Expression Of Autophagy Proteins Research Articles

Correlation between inflammatory factors, autophagy protein levels, and infection in granulation tissue of diabetic foot ulcer.

To observe the expression of inflammatory factors and autophagy-related proteins in granulation tissue of diabetic foot ulcer (DFU) patients and analyze their relationship with infection. This is a retrospective cohort study. One hundred and fifty-two patients with DFU in our hospital from July 2020 to March 2022 were selected as the DFU group, including 98 cases in infection stage group and 54 cases in infection control group. The patients were further graded as the mild (51 cases), the moderate (65 cases), and the severe infection group (36 cases) according to the Wagner grading criteria. Sixty-seven patients with foot burns during the same period were selected as the control group. The distribution of pathogenic bacteria on the ulcer surface was examined using fully automated bacterial analyzer. The expression of inflammatory factors (procalcitonin [PCT], tumor necrosis factor-α [TNF-α], and interleukin-6 [IL-6]) was valued by real-time fluorescence quantitative PCR (qRT-PCR). Protein expression was measured by immunohistochemistry (IHC). The correlation was analyzed by Pearson. The surface infection of DFU patients was mostly induced by gram-negative and gram-positive bacteria, with Pseudomonas aeruginosa predominating among the Gram-negative bacteria and Staphylococcus aureus among the gram-positive bacteria. The infection stage group had higher content of PCT, TNF-α, and IL-6 and lower content of Beclin-1 and LC3 than the infection control group (p < .001). The levels of PCT, TNF-α, and IL-6 in the DFU patients with cardiovascular events were higher than those in the nonoccurrence group (p < .001). Glycated hemoglobin in patients with DFU was positively correlated with PCT, TNF-α, and IL-6 levels (p < .05), and negatively correlated with Beclin-1 and LC3 levels (p < .001). P. aeruginosa and S. aureus were predominant bacterial in DFU infections. Inflammatory factor and autophagy protein expression were closely correlated with the degree of infection.

LAMP1 controls CXCL10-CXCR3 axis mediated inflammatory regulation of macrophage polarization during inflammatory stimulation

Increased expression of CXCL10 and its receptor CXCR3 represents an inflammatory response in cells and tissues. Macrophage polarization and autophagy are major functions in inflammatory macrophages; however, the cellular functions of the CXCL10-CXCR3 axis in macrophages are not well understood. Here, we examined the role of CXCL10-CXCR3-axis-regulated autophagy in macrophage polarization. First, in non-inflammatory macrophages, whereas CXCL10 promotes M2 polarization and inhibits M1 polarization, CXCR3 antagonist AMG487 induces the opposite macrophage polarization. Next, CXCL10 promotes the expression of autophagy proteins (Atg5-Atg12 complex, p62, LC3-II, and LAMP1) and AMG487 inhibits their expression. Knockdown of LAMP1 by short interfering RNA switches the CXCL10-induced polarization from M2 to M1 in non-inflammatory macrophages. Furthermore, in inflammatory macrophages stimulated by poly(I:C), CXCL10 induces M1 polarization and AMG487 induces M2 polarization in association with a decrease in LAMP1. Finally, AMG487 alleviates lung injury after poly(I:C) treatment in mice. In conclusion, CXCL10-CXCR3 axis differentially directs macrophage polarization in inflammatory and non-inflammatory states, and autophagy protein LAMP1 acts as the switch controlling the direction of macrophage polarization by CXCL10-CXCR3.

