content, lipid peroxidation parameters, ferroptosis markers, and ferritinophagy markers were evaluated in the two groups. Compared with the control group, PHN rats showed obvious proteinuria, hypoproteinemia, and hyperlipidemia. Besides, more severe renal pathological damage and higher Fe2+ levels were observed in PHN rats, and the levels of malondialdehyde (MDA) increased significantly, while the levels of superoxide Dismutase (SOD) and glutathione (GSH) decreased. In addition, the expression of glutathione peroxidase 4 (GPX4) in renal tissues of PHN rats decreased significantly, while the expression of transferrin receptor (TFR) and acyl-CoA synthetase long-chain family member 4 (ACSL4) increased. The expression of microtubule associated protein 1 light chain 3 (LC3) II/LC3I and nuclear receptor coactivator 4 (NCOA4) increased significantly. Therefore, our study shows that ferroptosis is involved in the pathological damage of MN, and companied by activation of ferritinophagy.

Saikosaponin D regulates apoptosis and autophagy of pancreatic cancer Panc-1 cells via Akt/mTOR pathway

This study aims to investigate the effect and mechanism of saikosaponin D on the proliferation, apoptosis, and autophagy of pancreatic cancer Panc-1 cells. The cell counting kit(CCK-8) was used to examine the effects of 7, 10, 13, 16, 19, 22, 25, and 28 μmol·L~(-1) saikosaponin D on the proliferation of Panc-1 cells. Three groups including the control(0 μmol·L~(-1)), low-concentration(10 μmol·L~(-1)) saikosaponin D, and high-concentration(16 μmol·L~(-1)) saikosaponin D groups were designed. The colony formation assay was employed to measure the effect of saikosaponin D on the colony formation rate of Panc-1 cells. The cells treated with saikosaponin D were stained with hematoxylin-eosin(HE), and the changes of cell morphology were observed. Hoechst 33258 fluorescent staining was used to detect the effect of saikosaponin D on the cell apoptosis. The autophagy staining assay kit with MDC was used to examine the effect of saikosaponin D on the autophagy of Panc-1 cells. Western blot and immunocytochemistry(ICC) were employed to examine the effect of saikosaponin D on the expression levels and distribution of B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), cysteine-aspartic acid protease-3(caspase-3), cleaved caspase-3, autophagy-associated protein Beclin1, microtubule-associated protein light chain 3(LC3), protein kinase B(Akt), phosphorylated protein kinase B(p-Akt), mammalian target of rapamycin(mTOR), and phosphorylated mammalian target of rapamycin(p-mTOR). The results showed that compared with the control group, saikosaponin D decreased the proliferation rate of Panc-1 cells in a dose-dependent and time-dependent manner. The colony formation rate of the cells significantly decreased after saikosaponin D treatment. Compared with the control group, the cells treated with saikosaponin D became small, accompanied by the formation of apoptotic bodies. The saikosaponin D groups showed increased apoptosis rate and autophagic vesicle accumulation. Compared with the control group, saikosaponin D up-regulated the expression of Bax, cleaved caspase3, Beclin1, LC3Ⅱ/LC3Ⅰ and down-regulated the expression of Bcl-2, caspase-3, p-Akt/Akt, and p-mTOR/mTOR. In addition, these proteins mainly existed in the cytoplasm. In conclusion, saikosaponin D can inhibit the proliferation and induce the apoptosis and autophagy of Panc-1 cells via inhibiting the Akt/mTOR pathway.

S-acylation of p62 promotes p62 droplet recruitment into autophagosomes in mammalian autophagy

