Ellagic Acid Prevents α-Synuclein Aggregation and Protects SH-SY5Y Cells from Aggregated α-Synuclein-Induced Toxicity via Suppression of Apoptosis and Activation of Autophagy

Parkinson’s disease (PD) is a neurodegenerative disease characterized by the loss of dopamine neurons and the deposition of misfolded proteins known as Lewy bodies (LBs), which contain α-synuclein (α-syn). The causes and molecular mechanisms of PD are not clearly understood to date. However, misfolded proteins, oxidative stress, and impaired autophagy are believed to play important roles in the pathogenesis of PD. Importantly, α-syn is considered a key player in the development of PD. The present study aimed to assess the role of Ellagic acid (EA), a polyphenol found in many fruits, on α-syn aggregation and toxicity. Using thioflavin and seeding polymerization assays, in addition to electron microscopy, we found that EA could dramatically reduce α-syn aggregation. Moreover, EA significantly mitigated the aggregated α-syn-induced toxicity in SH-SY5Y cells and thus enhanced their viability. Mechanistically, these cytoprotective effects of EA are mediated by the suppression of apoptotic proteins BAX and p53 and a concomitant increase in the anti-apoptotic protein, BCL-2. Interestingly, EA was able to activate autophagy in SH-SY5Y cells, as evidenced by normalized/enhanced expression of LC3-II, p62, and pAKT. Together, our findings suggest that EA may attenuate α-syn toxicity by preventing aggregation and improving viability by restoring autophagy and suppressing apoptosis.     

Autophagy Signaling by Neural-Induced Human Adipose Tissue-Derived Stem Cell-Conditioned Medium during Rotenone-Induced Toxicity in SH-SY5Y Cells

Rotenone (ROT) inhibits mitochondrial complex I, leading to reactive oxygen species formation, which causes neurodegeneration and alpha-synuclein (α-syn) aggregation and, consequently, Parkinson’s disease. We previously found that a neurogenic differentiated human adipose tissue-derived stem cell-conditioned medium (NI-hADSC-CM) was protective against ROT-induced toxicity in SH-SY5Y cells. In the present study, ROT significantly decreased the phospho (p)-mTORC1/total (t)-mTOR, p-mTORC2/t-mTOR, and p-/t-ULK1 ratios and the ATG13 level by increasing the DEPTOR level and p-/t-AMPK ratio. Moreover, ROT increased the p-/t-Akt ratio and glycogen synthase kinase-3β (GSK3β) activity by decreasing the p-/t-ERK1/2 ratios and beclin-1 level. ROT also promoted the lipidation of LC3B-I to LC3B-II by inducing autophagosome formation in Triton X-100-soluble and -insoluble cell lysate fractions. Additionally, the levels of ATG3, 5, 7, and 12 were decreased, along with those of lysosomal LAMP1, LAMP2, and TFEB, leading to lysosomal dysfunction. However, NI-hADSC-CM treatment increased the p-mTORC1, p-mTORC2, p-ULK1, p-Akt, p-ERK1/2, ATG13, and beclin-1 levels and decreased the p-AMPK level and GSK3β activity in response to ROT-induced toxicity. Additionally, NI-hADSC-CM restored the LC3B-I level, increased the p62 level, and normalized the ATG and lysosomal protein amounts to control levels. Autophagy array revealed that the secreted proteins in NI-hADSC-CM could be crucial in the neuroprotection. Taken together, our results showed that the neuroprotective effects of NI-hADSC-CM on the autophagy signaling pathways could alleviate the aggregation of α-syn in Parkinson’s disease and other neurodegenerative disorders.     