Abstract 4328: Erlotinib suppresses autophagy in colorectal cancer

Abstract Introduction: Oncogenic KRAS mutation is prevalent in approximately 45% of colorectal cancer (CRC) patients. Limited targeted therapies are available to inhibit the detrimental effects of KRAS mutation primarily pertaining to its unique structural intricacies. Erlotinib is an EGFR inhibitor which inducing autophagy in lung cancer cells. In this study, we followed the effects of Erlotinib treatment in mutant and wild-type colorectal cancer (CRC) cells. We report that Erlotinib exhibits selectivity between mutated KRAS and wild type, significantly modulating the expression of autophagy proteins ATG5, ULK1, Beclin1, and LC3A. Methodology: The 2 cell lines used in this study were HCT116 (KRAS mutant) and Hke3 (KRAS wild type). These cells were treated with Erlotinib for 24 hours and 48 hours, A cell viability assay was performed and subsequently followed by protein extraction. The expression of autophagy proteins ATG5, ULK1, Beclin1, and LC3A was determined. The TCGA COAD-READ patient dataset was utilized using GEPIA 2 software. A correlation was found between the autophagy proteins and the KRAS gene expression. Results: The cell viability assays showed that there was significantly more cell death in KRAS wild-type cells compared with KRAS mutant at time points ranging from 24 to 48 hours (p = 0.036). The experiment also showed that treatment of Erlotinib with concentrations of 20 uM, 35 uM, and 50 uM and significant down regulatory fold changes (p &amp;lt; 0.05). Both KRAS mutated and wild-type cells treated with Erlotinib expressed significant downregulation of ATG5, ULK1, Beclin1, and LC3A proteins at 48 hours (p &amp;lt; 0.05). The 20 uM treatment demonstrated a significance of p &amp;lt; 0.05 for all proteins. The 20 uM treatment resulted in fold changes of the KRAS mutated cells for ATG5, ULK1, Beclin1, and LC3A to be 1.42, 1.42, 1.81, and 2.09 respectively. A strong KRAS correlation was found with ATG5, ULK1, Beclin1, and LC3A using COAD &amp; READ tumor and COAD &amp; READ normal (R &amp;gt; 0.75, p &amp;lt; 0.05). Conclusion: This study highlights the effects of Erlotinib as a treatment for KRAS mutated CRC. Our results confirmed that Erlotinib had the opposite effect on CRC cells in relation to the expression of the autophagy proteins when compared to lung cancer cells. There were distinct effects of Erlotinib between KRAS wild-type and mutant cells in both cell viability and the expression of autophagy-related proteins. In both KRAS cell types, there was significant downregulation of autophagy proteins, however, the KRAS wild type had significantly more downregulation than the KRAS mutant. This data suggests that there is a relationship between KRAS mutation and the expression of the autophagy proteins. Our analysis of the patient datasets confirmed the strong correlation between KRAS mutation and the expression of autophagy proteins. These findings emphasize the need to explore further how Erlotinib can be augmented into the current treatment regimen as a benefit to CRC patients. Citation Format: Alexander Siegman, Aaron Shaykevich, Isaac Silverman, Sanjay Goel, Radhashree Maitra. Erlotinib suppresses autophagy in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4328.

Neuroprotective effects of cordycepin on MPTP-induced Parkinson's disease mice via suppressing PI3K/AKT/mTOR and MAPK-mediated neuroinflammation

Parkinson's disease (PD) is a prevalent progressive and multifactorial neurodegenerative disorder. Cordycepin is known to exhibit antitumor, anti-inflammatory, antioxidative stress, and neuroprotective effects; however, few studies have explored the neuroprotective mechanism of cordycepin in PD. Using a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model, we investigated the impact of cordycepin on PD and its underlying molecular mechanisms. The findings indicated that cordycepin significantly mitigated MPTP-induced behavior disorder and neuroapoptosis, diminished the loss of dopaminergic neurons in the striatum–substantia nigra pathway, elevated striatal monoamine levels and its metabolites, and inhibited the polarization of microglia and the expression of pro-inflammatory factors. Subsequent proteomic and phosphoproteomic analyses revealed the involvement of the MAPK, mTOR, and PI3K/AKT signaling pathways in the protective mechanism of cordycepin. Cordycepin treatment inhibited the activation of the PI3K/AKT/mTOR signaling pathway and enhanced the expression of autophagy proteins in the striatum and substantia nigra. We also demonstrated the in vivo inhibition of the ERK/JNK signaling pathway by cordycepin treatment. In summary, our investigation reveals that cordycepin exerts neuroprotective effects against PD by promoting autophagy and suppressing neuroinflammation and neuronal apoptosis by inhibiting the PI3K/AKT/mTOR and ERK/JNK signaling pathways. This finding highlights the favorable characteristics of cordycepin in neuroprotection and provides novel molecular insights into the neuroprotective role of natural products in PD.

MYC-dependent MiR-7-5p regulated apoptosis and autophagy in diffuse large B cell lymphoma by targeting AMBRA1.