p62 is a well-characterized autophagy receptor that recognizes and sequesters specific cargoes into autophagosomes for degradation. p62 promotes the assembly and removal of ubiquitinated proteins by forming p62-liquid droplets. However, it remains unclear how autophagosomes efficiently sequester p62 droplets. Herein, we report that p62 undergoes reversible S-acylation in multiple human-, rat-, and mouse-derived cell lines, catalyzed by zinc-finger Asp-His-His-Cys S-acyltransferase 19 (ZDHHC19) and deacylated by acyl protein thioesterase 1 (APT1). S-acylation of p62 enhances the affinity of p62 for microtubule-associated protein 1 light chain 3 (LC3)-positive membranes and promotes autophagic membrane localization of p62 droplets, thereby leading to theproduction of small LC3-positive p62 droplets and efficient autophagic degradation of p62-cargo complexes. Specifically, increasing p62 acylation by upregulating ZDHHC19 or by genetic knockout of APT1 accelerates p62 degradation and p62-mediated autophagic clearance of ubiquitinated proteins. Thus, the protein S-acylation-deacylation cycle regulates p62 droplet recruitment to the autophagic membrane and selective autophagic flux, thereby contributing to the control of selective autophagic clearance of ubiquitinated proteins.

Trapa Bispinosa Roxb. Inhibits the Insulin-Dependent AKT/WNK1 Pathway to Induce Autophagy in Mice with Type 2 Diabetes.

To elucidate the antiglycation activity of Trapa bispinosa Roxb. extract (TBE) and the related mechanism using a mouse model with type 2 diabetes. We prepared control mice by giving them a normal diet, leptin-deficient ob/ob mouse (ob/ob mice) with a normal diet (normal ob/ob mice), and ob/ob mice with a diet containing TBE (TBE ob/ob mice). The effect of TBE on diabetic retina was evaluated by immunohistochemical staining and quantitative real-time polymerase chain reaction (qPCR) analysis. In both groups with ob/ob mice, body weight and hyperglycemia levels increased over time. Immunohistochemical staining analysis revealed that glial fibrillary acidic protein (GFAP) and advanced glycation end products (AGEs) expression levels were higher in normal ob/ob mice than in control mice, and lower in the TBE ob/ob mice than in normal ob/ob mice. Light chain-3 (LC-3) expression levels reduced in normal ob/ob mice compared to the control mice, but increased in TBE ob/ob mice compared to normal ob/ob mice. In the qPCR analysis, LC-3 expression levels were significantly lower in normal ob/ob mice compared to control mice, and significantly higher in TBE ob/ob mice compared to normal ob/ob mice. Conversely, AKT1 and with-no-lysine kinases 1 (WNK1) expression levels were significantly higher in normal ob/ob mice compared to control mice, and significantly lower in TBE ob/ob mice than in normal ob/ob mice. In type 2 diabetes, it was suggested that TBE inhibits the insulin-dependent AKT/WNK1 pathway to induce autophagy, and thereby might promote anti-glycation and reduce retinal damage.

IL‑4 alleviates CIRI by suppressing autophagy via the HIF‑1α/Bcl‑2/BNIP3 pathway in rats.

This paper was designed for delving into the mechanism adopted by interleukin‑4 (IL‑4) to relieve cerebral ischemia‑reperfusion injury (CIRI) in rats via suppressing autophagy. Herein, rats stochastically fell into sham operation (sham), model (RI), model + IL‑4 intervention (IL‑4), model + HIF‑1α inhibitor (2‑methoxyestradiol, 2ME2) and model + IL‑4 + 2ME2 (IL‑4 + 2ME2) groups. Next, western blotting was utilized to examine the protein expressions of microtubule‑associated protein 1 light chain 3 (LC3), p62, hypoxia‑inducible factor 1‑alpha (HIF‑1α) and Bcl‑2/adenovirus E1B 19 kDa‑interacting protein 3 (BNIP3). Relative to RI group, IL‑4 group had a significantly lower neurological impairment scale (NIS) score and an overtly lower apoptosis rate of neurons as well as a strikingly smaller cerebral infarction volume and number of autophagosomes (P<0.05). The LC3II/LC3I ratio and HIF‑1α and BNIP3 protein expressions dropped, but p62 protein expression rose pronouncedly in IL‑4 group (P<0.05). In contrast to those in RI group, the NIS score, neuronal apoptosis rate, cerebral infarction volume and autophagosome number were strikingly reduced (P<0.05). The NIS score, cerebral infarction volume, neuronal apoptosis rate, autophagosome number, LC3II/LC3I ratio and protein expressions of HIF‑1α and BNIP3 plummeted, while p62 protein expression sharply rose in IL‑4 + 2ME2 group relative to those in IL‑4 group (P<0.05). IL‑4 suppresses cell autophagy by inhibiting the HIF‑1α/BNIP3 pathway, thus relieving CIRI in rats.

