ATG5 Expression Research Articles

Autophagy inhibitor 3-methyladenine mitigates the severity of colitis in aged mice by inhibiting autophagy.

The elderly ulcerative colitis (UC) patients pose unique challenges due to their comorbidities, diminished functional capacity, and heightened risk of treatment-related complications. Thus, finding a safe and effective treatment for this age group is crucial. This study investigates the role of autophagy in the pathogenesis of UC in young and elderly patients, and explores the therapeutic potential and mechanisms of autophagy modulators in aged mice with dextran sulfate sodium (DSS)-induced colitis. Colonic biopsies were collected from young and old UC patients as well as comparable healthy subjects. Young (6-8 weeks) and aged (56 weeks) C57BL/6 mice were treated with DSS to induce acute colitis model. The autophagy inhibitor 3-methyladenine was administered intraperitoneally to aged DSS-induced mice. The autophagy activity was detected by the protein expressions of LC3B-II, p62 and ATG5 by western blot and immunohistochemistry. The levels of TNF -α, IL-6, CCL4, CXCL12 and CD86 were measured by qRT-PCR. The transcriptional activity of NF-κB was measured by electrophoretic mobility shift assay (EMSA). Increased autophagy activity was observed in aged DSS-induced mice. Treatment with 3-methyladenine suppressed autophagy in intestinal epithelial cells (IECs) and alleviated colitis severity. Additionally, 3-methyladenine reduced macrophage recruitment, decreased IL-6 levels, and inhibited NF-κB signaling, thereby mitigating inflammation. Significant differences in autophagy activity were identified between young and aged DSS-induced mice. These findings underscore the potential therapeutic benefits of autophagy inhibition in elderly UC patients.

Rapamycin Increases the Development Competence of Yak (Bos grunniens) Oocytes by Promoting Autophagy via Upregulating 17β-Estradiol and HIF-1α During In Vitro Maturation

High-quality oocyte production strategies play an important role in animal-assisted reproductive biotechnologies, and rapamycin (Rap) has been commonly used to increase the development potential of mammalian oocytes. The purpose of this study is to evaluate the effects and possible molecular mechanisms of rap on the maturation of yak oocytes. Different concentrations of Rap were supplemented during in vitro maturation (IVM) of yak oocytes. The maturation rates of oocytes and development rates of parthenogenetically activated embryos were assessed. The levels of 17β-estradiol (E2) were detected via ELISA, and the expression of autophagy-related factors, steroidogenic enzymes, and HIF-1α was detected via qRT-PCR, western blotting, and fluorescence microscopy, respectively. In addition, the impacts of E2 and HIF-1α on Rap-mediated oocyte autophagy were investigated by investigating the activities of E2 and HIF-1α. Our results showed that 0.1 nM Rap substantially enhanced the developmental ability of yak oocytes. In this group, the levels of E2, CYP19A1, CYP17A1, and autophagy-related factors were also significantly increased, and the expression of ATG5 and BECN1 in subsequent embryos was also increased. Further analysis revealed that Rap tends to enhance the development competence of yak oocytes and that the levels of autophagy-related factors are reduced when the activity of E2 or HIF-1α is inhibited. Furthermore, the levels of E2, CYP19A1, and CYP17A1 were downregulated when the activity of HIF-1α was inhibited, and the levels of HIF-1α were also significantly reduced by the estrogen receptor antagonist G15. Nevertheless, the levels of CYP11A1 mRNA in mature yak COCs were not significantly different among these groups, a phenomenon which implies that the levels of E2 were not correlated with the CYP11A1 content in yak COCs. There was an increasing tendency for the development competence of yak oocytes at the optimum concentration of Rap during IVM. The potential underlying mechanism is that Rap can activate autophagy and upregulate the levels of E2 and HIF-1α in mature oocytes. Additionally, the levels of both E2 and HIF-1α are regulated by each other and involve Rap-regulated autophagy in oocytes.