Diffuse large B-cell lymphoma (DLBCL) is the leading cause of mortality from invasive hematological malignancies worldwide. MicroRNA-7-5p (miR-7-5p) has been shown to be a tumor suppressor in several types of tumors. However, its role in DLBCL is not fully understood. This study explored the role of miR-7-5p in the progression of DLBCL and pursued the underlying mechanism. Quantitative real-time PCR and transfection of miRNA mimic and inhibitors were used to assess the effects of miR-7-5p on autophagy and apoptosis in SU-DHL-4 and SU-DHL-10 cells. Dual-luciferase reporter assay was used to identify target genes of miR-7-5p. Immunofluorescence, flow cytometry, and western blotting (WB) were performed to explore the underlying mechanism and downstream pathways of miR-7-5p and AMBRA1 in DLBCL cells. MiR-7-5p was upregulated in DLBCL cells. Luciferase reporter assays implicated AMBRA1 as a downstream target of miR-7-5p in DLBCL. WB and flow cytometry showed that an increase in miR-7-5p level and a decrease in AMBRA1 expression led to a decrease in autophagy and apoptosis-related protein expression. Furthermore, miR-7-5p prevented c-MYC dephosphorylation through AMBRA1 downregulation. On the contrary, c-MYC increased the expression of miR-7-5p, thereby establishing positive feedback on miR-7-5p transcription. The addition of hydroxychloroquine, an autophagy inhibitor, reduced autophagy and increased apoptosis in DLBCL cells. In vivo experiments further proved that the increase of miR-7-5p played a regulatory role in the expression of downstream AMBRA1 and c-MYC. These results demonstrate that c-MYC-dependent MiR-7-5p suppressed autophagy and apoptosis by targeting AMBRA1 in DLBCL cells. MiR-7-5p also suppressed autophagy and apoptosis by targeting AMBRA1 in DLBCL cells. Therefore, these data suggest that targeting miR-7-5p may be a promising strategy in DLBCL therapy.

Toll-like receptor 4 mediated autophagy regulates airway smooth muscle cells behavior

Objectives Airway remodeling, a prominent feature of asthma, involves aberrant proliferation, apoptosis, and migration of airway smooth muscle cells (ASMCs). Toll-like receptors (TLRs) are implicated in the regulation of the autophagy pathway. In this study, we aimed to investigate the influence of Toll-like receptor 4 (TLR4) on autophagy and its underlying mechanism in ASMC proliferation, apoptosis, and migration. Methods Histopathological changes in the lungs of asthmatic mice assessed by Hematoxylin-Eosin (HE) and Masson staining. Cell proliferation, apoptosis and migration were evaluated utilizing CCK8, Edu, Flow cytometry and wound heading assays. The effectiveness of siRNA transfection and the expression of TLR4, autophagy, and proliferation-related proteins after siRNA treatment were examined through RT-PCR and Western blot (WB). Conclusion We observed an increase in TLR4 expression and autophagy in a mouse model of OVA-induced asthma. In vitro experiments showed that siRNA-mediated inhibition of TLR4 suppressed autophagy, proliferation, and migration of ASMCs, whereas TLR4 activation by lipopolysaccharide (LPS) had the opposite effect. Furthermore, the autophagy inhibitor 3-Methyladenine (3MA) inhibited ASMCs proliferation and migration while promoting apoptosis. Significantly, our study demonstrated that autophagy inhibition reversed the promotion effect of LPS on ASMC proliferation and migration. These findings suggest that TLR4 may modulate ASMC behavior through the regulation of autophagy.

RING1 Inhibition Has a Cell-Specific Antitumoral Role by Promoting Autophagy in Endometrial Cancer Cells.

Factors influencing gene expression through chemical modifications of histones may play an important role in the regulation of the autophagy process in cancers. RING1A or RING1B are responsible for the catalytical activity of Polycomb repressive complex 1 (PRC1) which monoubiquitylate histone H2A. The aim of the study was to determine the effect of the RING1A/B protein inhibition on the autophagy process in endometrial cancer cells and the anticancer effectiveness of RING1 inhibitor PRT4165 in combination with autophagy inhibitors. The expression of autophagy genes and proteins were analyzed in endometrial cancer cells HEC-1A and Ishikawa grown in different glucose concentrations and treated with PRT4165. To assess the effectiveness of PRT4165 used alone or in combination with HCQ or Lys05, IC50 and the combination index (CI) were calculated. Flow cytometry method was used to estimate apoptotic cells after treatment. The results confirm the impact of RINGs on autophagy and apoptosis in endometrial cancer cells. PRT4165 inhibitor causes changes in the expression of ATG genes and autophagy markers and the effect depends on glucose concentration and cell types. However, the anticancer effectiveness of PRT4165 was lower when it was used in combination with autophagy inhibitors, suggesting that such a combination is not a promising anticancer strategy. The results indicate the importance of the RINGs in the process of autophagy and apoptosis. Further potentially more effective combinations of PRT4165 with autophagy modulators should be sought.

Alpha-mangostin decreases high glucose-induced damage on human umbilical vein endothelial cells by increasing autophagic protein expression.