VEGF-A ameliorates ischemia hippocampal neural injury via regulating autophagy and Akt/CREB signaling in a rat model of chronic cerebral hypoperfusion

ObjectiveChronic cerebral hypoperfusion (CCH) can cause a series of pathophysiological processes, including neuronal autophagy and apoptosis. VEGF-A has been reported to affect angiogenesis and neurogenesis in many CNS diseases. However, its effects on neuronal autophagy and apoptosis, as well as the underlying mechanisms in CCH remain unclear. MethodsTo address these issues, the CCH model was established by permanent bilateral common carotid artery occlusion (2VO). Rats were sacrificed at different stages of CCH. Hippocampal morphological and ultrastructural changes were detected using HE staining and electron microscopy. The immunoreactivities of microtubule-associated protein 1 light chain 3 (LC3) and phospho-cAMP response element binding protein (p-CREB) were examined by immunofluorescence staining. The neuronal apoptosis was detected via TUNEL staining. The levels of LC3-II, Beclin-1, Akt, p-Akt, CREB, p-CREB, Caspase-3, and Bad were accessed by Western blotting. Furthermore, mouse hippocampal HT22 neurons received the oxygen and glucose deprivation (OGD) treatment, VEGF-A treatment, and GSK690693 (an Akt inhibitor) treatment, respectively. ResultsLC3-II protein started to increase at 3 days of CCH, peaked at 4 weeks of CCH, then decreased. CCH increased the levels of LC3-II, Caspase-3, and Bad, and decreased the levels of p-Akt, CREB, and p-CREB, which were reversed by VEGF-A treatment. VEGF-A also improved CCH-induced neuronal ultrastructural injuries and apoptosis in the hippocampus in vitro. In HT22, the anti-apoptosis and pro-phosphorylation of VEGF-A were reversed by GSK690693. ConclusionPresent results provide a novel neuroprotective effect of VEGF-A in CCH that is related to the inhibition of neuronal autophagy and activation of the Akt/CREB signaling, suggesting a potential therapeutic strategy for ischemic brain damage.

Meta‑analysis of the autophagy‑associated protein LC3 as a prognostic marker in colorectal cancer.

Microtubule-associated protein 1 light chain 3 (LC3) is an autophagy-associated gene, which is involved in the progression of a number of human malignancies. Such as Breast Cancer, Liver Cancer, and Lung Cancer. However, the role of LC3 in colorectal cancer (CC) remains to be fully elucidated. Therefore, the prognostic role of LC3 expression in CC was evaluated in the present study, with an emphasis on the clinicopathology and prognosis. Expression of LC3 in CC was examined using PubMed, Cochrane Library, Excerpta Medica Database, China Knowledge Infrastructure and Wanfang Data. Newcastle-Ottawa scale was used to screen the literature quality, and RevMan 5.4 and STATA 14.0 were used for the meta-analysis. A total of 1,689 patients from 10 studies were included in the present meta-analysis. The findings of the present study suggested that increased LC3 expression levels were associated with histological grade [odds ratio (OR)=0.91, 95% confidence interval (CI) (0.47, 1.77), P<0.001] and TNM stage [OR=0.91, 95% CI (0.47, 1.77), P<0.001], but were not associated with sex [OR=1.14, 95% CI (0.90, 1.51)], age [OR=0.89, 95% CI (0.67, 1.20)], tumor size [OR=0.78, 95% CI (0.30, 2.34)], histological grade [OR=0.82, 95% CI (0.43, 1.95)] and lymph node metastasis [OR=2.05, 95% CI (1.19, 3.60)] in CC. In addition, the increased expression of LC3 was revealed to be a prognostic factor for the overall survival of patients with CC. In conclusion, the autophagy-associated protein LC3 may be a prognostic indicator of human CC.