Alteration of Dynein Function Affects α-Synuclein Degradation via the Autophagosome-Lysosome Pathway

Growing evidence suggests that dynein dysfunction may be implicated in the pathogenesis of neurodegeneration. It plays a central role in aggresome formation, the delivery of autophagosome to lysosome for fusion and degradation, which is a pro-survival mechanism essential for the bulk degradation of misfolded proteins and damaged organells. Previous studies reported that dynein dysfuntion was associated with aberrant aggregation of α-synuclein, which is a major component of inclusion bodies in Parkinson’s disease (PD). However, it remains unclear what roles dynein plays in α-synuclein degradation. Our study demonstrated a decrease of dynein expression in neurotoxin-induced PD models in vitro and in vivo, accompanied by an increase of α-synuclein protein level. Dynein down-regulation induced by siRNA resulted in a prolonged half-life of α-synuclein and its over-accumulation in A53T overexpressing PC12 cells. Dynein knockdown also prompted the increase of microtubule-associated protein 1 light chain 3 (LC3-II) and sequestosome 1 (SQSTM1, p62) expression, and the accumulation of autophagic vacuoles. Moreover, dynein suppression impaired the autophagosome fusion with lysosome. In summary, our findings indicate that dynein is critical for the clearance of aberrant α-synuclein via autophagosome-lysosome pathway.     

The Potential Role of Electronegative High-Density Lipoprotein H5 Subfraction in RA-Related Atherosclerosis

Although the heterogeneity of high-density lipoprotein-cholesterol (HDL-c) composition is associated with atherosclerotic cardiovascular risk, the link between electronegative subfractions of HDL-c and atherosclerosis in rheumatoid arthritis (RA) remains unknown. We examined the association of the percentage of the most electronegative subfraction of HDL-c (H5%) and RA-related atherosclerosis. Using anion-exchange purification/fast-protein liquid chromatography, we demonstrated significantly higher H5% in patients (median, 7.2%) than HC (2.8%, p < 0.005). Multivariable regression analysis revealed H5% as a significant predictor for subclinical atherosclerosis. We subsequently explored atherogenic role of H5 using cell-based assay. The results showed significantly higher levels of IL-1β and IL-8 mRNA in H5-treated (mean ± SD, 4.45 ± 1.22 folds, 6.02 ± 1.43-folds, respectively) than H1-treated monocytes (0.89 ± 0.18-folds, 1.03 ± 0.26-folds, respectively, both p < 0.001). In macrophages, H5 upregulated the mRNA and protein expression of IL-1β and IL-8 in a dose-dependent manner, and their expression levels were significantly higher than H1-treated macrophages (all p < 0.001). H5 induced more foam cell formation compared with H1-treated macrophages (p < 0.005). In addition, H5 has significantly lower cholesterol efflux capacity than H1 (p < 0.005). The results of nanoLC-MS/MS approach reveal that the best discriminator between high-H5% and normal-H5% is Apo(a), the main constituent of Lp(a). Moreover, Lp(a) level is a significant predictor for high-H5%. These observations suggest that H5 is involved in RA-related atherosclerosis.     

Apigenin Induces Autophagy and Cell Death by Targeting EZH2 under Hypoxia Conditions in Gastric Cancer Cells

Hypoxia is a major obstacle to gastric cancer (GC) therapy and leads to chemoresistance as GC cells are frequently exposed to the hypoxia environment. Apigenin, a flavonoid found in traditional medicine, fruits, and vegetables and an HDAC inhibitor, is a powerful anti-cancer agent against various cancer cell lines. However, detailed mechanisms involved in the treatment of GC using APG are not fully understood. In this study, we investigated the biological activity of and molecular mechanisms involved in APG-mediated treatment of GC under hypoxia. APG promoted autophagic cell death by increasing ATG5, LC3-II, and phosphorylation of AMPK and ULK1 and down-regulating p-mTOR and p62 in GC. Furthermore, our results show that APG induces autophagic cell death via the activation of the PERK signaling, indicating an endoplasmic reticulum (ER) stress response. The inhibition of ER stress suppressed APG-induced autophagy and conferred prolonged cell survival, indicating autophagic cell death. We further show that APG induces ER stress- and autophagy-related cell death through the inhibition of HIF-1α and Ezh2 under normoxia and hypoxia. Taken together, our findings indicate that APG activates autophagic cell death by inhibiting HIF-1α and Ezh2 under hypoxia conditions in GC cells.     