Diabetes is a chronic disorder that occurs as a result of impaired glucose metabolism. In hyperglycaemic states, the balance between oxidative stress and antioxidant enzymes is disrupted leading to oxidative damage and cell death. In addition, impaired autophagy leads to the storage of dysfunctional proteins and cellular organelles in the cell. Hence, the cytoprotective function of autophagy may be disrupted by high glucose conditions. Alpha-mangostin (A-MG) is an essential xanthone purified from the mangosteen fruit. The different pharmacological benefits of alpha-mangostin, including antioxidant, anti-obesity, and antidiabetic, were demonstrated. We evaluated the protective influence of A-MG on autophagic response impaired by high concentrations of glucose in human umbilical vein endothelial cells (HUVECs). The HUVECs were treated with various glucose concentrations (5-60 mM) and A-MG (1.25-10 μM) for three days. Then, HUVECs were treated with 60 mM of glucose+2.5 μM of A-MG to measure viability, ROS, and NO content. Finally, the levels of autophagic proteins including LC3, SIRT1, and beclin 1 were evaluated by western blot. The results expressed that high glucose condition (60 mM) decreased viability and increased ROS and NO content in HUVECs. In addition, LC3, SIRT1, and beclin 1 protein levels declined when HUVECs were exposed to the high concentration of glucose. A-MG reversed these detrimental effects and elevated autophagic protein levels. Our data represent that A-MG protects HUVECs against high glucose conditions by decreasing ROS and NO generation as well as increasing the expression of autophagy proteins.

Perilipin2 inhibits the replication of hepatitis B virus deoxyribonucleic acid by regulating autophagy under high-fat conditions.

Chronic hepatitis B virus (HBV) infection is often associated with increased lipid deposition in hepatocytes. However, when combined with non-alcoholic fatty liver disease or hyperlipidemia, it tends to have a lower HBV deoxyribonucleic acid (DNA) load. The relationship between lipid metabolism and HBV DNA replication and its underlying mechanisms are not well understood. To investigate the relationship between lipid metabolism and HBV DNA replication and its underlying mechanisms. 1603 HBsAg-seropositive patients were included in the study. We first explored the relationship between patients' lipid levels, hepatic steatosis, and HBV DNA load. Also, we constructed an HBV infection combined with a hepatic steatosis cell model in vitro by fatty acid stimulation of HepG2.2.15 cells to validate the effect of lipid metabolism on HBV DNA replication in vitro. By knocking down and overexpressing Plin2, we observed whether Plin2 regulates autophagy and HBV replication. By inhibiting both Plin2 and cellular autophagy under high lipid stimulation, we examined whether the Plin2-autophagy pathway regulates HBV replication. The results revealed that serum triglyceride levels, high-density lipoprotein levels, and hepatic steatosis ratio were significantly lower in the HBV-DNA high load group. Logistic regression analysis indicated that hepatic steatosis and serum triglyceride levels were negatively correlated with HBV-DNA load. Stratified analysis by HBeAg showed significant negative correlations between HBV-DNA load and hepatic steatosis ratio in both HBeAg-positive and HBeAg-negative groups. An in vitro cell model was developed by stimulating HepG2.2.15 cells with palmitic acid and oleic acid to study the relationship between HBV-DNA load and lipid metabolism. The results of the in vitro experiments suggested that fatty acid treatment increased lipid droplet deposition and decreased the expression of cell supernatant HBsAg, HBeAg, and HBV DNA load. Western blot and polymerase chain reaction analysis showed that fatty acid stimulation significantly induced Plin2 protein expression and inhibited the expression of hepatocyte autophagy proteins. Inhibition of Plin2 protein expression under fatty acid stimulation reversed the reduction in HBsAg and HBeAg expression and HBV DNA load induced by fatty acid stimulation and the inhibition of cellular autophagy. Knocking down Plin2 and blocking autophagy with 3-methyladenine (3-MA) inhibited HBV DNA replication. In conclusion, lipid metabolism is a significant factor affecting HBV load in patients with HBV infection. The in vitro experiments established that fatty acid stimulation inhibits HBV replication via the Plin2-autophagy pathway.

Rhapontigenin Improves Non-alcoholic Fatty Liver Disease by Inducing Lipophagy Through the ACOX1 and mTOR/ULK1 Pathways