The Effect of Maternal Obesity on Placental Autophagy in Lean Breed Sows

This study aimed to evaluate the influence of back-fat thickness (BF), at mating of sows, on autophagy in placenta and the potential mechanism. The sows were divided into two groups according to their BF at mating: BFI (15–20 mm, n = 14) and BFII (21–27 mm, n = 14) as the maternal obesity group. The placental samples used for investigating autophagic function and fatty acid profiles were obtained by vaginal delivery. Our results demonstrated that autophagy defects were observed in placenta from BFII sows along with altered circulating and placental fatty acid profiles. Indicative of impaired autophagy, reduced autophagic vesicles as well as LC3-positive puncta were linked to decreased mRNA or protein expression of autophagy-related genes, including ATG5, ATG7, Beclin1, ATG12, LC3, LAMP1 and LAMP2 in the placenta of BFII sows (p < 0.05). Meanwhile, we found reduced conversion of LC3-I to LC3-II and up-regulated protein content of p62 in the placenta from BFII group (p < 0.05). Furthermore, excessive back-fat was also associated with increased activation of AKT/mTOR signaling and decreased mRNA content of transcription factors regulating the autophagic pathway, including PPARα and PGC1α, but increased mRNA expression of NcoR1 in placenta. Together, these findings indicate that maternal obesity incites autophagy injury in pig term placenta, which may contribute to augmented placental lipid accumulation and therefore impaired placental function.

Synergistic anticancer effects of cisplatin and phenolic aglycones of the aerial part of Rumex dentatus L. in tongue squamous cell carcinoma: insights from network pharmacology and biological verification

BackgroundOral squamous cell carcinoma (OSCC) ranks as the sixth most common malignancy globally. Cisplatin is the standard chemotherapy for OSCC, but resistance often reduces its efficacy, necessitating new treatments with fewer side effects. Rumex dentatus L., from the Polygonaceae family, is known for its medicinal properties, but its anticancer potential has not been thoroughly explored. This study aimed to investigate the synergy between cisplatin and an extract from the aerial parts of R. dentatus L. in treating tongue carcinoma (HNO97) in vitro, using network pharmacology, biological verification, and phytochemical analysis.MethodsThe study included UPLC-ESI–MS/MS analysis, cytotoxicity assays, cell cycle analysis, apoptosis assessment, and RT-qPCR for gene expression of Bcl2, p53, and ATG7. Potential targets were identified, and mechanisms of action were examined through online databases and enrichment analyses.ResultsThe R. dentatus L. extract contained 14 phenolic aglycons. Combining cisplatin and R. dentatus L. was more effective in inhibiting proliferation, inducing cell cycle arrest and apoptosis, and reducing autophagy in HNO97 cells than cisplatin alone. KEGG analysis indicated that the drug combination might work through pathways like PI3K-Akt signaling, microRNAs in cancer, and EGFR tyrosine kinase inhibitor resistance.ConclusionsCombining cisplatin with R. dentatus L. may be a promising approach for treating tongue carcinoma by affecting multiple pathways, providing a new perspective for developing more effective treatments for OSCC.

A Functional Variant rs2122031 in ATG7 Is Associated with the Risk of Radiation Pneumonitis.

Radiation pneumonitis (RP) is characterized by inflammation and is associated with autophagy. However, the relationship between functional genetic variants of autophagy-related genes and radiation pneumonitis remains unknow. In this study we aimed to investigate whether genetic variants of genes involved in autophagy are associated with radiation pneumonitis. Genotyping was conducted on a total of 301 patients for thirteen single nucleotide polymorphisms (SNPs) of 5 genes in the autophagy pathway using MassArray and Sanger sequencing. Two radiation oncologists independently measured the degree of RP by chest X-ray or CT. The multivariate Cox hazard analysis and multiple testing showed that ATG7: rs2122031 GA/GG significantly decreased the risk of RP ≥ grade 3 (HR=0.369, 95% CI: 0.189-0.720, P=0.003, Pc=0.039). Furthermore, qRT-PCR and immunohistochemical analysis demonstrated that the ATG7: rs2122031 AA genotypes were related to decreased expression of ATG7. Loss of autophagy by deletion of ATG7 in fibroblasts or conditional ATG7 knockout mice was proven to increase radiation pneumonitis. Single-cell RNA-seq revealed regulation of autophagy related genes enriched after irradiation stress in conditional ATG7 knockout mice. Our findings indicated that genetic variants of ATG7 were associated with RP and may therefore be used to predict RP before radiotherapy. Loss of ATG7 was also shown to promote RP, which suggested that ATG7 may be an intervention target for RP.

Effects of Citrus-derived Diosmetin on Melanoma: Induction of Apoptosis and Autophagy Mediated by PI3K/Akt/mTOR Pathway Inhibition.