Glucolipotoxicity Suppressed Autophagy and Insulin Contents in Human Islets, and Attenuation of PERK Activity Enhanced Them in an ATG7-Dependent Manner.

Administration of pancreatic endoplasmic reticulum kinase inhibitor (PERKi) improved insulin secretion and hyperglycemia in obese diabetic mice. In this study, autophagic balance was studied whether to mediate it. Human islets were isolated from living patients without diabetes. PERKi GSK2606414 effects were evaluated in the islets under glucolipotoxicity by palmitate. Islet insulin contents and secretion were measured. Autophagic flux was assessed by microtubule associated protein 1 light chain 3 (LC3) conversion, a red fluorescent protein (RFP)-green fluorescent protein (GFP)- LC3 tandem assay, and P62 levels. For mechanical analyses, autophagy was suppressed using 3-methyladenine in mouse islets. Small interfering RNA for an autophagy-related gene autophagy related 7 (Atg7) was transfected to interfere autophagy. PERKi administration to mice decreased diabetes-induced P62 levels in the islets. Glucolipotoxicity significantly increased PERK phosphorylation by 70% and decreased insulin contents by 50% in human islets, and addition of PERKi (40 to 80 nM) recovered both. PERKi also enhanced glucose-stimulated insulin secretion (6-fold). PERKi up-regulated LC3 conversion suppressed by glucolipotoxicity, and down-regulated P62 contents without changes in P62 transcription, indicating enhanced autophagic flux. Increased autophagosome-lysosome fusion by PERKi was visualized in mouse islets, where PERKi enhanced ATG7 bound to LC3. Suppression of Atg7 eliminated PERKi-induced insulin contents and secretion. This study provided functional changes of human islets with regard to autophagy under glucolipotoxicity, and suggested modulation of autophagy as an anti-diabetic mechanism of PERKi.

Iron oxide nanoparticles induce ferroptosis via the autophagic pathway by synergistic bundling with paclitaxel.

In recent years, inhibiting tumor cell activity by triggering cell ferroptosis has become a research hotspot. The development of generic targeted nanotherapeutics might bring new ideas for non‑invasive applications. Currently, the potential mechanism underlying the universal application of paclitaxel (PTX)‑loaded iron oxide nanoparticles (IONP@PTX) to different types of tumors is unclear. The present study aimed to prepare IONP@PTX for targeted cancer therapy and further explore the potential mechanisms underlying the inhibitory effects of this material on the NCI‑H446 human small cell lung cancer and brain M059K malignant glioblastoma cell lines. First, a CCK‑8 assay was performed to determine cell viability, and then the combination index for evaluating drug combination interaction effect was evaluated. Intracellular reactive oxygen species (ROS) and lipid peroxidation levels were monitored using a DCFH‑DA fluorescent probe and a C11‑BODIPY™ fluorescent probe, respectively. Furthermore, western blotting assay was performed to determine the expression of autophagy‑ and iron death‑related proteins. The experimental results showed that, compared with either IONP monotherapy, PTX monotherapy, or IONP + PTX, IONP@PTX exerted a synergistic effect on the viability of both cell types, with significantly increased total iron ion concentration, ROS levels and lipid peroxidation levels. IONP@PTX significantly increased the expression of autophagy‑related proteins Beclin 1 and histone deacetylase 6 (HDAC6) in both cell lines (P<0.05), increased the expression of light chain 3 (LC3)‑II/I in NCI‑H446 cells (P<0.05) and decreased that of sequestosome1 (p62) in M059K cells (P<0.05). Moreover, the addition of rapamycin enhanced the IONP@PTX‑induced the upregulation of Beclin 1, LC3‑II/I and HDAC6 and the downregulation of mTORC1 protein in both cell lines (P<0.05). Moreover, rapamycin enhanced the IONP@PTX‑induced downregulation of p62 protein in NCI‑H446 cells (P<0.05), suggesting that IONP@PTX induces ferroptosis, most likely through autophagy. Collectively, the present findings show that IONP works synergistically with PTX to induce ferroptosis via the autophagic pathway.