Background Non-alcoholic fatty liver disease (NAFLD) is severely affecting the quality of people’s life. Rhapontigenin (RA) is a stilbene compound isolated from Rhubarb L., which has been reported to have an effect on cholesterol diet-induced hyperlipidemia in rats. This study aims to explore the pharmacodynamics and the mechanism of RA obtained from Rheum franzenbachii Munt. against NAFLD. RA was extracted from the roots of the Rheum L. (Polygonaceae) plant Rheum franzenbachii Munt. Materials and Methods RA was extracted by Sephadex-gel column and identified by high-performance liquid chromatography (HPLC)-UV and HR-ESI-MS. The pharmacodynamic indexes of L-02 cells and mice treated with RA were determined by histological staining and ELISA, while the expression of autophagy-related proteins was analyzed by Western blot. Results The results in vivo showed that the liver structure of RA-treatment mice was normal, and the organ coefficient was significantly decreased. It also showed that RA could significantly reduce the expression of reactive oxygen species (ROS) in the liver as well as inhibit oxidative stress and inflammatory response. Interestingly, the autophagy inhibitor 3-methyladenine (3-MA) could reverse the effect of RA on NAFLD, which further confirmed that RA plays an anti-NAFLD role through activating lipophagy. Conclusion It suggested that RA has an effect on NAFLD by down-regulating the expression of Acyl-CoA oxidase 1 (ACOX1), p-mTOR, and p-Unc-51-like kinase 1 (ULK1), then inhibiting Acetyl-CoA (A-CoA) production, and up-regulating the expression of autophagy protein 5 (Atg5) to promote the lipophagy of lipocytes.

Inhibition of Autophagy in Heat-Stressed Sperm of Adult Mice: A Possible Role of Catsper1, 2 Channel Proteins.

Various phenomena guarantee gamete maturation and formation at all stages of evolution, one of which is autophagy playing a critical role in the final morphology of gametes, particularly sperms. Autophagy is influenced by oxidative stress, disturbances of calcium homeostasis, and hyperthermia conditions. The current study aimed to assess the autophagy-related proteins along with the activity of sperm calcium channel (CatSper) proteins following the induction of heat stress (HS). The study sample includes two groups of adult mice: sham and HS groups. In the HS group, the right testis was transferred to the abdominal cavity for 120 hours and then returned to the scrotum where it remained for 7 days. After 7 days, the testis and epididymis were removed to conduct real-time, immunohistochemical studies, sperm parameter evaluation, and seminiferous tubule assessment. In this study, the expression and distribution of autophagy proteins were measured. Plus, CatSper1 and CatSper2 were evaluated as proteins of calcium channels. The results of the present study demonstrated that the expression intensity of autophagy indices in seminiferous tubules decreased significantly after HS induction, which was associated with a decrease in the distribution of CatSper proteins in the sperms. HS led to morphological changes in sperm, reduced motility and viability of sperm, and decreased spermatogenesis indices. In this study, following heat stress, the decrease in CatSper protein distribution may lead to the structural disorder of CatSper channels, which could strongly affect autophagic activity. Also, disruption of spermatogenesis and sperm parameters may be the consequence of decreased autophagy activity.

Msi2 enhances muscle dysfunction in a myotonic dystrophy type 1 mouse model

BackgroundMyotonic dystrophy type 1 (DM1) is a rare neuromuscular disease caused by a CTG repeat expansion in the 3′ untranslated region of the DM1 protein kinase gene. Characteristic degenerative muscle symptoms include myotonia, atrophy, and weakness. We previously proposed an MSI2>miR-7>autophagy axis whereby MSI2 overexpression repressed miR-7 biogenesis that subsequently de-repressed muscle catabolism through excessive autophagy. Because the DM1 HSALR mouse model expressing expanded CUG repeats shows weak muscle-wasting phenotypes, we hypothesized that MSI2 overexpression was sufficient to promote muscle dysfunction in vivo. MethodsBy means of recombinant AAV murine Msi2 was overexpressed in neonates HSALR mice skeletal muscle to induce DM1-like phenotypes. ResultsSustained overexpression of the murine Msi2 protein in HSALR neonates induced autophagic flux and expression of critical autophagy proteins, increased central nuclei and reduced myofibers area, and weakened muscle strength. Importantly, these changes were independent of Mbnl1, Mbnl2, and Celf1 protein levels, which remained unchanged upon Msi2 overexpression. ConclusionsGlobally, molecular, histological, and functional data from these experiments in the HSALR mouse model confirms the pathological role of Msi2 expression levels as an atrophy-associated component that impacts the characteristic muscle dysfunction symptoms in DM1 patients.

MANF inhibits Sjögren's syndrome salivary gland epithelial cell apoptosis and antigen expression of Ro52/SSA through endoplasmic reticulum stress/autophagy pathway.