Suppression of Ribose-5-Phosphate Isomerase a Induces ROS to Activate Autophagy,Apoptosis, and Cellular Senescence in Lung Cancer

Ribose-5-phosphate isomerase A (RPIA) regulates tumorigenesis in liver and colorectal cancer. However, the role of RPIA in lung cancer remains obscure. Here we report that the suppression of RPIA diminishes cellular proliferation and activates autophagy, apoptosis, and cellular senescence in lung cancer cells. First, we detected that RPIA protein was increased in the human lung cancer versus adjust normal tissue via tissue array. Next, the knockdown of RPIA in lung cancer cells displayed autophagic vacuoles, enhanced acridine orange staining, GFP-LC3 punctae, accumulated autophagosomes, and showed elevated levels of LC3-II and reduced levels of p62, together suggesting that the suppression of RPIA stimulates autophagy in lung cancer cells. In addition, decreased RPIA expression induced apoptosis by increasing levels of Bax, cleaved PARP and caspase-3 and apoptotic cells. Moreover, RPIA knockdown triggered cellular senescence and increased p53 and p21 levels in lung cancer cells. Importantly, RPIA knockdown elevated reactive oxygen species (ROS) levels. Treatment of ROS scavenger N-acetyl-L-cysteine (NAC) reverts the activation of autophagy, apoptosis and cellular senescence by RPIA knockdown in lung cancer cells. In conclusion, RPIA knockdown induces ROS levels to activate autophagy, apoptosis, and cellular senescence in lung cancer cells. Our study sheds new light on RPIA suppression in lung cancer therapy.     

Defective Autophagosome Formation in p53-Null Colorectal Cancer Reinforces Crocin-Induced Apoptosis

Crocin, a bioactive molecule of saffron, inhibited proliferation of both HCT116 wild-type and HCT116 p53−/− cell lines at a concentration of 10 mM. Flow cytometric analysis of cell cycle distribution revealed that there was an accumulation of HCT116 wild-type cells in G₁ (55.9%, 56.1%) compared to the control (30.4%) after 24 and 48 h of crocin treatment, respectively. However, crocin induced only mild G2 arrest in HCT116 p53−/− after 24 h. Crocin induced inefficient autophagy in HCT116 p53−/− cells, where crocin induced the formation of LC3-II, which was combined with a decrease in the protein levels of Beclin 1 and Atg7 and no clear p62 degradation. Autophagosome formation was not detected in HCT116 p53−/− after crocin treatment predicting a nonfunctional autophagosome formation. There was a significant increase of p62 after treating the cells with Bafilomycin A1 (Baf) and crocin compared to crocin exposure alone. Annexin V staining showed that Baf-pretreatment enhanced the induction of apoptosis in HCT116 wild-type cells. Baf-exposed HCT116 p53−/− cells did not, however, show any enhancement of apoptosis induction despite an increase in the DNA damage-sensor accumulation, γH2AX indicating that crocin induced an autophagy-independent classical programmed cell death.     

COX-2/sEH Dual Inhibitor Alleviates Hepatocyte Senescence in NAFLD Mice by Restoring Autophagy through Sirt1/PI3K/AKT/mTOR

We previously found that the disorder of soluble epoxide hydrolase (sEH)/cyclooxygenase-2 (COX-2)-mediated arachidonic acid (ARA) metabolism contributes to the pathogenesis of the non-alcoholic fatty liver disease (NAFLD) in mice. However, the exact mechanism has not been elucidated. Accumulating evidence points to the essential role of cellular senescence in NAFLD. Herein, we investigated whether restoring the balance of sEH/COX-2-mediated ARA metabolism attenuated NAFLD via hepatocyte senescence. A promised dual inhibitor of sEH and COX-2, PTUPB, was used in our study to restore the balance of sEH/COX-2-mediated ARA metabolism. In vivo, NAFLD was induced by a high-fat diet (HFD) using C57BL/6J mice. In vitro, mouse hepatocytes (AML12) and mouse hepatic astrocytes (JS1) were used to investigate the effects of PTUPB on palmitic acid (PA)-induced hepatocyte senescence and its mechanism. PTUPB alleviated liver injury, decreased collagen and lipid accumulation, restored glucose tolerance, and reduced hepatic triglyceride levels in HFD-induced NAFLD mice. Importantly, PTUPB significantly reduced the expression of liver senescence-related molecules p16, p53, and p21 in HFD mice. In vitro, the protein levels of γH2AX, p53, p21, COX-2, and sEH were increased in AML12 hepatocytes treated with PA, while Ki67 and PCNA were significantly decreased. PTUPB decreased the lipid content, the number of β-gal positive cells, and the expression of p53, p21, and γH2AX proteins in AML12 cells. Meanwhile, PTUPB reduced the activation of hepatic astrocytes JS1 by slowing the senescence of AML12 cells in a co-culture system. It was further observed that PTUPB enhanced the ratio of autophagy-related protein LC3II/I in AML12 cells, up-regulated the expression of Fundc1 protein, reduced p62 protein, and suppressed hepatocyte senescence. In addition, PTUPB enhanced hepatocyte autophagy by inhibiting the PI3K/AKT/mTOR pathway through Sirt1, contributing to the suppression of senescence. PTUPB inhibits the PI3K/AKT/mTOR pathway through Sirt1, improves autophagy, slows down the senescence of hepatocytes, and alleviates NAFLD.     