Diosmetin (DIOS) is a naturally abundant flavonoid and possesses various biological activities that hold promise as an anti-cancer agent. However, the anti-cancer activities and underlying mechanism of DIOS on cutaneous melanoma remain unclear. This study seeks to explore the anti-tumor effect and mechanism of DIOS in cutaneous melanoma. Here, a variety of in vitro and in vivo experiments, combined with RNA sequencing (RNA-seq), were employed to ascertain the potential anti-cutaneous melanoma capacity and mechanism of DIOS. The results demonstrated that DIOS considerably impeded cell proliferation and triggered cell apoptosis in a dose- and time-dependent manner. Concurrently, DIOS markedly elevated the expression of pro-apoptotic proteins (Cleaved caspase-3, Bax, Cleaved PARP, and Cleaved caspase-9) and downregulated the expression of Bcl-2. Additionally, DIOS markedly upregulated the protein expressions of LC3B-II and Atg5, while downregulating p62 protein expression. Notably, pre-treatment with an autophagy inhibitor significantly inhibited DIOSinduced cell apoptosis and autophagy. Mechanistically, DIOS was identified to repress the PI3K/Akt/mTOR signaling pathway by western blot analyses and RNA-seq. Finally, in vivo experiments using a syngeneic mouse model confirmed the anti-tumor effect of DIOS, which exhibited high levels of apoptosis and autophagy. These findings propose that DIOS acts as a potential melanoma therapy that exerts its anti-tumor effects by triggering apoptosis and autophagy via inhibition of the PI3K/Akt/mTOR pathway.

Icariin mediates autophagy and apoptosis of hepatocellular carcinoma cells induced by the β-catenin signaling pathway through lncRNA LOXL1-AS1.

To investigate the effect of icariin (ICA) on hepatocellular carcinoma (HCC) and its autophagy/apoptosis mechanism in HCC. The anti-HCC mechanism of ICA was investigated using HCC cells treated with 20 µmol/L ICA. Cell viability and proliferation were assessed using CCK-8 and colony formation assays, respectively, while TUNEL staining evaluated anti-apoptotic effects. DHE staining quantified intracellular ROS levels, and JC-1 staining assessed mitochondrial membrane potential. The expression of LC3 was detected by immunofluorescence staining. Additionally, HepG2 cells (2.0 × 106) were implanted into the thymus of BALB/c nude mice, which received intraperitoneal injections of 40 mg/kg ICA. Western blotting was used to evaluate the expression of proteins related to apoptosis and autophagy. ICA effectively inhibited the proliferation and invasion of HCC cells, enhancing autophagy and apoptosis. Silencing of lncRNA LOXL1-AS1 reduced β-catenin expression and downregulated PI3K/AKT/mTOR pathway phosphorylation. Targeting β-catenin with siRNA augmented apoptosis in HepG2 cells through elevated levels of Bax and caspase-3/8/9 and boosted autophagy via increased expression of LC3-II, Atg5, Atg7, Atg8, and Beclin-1. ICA reversed this autophagic effect, while rapamycin enhanced ICA's efficacy. In vivo, ICA suppressed tumor growth and promoted autophagy and apoptosis in mice. Icariin induces autophagy and apoptosis in HCC cells via the β-catenin signaling pathway mediated by lncRNA LOXL1-AS1, offering a novel approach to HCC clinical management.

Relationship of Autophagic Dysfunction With the Quality of Life and Sleep, Depression and Disease Severity in Patients With Restless Legs Syndrome.

Restless legs syndrome (RLS) is a frequently encountered neurological illness that has no effective treatment and imposes an enormous socioeconomic burden. Autophagy is essential for the maintenance of healthy cellular physiology, cell viability, and defense against pathogenic conditions. However, there is no study investigating the possible role of autophagy-related proteins (ATGs) in RLS patients. Therefore, we aimed to investigate the expression and diagnostic potential of ATG3 and ATG5, as well as the relationships between these proteins and laboratory markers, depression, disease score, quality of life, and sleep in RLS patients. A total of 49 patients with RLS and 26 healthy individuals were recruited for the current study. The severity of the disease was assessed using the international RLS rating scale. All participants were administered the Pittsburgh Sleep Quality Index, the Quality-of-Life Scale, and the Beck Depression Inventory. The enzyme-linked immunosorbent assay was employed to quantify the expressions of ATG3 and ATG5. Serum ATG3 and ATG5 expressions were significantly upregulated in RLS patients compared to healthy controls (p = 0.005) and upregulated ATG3 and ATG5 expressions were relationship with the severity of the disease (p < 0.05). ATG3 was substantially correlated with the quality of sleep, whereas ATG5 was correlated with the quality of life and depression status (p < 0.05). The ROC curve analysis demonstrated that ATG3 expressions over 3146.5ng/mL and ATG5 expressions over 4732.5ng/mL may predict the presence of RLS (p < 0.01). We report for the first time that autophagy may have a significant impact on the development of RLS.