Sjögren's syndrome (SS) is a typical autoimmune disease characterized by lymphocyte infiltration accompanied by the production of Ro52/SSA and La/SSB autoantibodies against whole body ribonucleoprotein particles. The release of type I IFN can induce endoplasmic reticulum stress (ERS) in submandibular gland cells. ERS not only produces a large number of Ro52/SSA antigens and changes their location, but also down-regulates autophagy and increases apoptosis. We collected human submandibular gland tissue samples, established an Experimental Sjögren's syndrome (ESS) mouse model, and used submandibular gland cells to test whether Mesencephalic astrocyte-derived neurotrophic factor (MANF) could reverse ERS-induced autophagy downregulation and reduce apoptosis and Ro52/SSA antigen expression. It was found that MANF could reduce lymphocyte infiltration and the proportion of CD4+ T cell subsets in the salivary glands, reduce the phosphorylation of AKT and mTOR proteins and the expression of ERS-related proteins, and increase the expression of autophagy proteins. We also found that MANF can reduce the expression of Ro52/SSA antigen on the cell membrane and reduce apoptosis. In short, we found that MANF can activate autophagy, inhibit apoptosis and reduce the expression of Ro52/SSA by regulating the AKT/mTOR/LC3B signaling pathway. The above results suggest that MANF may be a protective factor against SS.

Autophagy-dependent ferroptosis is involved in the development of endometriosis

Objective Endometriosis (EMS) is an estrogen-dependent condition with unclear pathogenesis. Recent findings suggest implicate autophagy and ferroptosis in EMS development. Methods We assessed autophagy and ferroptosis proteins in EMS patients using immunohistochemistry and western blot and established an EMS rat model through allograft endometrial transplantation, confirmed via hematoxylin and eosin staining and epithelial–mesenchymal transition -related proteins. Primary EMS cells were isolated from the model rats and cultured under five conditions: control, EMS, EMS with Rapamycin (autophagy inducer), EMS with si-Atg5 (autophagy inhibitor), and EMS with si-Atg5 plus Erastin (ferroptosis inducer). We evaluated cell viability, iron content, oxidative stress, and mitochondrial morphologyin EMS cells, and detected autophagy and ferroptosis proteins through immunofluorescence, western blot, and monodansylcadaverine staining. Results Autophagy proteins Beclin1 and LC3 were highly expressed, whereas p62, glutathione peroxidase 4, and p53 were lowly expressed in EMS patients. Rapamycin decreased cell viability but increased iron content, reactive oxygen species, lipid peroxide production, the number of ferroptotic mitochondria, and the expression of autophagy proteins in EMS cells, while si-Atg5 showed opposite effects. Additionally, Erastin reversed the impact of si-Atg5 on EMS cells. Conclusion Our findings suggest that autophagy-dependent ferroptosis plays a role in EMS progression.

Synthetic phenolic antioxidants evoked hepatoxicity in grass carp (Ctenopharyngodon idella) through modulating the ROS-PI3K/mTOR/AKT pathway: Apoptosis-autophagy crosstalk.

Synthetic phenolic antioxidants (SPAs) are an environmental concern due to their persistence nature and bioaccumulation. However, the hepatoxicity and mechanisms of SPAs in aquatic organisms remain poorly understood. In this study, grass carp were exposed to two representative SPAs (BHA and BHT) at environmentally relevant levels (0.1 μM) for 30 days. We observed that BHA and BHT exposure significantly increased the levels of serum aminotransferase (ALT) and aspartate aminotransferase (AST) in grass carp, accompanied by mild inflammatory cell infiltration and irregularity in the shape of hepatocytes. Dihydro ethylenediamine staining showed that BHA and BHT exposure resulted in elevated levels of superoxide levels, accompanied by increased antioxidant enzyme activities (T-AOC, SOD, CAT, GSH-PX) and MDA levels, which is suggestive of oxidative stress responses in the liver of grass carp. Besides, BHA and BHT could dock into the pocket of phosphatidylinositol 3-kinases (PI3K) and thereby inhibiting PI3K/mammalian target of rapamycin (mTOR)/protein kinase B (AKT) signaling cascades. Meanwhile, our results clarified that BHA and BHT could promote autophagosome production and increase the expression of key autophagy proteins, likely due to inhibition of PI3K/mTOR/AKT signaling pathway. Moreover, BHA and BHT could induce apoptotic process by upregulating the expression of Bax, Caspase3 and Caspase8 and downregulating Bcl2 expression. Notably, BHT exhibited more hepatoxicity on the indicators of the apoptosis and oxidative stress than BHA. In summary, our findings demonstrated that BHA and BHT exposure could induce liver damage induced via regulating ROS/PI3K-mediated autophagic hyperactivation, which is a crucial step in triggering hepatocyte death. This study provides novel insight into the potential mechanisms underlying liver damage caused by BHA and BHT in aquatic organisms, and offers a new theoretical basis for ecological risk assessment of SPAs.

P7C3-A20 treats traumatic brain injury in rats by inhibiting excessive autophagy and apoptosis.