Activation of Autophagy by Low-Dose Silica Nanoparticles Enhances Testosterone Secretion in Leydig Cells

Silica nanoparticles (SNPs) can cause abnormal spermatogenesis in male reproductive toxicity. However, the toxicity and toxicological mechanisms of SNPs in testosterone synthesis and secretion in Leydig cells are not well known. Therefore, this study aimed to determine the effect and molecular mechanism of low doses of SNPs in testosterone production in Leydig cells. For this, mouse primary Leydig cells (PLCs) were exposed to 100 nm Stöber nonporous spherical SNPs. We observed significant accumulation of SNPs in the cytoplasm of PLCs via transmission electron microscopy (TEM). CCK-8 and flow cytometry assays confirmed that low doses (50 and 100 μg/mL) of SNPs had no significant effect on cell viability and apoptosis, whereas high doses (more than 200 μg/mL) decreased cell viability and increased cell apoptosis in PLCs. Monodansylcadaverine (MDC) staining showed that SNPs caused the significant accumulation of autophagosomes in the cytoplasm of PLCs. SNPs activated autophagy by upregulating microtubule-associated protein light chain 3 (LC3-II) and BCL-2-interacting protein (BECLIN-1) levels, in addition to downregulating sequestosome 1 (SQSTM1/P62) level at low doses. In addition, low doses of SNPs enhanced testosterone secretion and increased steroidogenic acute regulatory protein (StAR) expression. SNPs combined with rapamycin (RAP), an autophagy activator, enhanced testosterone production and increased StAR expression, whereas SNPs combined with 3-methyladenine (3-MA) and chloroquine (CQ), autophagy inhibitors, had an opposite effect. Furthermore, BECLIN-1 depletion inhibited testosterone production and StAR expression. Altogether, our results demonstrate that low doses of SNPs enhanced testosterone secretion via the activation of autophagy in PLCs.     

In Vitro SARS-CoV-2 Infection of Microvascular Endothelial Cells: Effect on Pro-Inflammatory Cytokine and Chemokine Release

In the novel pandemic of Coronavirus Disease 2019, high levels of pro-inflammatory cytokines lead to endothelial activation and dysfunction, promoting a pro-coagulative state, thrombotic events, and microvasculature injuries. The aim of the present work was to investigate the effect of SARS-CoV-2 on pro-inflammatory cytokines, tissue factor, and chemokine release, with Human Microvascular Endothelial Cells (HMEC-1). ACE2 receptor expression was evaluated by western blot analysis. SARS-CoV-2 infection was assessed by one-step RT-PCR until 7 days post-infection (p.i.), and by Transmission Electron Microscopy (TEM). IL-6, TNF-α, IL-8, IFN-α, and hTF mRNA expression levels were detected by RT-PCR, while cytokine release was evaluated by ELISA. HMEC-1 expressed ACE2 receptor and SARS-CoV-2 infection showed a constant viral load. TEM analysis showed virions localized in the cytoplasm. Expression of IL-6 at 24 h and IFN-α mRNA at 24 h and 48 h p.i. was higher in infected than uninfected HMEC-1 (p < 0.05). IL-6 levels were significantly higher in supernatants from infected HMEC-1 (p < 0.001) at 24 h, 48 h, and 72 h p.i., while IL-8 levels were significantly lower at 24 h p.i. (p < 0.001). These data indicate that in vitro microvascular endothelial cells are susceptible to SARS-CoV-2 infection but slightly contribute to viral amplification. However, SARS-CoV-2 infection might trigger the increase of pro-inflammatory mediators.     