Traumatic brain injury is a severe health problem leading to autophagy and apoptosis in the brain. 3,6-Dibromo-beta-fluoro-N-(3-methoxyphenyl)-9H-carbazole-9-propanamine (P7C3-A20) can be neuroprotective in various diseases, including ischemic stroke and neurodegenerative diseases. However, whether P7C3-A20 has a therapeutic effect on traumatic brain injury and its possible molecular mechanisms are unclear. Therefore, in the present study, we investigated the therapeutic effects of P7C3-A20 on traumatic brain injury and explored the putative underlying molecular mechanisms. We established a traumatic brain injury rat model using a modified weight drop method. P7C3-A20 or vehicle was injected intraperitoneally after traumatic brain injury. Severe neurological deficits were found in rats after traumatic brain injury, with deterioration in balance, walking function, and learning memory. Furthermore, hematoxylin and eosin staining showed significant neuronal cell damage, while terminal deoxynucleotidyl transferase mediated dUTP nick end labeling staining indicated a high rate of apoptosis. The presence of autolysosomes was observed using transmission electron microscope. P7C3-A20 treatment reversed these pathological features. Western blotting showed that P7C3-A20 treatment reduced microtubule-associated protein 1 light chain 3-II (LC3-II) autophagy protein, apoptosis-related proteins (namely, Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 [BNIP3], and Bcl-2 associated x protein [Bax]), and elevated ubiquitin-binding protein p62 (p62) autophagy protein expression. Thus, P7C3-A20 can treat traumatic brain injury in rats by inhibiting excessive autophagy and apoptosis.

Seawater pearl hydrolysate inhibits photoaging via decreasing oxidative stress, autophagy and apoptosis of Ultraviolet B-induced human skin keratinocytes.

Ultraviolet (UV) is the main reason to cause photoaging skin which not only hinders beauty, brings the patients with psychological burden, but also pathologically leads to the occurrence of tumors in skin. This study goes into the inhibitory effect and mechanism of seawater pearl hydrolysate (SPH) to address human skin keratinocytes photoaging induced by UVB. The photoaging model of Hacat cell was constructed by UVB irradiation, the levels of oxidative stress, apoptosis, aging, autophagy and autophagy-related protein and signal pathway expression were assessed to characterize the inhibitory effect and mechanism of SPH on photoaging Hacat cell. Seawater pearl hydrolysate significantly accelerated (p < 0.05) the activities of superoxide dismutase, catalase, and glutathione peroxidase, and markedly reduced (p < 0.05) the contents of reactive oxygen species (ROS), malondialdehyde, protein carbonyl compound and nitrosylated tyrosine protein, aging level, apoptosis rate in Hacat cell induced by 200 mJ cm-2 UVB after 24 and 48 h of culture; high dose SPH significantly raised (p < 0.05) relative expression level of p-Akt, p-mTOR proteins, and markedly decreased (p < 0.05) relative expression level of LC3II protein, p-AMPK, and autophagy level in Hacat cell induced by 200 mJ cm-2 UVB, or in combination with the intervention of PI3K inhibitor or AMPK overexpression after 48 h of culture. Seawater pearl hydrolysate can effectively inhibit 200 mJ cm-2 UVB-induced photoaging of Hacat cells. The mechanism indicates removing the excessive ROS through increasing the antioxidation of photoaging Hacat cells. Once redundant ROS is eliminated, SPH works to reduce AMPK, increase PI3K-Akt pathway expression, activate mTOR pathway to lowdown autophagy level, and as a result, inhibit apoptosis and aging in photoaging Hacat cells.

Botulinum toxin type A activates protective autophagy by modulating endoplasmic reticulum stress in hypoxia/reoxygenation-treated endothelial cells

Botulinum toxin type A (BTXA) previously protected endothelial cells in free skin flaps from ischemia/reperfusion injury by inducing autophagy. Endoplasmic reticulum (ER) stress-autophagy activation may have a role in BTXA-antagonized ischemia/reperfusion damage in human dermal microvascular endothelial cells (HDMECs), however, this has yet to be proven. HDMECs were pretreated with BTXA at various concentrations for 12 h before being subjected to hypoxia and reoxygenation (H/R). Cell Count Kit 8 (CCK8) and Western blot (WB) data showed that H/R treatment significantly increased the expression of ER stress (GRP78, CHOP) and autophagy (LC3II/I, Beclin-1) proteins. The optimal BTXA pretreatment concentration was 1.6 U/mL, which resulted in the highest levels of cell survival and expression of ER stress and autophagy. Following pretreatment with 1.6 U/mL BTXA and the addition of the ER stress inducer Thapsigargin (Tg), the ER stress inhibitor 4-phenylbutyrate (4-PBA), and the inhibitor of autophagy Bafilomycin A1 (Baf A1), respectively, HDMECs were then subjected to H/R treatment. Cell survival and the percentage of ethynyldeoxyuridine-labeled cells in the BTXA pretreatment groups were reduced by the addition of Tg, 4-PBA, and Baf A1. While the expression of GRP78, CHOP, and LC3 was upregulated by Tg and Baf A1, it was downregulated by 4-PBA. The findings showed that ER stress produced by BTXA pretreatment triggers protective autophagy and protects HDMECs from H/R damage. There were no cytoprotective effects from either excessive activation or reduction of ER stress. Our results are consistent with the notion that autophagy and moderate ER stress are critical for cellular longevity and, consequently, functional integrity and may represent a potential therapeutic target.