Colorectal Cancer Apoptosis Induced by Dietary δ-Valerobetaine Involves PINK1/Parkin Dependent-Mitophagy and SIRT3

Understanding the mechanisms of colorectal cancer progression is crucial in the setting of strategies for its prevention. δ-Valerobetaine (δVB) is an emerging dietary metabolite showing cytotoxic activity in colon cancer cells via autophagy and apoptosis. Here, we aimed to deepen current knowledge on the mechanism of δVB-induced colon cancer cell death by investigating the apoptotic cascade in colorectal adenocarcinoma SW480 and SW620 cells and evaluating the molecular players of mitochondrial dysfunction. Results indicated that δVB reduced cell viability in a time-dependent manner, reaching IC50 after 72 h of incubation with δVB 1.5 mM, and caused a G2/M cell cycle arrest with upregulation of cyclin A and cyclin B protein levels. The increased apoptotic cell rate occurred via caspase-3 activation with a concomitant loss in mitochondrial membrane potential and SIRT3 downregulation. Functional studies indicated that δVB activated mitochondrial apoptosis through PINK1/Parkin pathways, as upregulation of PINK1, Parkin, and LC3B protein levels was observed (p < 0.0001). Together, these findings support a critical role of PINK1/Parkin-mediated mitophagy in mitochondrial dysfunction and apoptosis induced by δVB in SW480 and SW620 colon cancer cells.     

Autophagy Activation Protects Ocular Surface from Inflammation in a Dry Eye Model In Vitro

Inflammation is the main pathophysiology of dry eye, characterized by tear film instability and hyperosmolarity. The aim of this study was to investigate the association of inflammation and cellular autophagy using an in vitro dry eye model with primary cultured human corneal epithelial cells (HCECs). Primary HCECs cultured with fresh limbal explants from donors were switched to a hyperosmotic medium (450 mOsM) by adding sodium chloride into the culture medium. We observed the stimulated inflammatory mediators, TNF-α, IL-1β, IL-6 and IL-8, as well as the increased expression of autophagy related genes, Ulk1, Beclin1, Atg5 and LC3B, as evaluated by RT-qPCR and ELISA. The immunofluorescent staining of LC3B and Western blotting revealed the activated autophagosome formation and autophagic flux, as evidenced by the increased LC3B autophagic cells with activated Beclin1, Atg5, Atg7 and LC3B proteins, and the decreased levels of P62 protein in HCECs. Interestingly, the autophagy activation was later at 24 h than inflammation induced at 4 h in HCECs exposed to 450 mOsM. Furthermore, application of rapamycin enhanced autophagy activation also reduced the inflammatory mediators and restored cell viability in HCECs exposed to the hyperosmotic medium. Our findings for the first time demonstrate that the autophagy activation is a late phase response to hyperosmotic stress, and is enhanced by rapamycin, which protects HCECs by suppressing inflammation and promoting cells survival, suggesting a new therapeutic potential to treat dry eye diseases.     

Evaluating Whether Radiofrequency Irradiation Attenuated UV-B-Induced Skin Pigmentation by Increasing Melanosomal Autophagy and Decreasing Melanin Synthesis

Autophagy is involved in the degradation of melanosomes and the determination of skin color. TLR4 and tumor necrosis factor (TNF) signaling upregulates NF-kB expression, which is involved in the upregulation of mTOR. The activation of mTOR by UV-B exposure results in decreased autophagy, whereas radiofrequency (RF) irradiation decreases TLR4 and TNF receptor (TNFR) expression. We evaluated whether RF decreased skin pigmentation by restoring autophagy by decreasing the expression of TLR4 or TNFR/NF-κB/mTOR in the UV-B-irradiated animal model. UV-B radiation induced the expressions of TNFR, TLR, and NF-κB in the skin, which were all decreased by RF irradiation. RF irradiation also decreased phosphorylated mTOR expression and upregulated autophagy initiation factors such as FIP200, ULK1, ULK2, ATG13, and ATG101 in the UV-B-irradiated skin. Beclin 1 expression and the expression ratio of LC3-I to LC3-II were increased by UV-B/RF irradiation. Furthermore, melanin-containing autophagosomes increased with RF irradiation. Fontana-Masson staining showed that the amount of melanin deposition in the skin was decreased by RF irradiation. This study showed that RF irradiation decreased skin pigmentation by restoring melanosomal autophagy, and that the possible signal pathways which modulate autophagy could be TLR4, TNFR, NF-κB, and mTOR.     

Cathepsin B Regulates Mice Granulosa Cells’ Apoptosis and Proliferation In Vitro

Cathepsin B (CTSB), a lysosomal cysteine protease’s high expression and activity, has been reported to cause poor-quality embryos in porcine and bovine. Nevertheless, CTSB functions in mice granulosa cells remain to explore. To discuss the CTSB functional role in follicular dynamics, we studied apoptosis, proliferation, cell cycle progression, and related signaling pathways in primary mouse granulosa cells transfected with small interference RNA specific to CTSB (siCTSB) for 48 h. Further, mRNA and protein expression of cell proliferation regulators (Myc and cyclin D2), apoptosis regulators (caspase 3, caspase 8, TNF-α, and Bcl2), steroidogenesis-related genes (FSHR and CYP11A1), and autophagy markers (LC3-I and ATG5) were investigated. In addition, the effect of CTSB on steroidogenesis and autophagy was also examined. Flow cytometry analysis assay displayed that silencing of CTSB decreased the early and total apoptosis rate by downregulating TNF-α, caspase 8, and caspase 3, and upregulating Bcl2. By regulating Myc and cyclin D2 expression and activating the p-Akt and p-ERK pathways, CTSB knockdown increased GC proliferation and number. A significant decline in estradiol and progesterone concentrations was observed parallel to a significant decrease in autophagy-related markers LC3-I and ATG5 compared to the control group. Herein, we demonstrated that CTSB serves as a proapoptotic agent and plays a critical role in folliculogenesis in female mice by mediating apoptosis, autophagy, proliferation, and steroidogenesis. Hence, CTSB could be a potential prognostic agent for female infertility.     

Decursinol Angelate Arrest Melanoma Cell Proliferation by Initiating Cell Death and Tumor Shrinkage via Induction of Apoptosis

Melanoma is known to aggressively metastasize and is one of the prominent causes of skin cancer mortality. This study was designed to assess the molecular mechanism of decursinol angelate (DA) against murine melanoma cell line (B16F10 cells). Treatment of DA resulted in growth inhibition and cell cycle arrest at G0/G1 (p < 0.001) phase, evaluated through immunoblotting. Moreover, autophagy-related proteins such as ATG-5 (p < 0.0001), ATG-7 (p < 0.0001), beclin-1 (p < 0.0001) and transition of LC3-I to LC3-II (p < 0.0001) were markedly decreased, indicating autophagosome inhibition. Additionally, DA treatment triggered apoptotic events which were corroborated by the occurrence of distorted nuclei, elevated reactive oxygen species (ROS) levels and reduction in the mitochondrial membrane potential. Subsequently, there was an increase in the expression of pro-apoptotic protein Bax in a dose-dependent manner, with the corresponding downregulation of Bcl-2 expression and cytochrome C expression following 24 h DA treatment in A375.SM and B16F10 cells. We substantiated our results for apoptotic occurrence through flow cytometry in B16F10 cells. Furthermore, we treated B16F10 cells with N-acetyl-L-cysteine (NAC). NAC treatment upregulated ATG-5 (p < 0.0001), beclin-1 (p < 0.0001) and LC3-I to LC3-II (p < 0.0001) conversion, which was inhibited in the DA treatment group. We also noticed a systematic upregulation of important markers for progression of G1 cell phase such as CDK-2 (p < 0.029), CDK-4 (p < 0.036), cyclin D1 (p < 0.0003) and cyclin E (p < 0.020) upon NAC treatment. In addition, we also observed a significant fold reduction (p < 0.05) in ROS fluorescent intensity and the expression of Bax (p < 0.0001), cytochrome C (p < 0.0001), cleaved caspase-9 (p > 0.010) and cleaved caspase-3 (p < 0.0001). NAC treatment was able to ameliorate DA-induced apoptosis and cell cycle arrest to support our finding. Our in vivo xenograft model also revealed similar findings, such as downregulation of CDK-2 (p < 0.0001) and CDK-4 (p < 0.0142) and upregulation of Bax (p < 0.0001), cytochrome C (p < 0.0001), cleaved caspase 3 (p < 0.0001) and cleaved caspase 9 (p < 0.0001). In summary, our study revealed that DA is an effective treatment against B16F10 melanoma cells and xenograft mice model.     