MiR-506-3p induces autophagy of renal tubular epithelial cells in sepsis through targeting PI3K pathway.

To explore the effect of micro ribonucleic acid (miR)-506-3p on autophagy of renal tubular epithelial cells in sepsis and its mechanism. It was found through bioinformatics analysis that phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) was expressed at a low level in sepsis, and miR-506-3p had a targeted regulatory effect on PIK3CA. 40 8-week-old male C57BL/6 mice were randomly divided into control miR-506-3p NC group, control miR-506-3p OE group, sepsis miR-506-3p NC group, sepsis miR-506-3p OE group and sepsis miR-506-3p KD group. The pathological changes in kidney tissues of mice in each group were observed by hematoxylin-eosin (HE) staining and TUNEL staining, and mitochondria and autophagosomes were visualized by transmission electron microscopy. CCK8 assay was performed to detect the effect of miR-506-3p on the proliferation capacity of renal tubular epithelial cells. The changes in the expression of PI3K-Akt pathway proteins, mTOR and autophagy proteins were tested by Western blotting. The injury and apoptotic positive cells were suppressed and decreased in miR-506-3p OE mice vs. NC group. miR-506-3p could increase the number of mitochondria and autophagosomes in kidney tissues. After introduction of exogenous miR-506-3p OE into renal tubular epithelial cells, the expressions of PI3K pathway proteins were significantly inhibited, while the expressions of autophagy proteins were significantly enhanced. After 740Y-P was added, the expressions of associated proteins had no significant changes in each group. Overexpression of miR-506-3p can enhance the autophagy of renal tubular epithelial cells in sepsis through inhibiting the PI3K signaling pathway.

Hydrogen inhalation enhances autophagy via the AMPK/mTOR pathway, thereby attenuating doxorubicin-induced cardiac injury.

Doxorubicin is a drug widely used in clinical cancer treatment, but severe cardiotoxicity limits its clinical application. Autophagy disorder is an important factor in the mechanism of doxorubicin-induced cardiac injury. As the smallest molecule in nature, hydrogen has various biological effects such as anti-oxidation, anti-apoptosis and regulation of autophagy. Hydrogen therapy is currently considered to be an emerging therapeutic method, but the effect and mechanism of hydrogen on doxorubicin-induced myocardial injury have not been determined. The purpose of this study was to investigate the protective effect of hydrogen inhalation on doxorubicin-induced chronic myocardial injury and its effect and mechanism on autophagy. In this study, we established a chronic heart injury model by intraperitoneal injection of doxorubicin in rats for 30days, accumulating 20mg/kg. The effect of hydrogen inhalation on the cardiac function in rats was explored by echocardiography, Elisa, and H&E staining. To clarify the influence of autophagy, we detected the expression of LC3 and related autophagy proteins in vivo and in vitro by immunofluorescence and western blot.In order to further explore the mechanism of autophagy, we added pathway inhibitors and used western blot to preliminarily investigate the protective effect of hydrogen inhalation on myocardial injury caused by doxorubicin. Hydrogen inhalation can improve doxorubicin-induced cardiac function decline and pathological structural abnormalities in rats. It was confirmed by immunofluorescence that hydrogen treatment could restore the expression of autophagy marker protein LC3 (microtubule-associated protein 1 light chain 3) in cardiomyocytes reduced by doxorubicin, while reducing cardiomyocyte apoptosis. Mechanistically, Western blot results consistently showed that hydrogen treatment up-regulated the ratio of p-AMPK (phosphorylated AMP-dependent protein kinase) to AMPK and down-regulated p-mTOR (phosphorylated mammalian target of rapamycin) and mTOR ratio. These results suggest that hydrogen inhalation can activate autophagy through the AMPK/mTOR pathway and protect against myocardial injury induced by doxorubicin. Hydrogen inhalation therapy may be a potential treatment for doxorubicin-induced myocardial injury.