Empagliflozin Attenuates Non-Alcoholic Fatty Liver Disease (NAFLD) in High Fat Diet Fed ApoE(-/-) Mice by Activating Autophagy and Reducing ER Stress and Apoptosis

Aims/hypothesis: SGLT-2 inhibitors (SGLT-2i) have been studied as potential treatments against NAFLD, showing varying beneficial effects. The molecular mechanisms mediating these effects have not been fully clarified. Herein, we investigated the impact of empagliflozin on NAFLD, focusing particularly on ER stress, autophagy and apoptosis. Methods: Five-week old ApoE^(-/-) mice were switched from normal to a high-fat diet (HFD). After five weeks, mice were randomly allocated into a control group (HFD + vehicle) and Empa group (HFD + empagliflozin 10 mg/kg/day) for five weeks. At the end of treatment, histomorphometric analysis was performed in liver, mRNA levels of Fasn, Screbp-1, Scd-1, Ppar-γ, Pck-1, Mcp-1, Tnf-α, Il-6, F4/80, Atf4, Elf2α, Chop, Grp78, Grp94, Χbp1, Ire1α, Atf6, mTor, Lc3b, Beclin-1, P62, Bcl-2 and Bax were measured by qRT-PCR, and protein levels of p-EIF2α, EIF2a, CHOP, LC3II, P62, BECLIN-1 and cleaved CASPASE-8 were assessed by immunoblotting. Results: Empagliflozin-treated mice exhibited reduced fasting glucose, total cholesterol and triglyceride serum levels, as well as decreased NAFLD activity score, decreased expression of lipogenic enzymes (Fasn, Screbp-1c and Pck-1) and inflammatory molecules (Mcp-1 and F4/80), compared to the Control group. Empagliflozin significantly decreased the expression of ER stress molecules Grp78, Ire1α, Xbp1, Elf2α, Atf4, Atf6, Chop, P62(Sqstm1) and Grp94; whilst activating autophagy via increased AMPK phosphorylation, decreased mTOR and increased LC3B expression. Finally, empagliflozin increased the Bcl2/Bax ratio and inhibited CASPASE-8 cleavage, reducing liver cell apoptosis. Immunoblotting analysis confirmed the qPCR results. Conclusion: These novel findings indicate that empagliflozin treatment for five weeks attenuates NAFLD progression in ApoE^(-/-) mice by promoting autophagy, reducing ER stress and inhibiting hepatic apoptosis.     

Autophagy in DNA Damage Response

DNA damage response (DDR) involves DNA repair, cell cycle regulation and apoptosis, but autophagy is also suggested to play a role in DDR. Autophagy can be activated in response to DNA-damaging agents, but the exact mechanism underlying this activation is not fully understood, although it is suggested that it involves the inhibition of mammalian target of rapamycin complex 1 (mTORC1). mTORC1 represses autophagy via phosphorylation of the ULK1/2–Atg13–FIP200 complex thus preventing maturation of pre-autophagosomal structures. When DNA damage occurs, it is recognized by some proteins or their complexes, such as poly(ADP)ribose polymerase 1 (PARP-1), Mre11–Rad50–Nbs1 (MRN) complex or FOXO3, which activate repressors of mTORC1. SQSTM1/p62 is one of the proteins whose levels are regulated via autophagic degradation. Inhibition of autophagy by knockout of FIP200 results in upregulation of SQSTM1/p62, enhanced DNA damage and less efficient damage repair. Mitophagy, one form of autophagy involved in the selective degradation of mitochondria, may also play role in DDR. It degrades abnormal mitochondria and can either repress or activate apoptosis, but the exact mechanism remains unknown. There is a need to clarify the role of autophagy in DDR, as this process may possess several important biomedical applications, involving also cancer therapy.