Actl6a regulates autophagy via Sox2-dependent Atg5 and Atg7 expression to inhibit apoptosis in spinal cord injury.

Spinal cord injury (SCI) is a severe central nervous system disorder with limited treatment options. While autophagy plays a protective role in neural repair, its regulatory mechanisms in SCI remain unclear. Actin-like protein 6A (Actl6a) influences cell fate and neural development, yet its specific role in SCI repair is not well understood. This study investigates Actl6a's function in regulating autophagy and apoptosis via the transcription factor Sox2 in SCI. This study aims to determine if Actl6a promotes neural survival post-SCI by regulating autophagy-related genes Atg5 and Atg7 through Sox2. It also examines how the demethylase Fto modulates Actl6a mRNA stability via m6A methylation. In vitro experiments were conducted using primary neurons and HT-22 hippocampal cells exposed to hydrogen peroxide (H2O2)-induced oxidative stress. Actl6a expression was manipulated by knockdown or overexpression. For in vivo studies, a rat SCI model was established with AAV-Actl6a injected at the injury site to induce Actl6a overexpression. Autophagy and apoptosis markers were analyzed using immunofluorescence, Western blotting, and qPCR. Additionally, m6A dot blot and RNA immunoprecipitation (RIP) assays were performed to assess Fto's role in regulating Actl6a mRNA methylation and stability. Actl6a expression significantly decreased after SCI, resulting in increased apoptosis. Overexpressing Actl6a enhanced autophagy, reduced apoptosis, and improved neurological function in SCI models. Mechanistically, Actl6a and Sox2 collaboratively upregulated Atg5 and Atg7 expression, promoting autophagy. Fto's modulation of Actl6a mRNA stability via m6A demethylation further influenced autophagy and apoptosis. Actl6a, through interaction with Sox2, plays a critical role in modulating autophagy and reducing apoptosis in SCI, with Fto's m6A modification affecting Actl6a stability. This Fto/Actl6a/Sox2 axis is a promising therapeutic target for SCI repair.

Gold Nanoparticle Inhibits the Tumor-Associated Macrophage M2 Polarization by Inhibiting m6A Methylation-Dependent ATG5/Autophagy in Prostate Cancer.

Background: This study aims to study how gold nanoparticles (AuNPs) function in the recruitment and polarization of tumor-associated macrophages (TAMs) in hormone-sensitive prostate cancer (HSPC) and castration-resistant prostate cancer (CRPC). Methods: Phorbol ester (PMA)-treated THP-1 cells were cocultured with LNCaP or PC3 cells to simulate TAMs. Macrophage M2 polarization levels were detected using flow cytometry and M2 marker determination. ATG5 expression was detected by western blotting. Luciferase reporter assay was used to analyze the N6-methyladenosine (m6A) site activity of ATG5 3' untranslated regions (3'-UTRs). Methylated RNA immune precipitation (MeRIP)-quantitative polymerase chain reaction (qPCR) was performed to determine the m6A levels at ATG5 3'-UTR. Xenograft mouse models were used to determine the function of AuNPs in vivo. Results: Macrophages exhibited reduced M2 polarization in both HSPC and CRPC cells after AuNP treatment which was prevented by induction of autophagy. AuNP treatment decreased the m6A levels in the 3'-UTR of ATG5. Mutational analysis of potential m6A sites within ATG5 3'-UTR revealed that these sites were required for AuNP regulation, indicating that AuNPs inhibited ATG5 levels in an m6A-dependent manner. The mouse model revealed that AuNPs significantly reduced the M2 polarization of TAMs in an autophagy-dependent manner in vivo. This suggests that AuNPs inhibit tumor growth in vivo partially through targeting M2 TAM. Conclusion: The ATG5/autophagy pathway is inhibited by AuNP treatment in an METTL3/m6A-dependent manner. AuNPs inhibit the TAM M2 polarization in HSPC and CRPC by inhibiting ATG5/autophagy.

Angong Niuhuang Pill pretreatment alleviates cerebral ischemia-reperfusion injury by inhibiting excessive autophagy through the SIRT1-H4K16ac axis

Ethnopharmacological relevanceCerebral ischemia-reperfusion injury (CIRI) is an important pathological process in stroke treatment. Angong Niuhuang Pill (ANP), originating from Wenbing Tiaobian, has been shown to have neuroprotective effects, but its mechanism in alleviating CIRI remains unclear. Aim of the studyThis study aimed to elucidate the mechanism by which ANP alleviates CIRI using acetylomics and proteomics. Materials and methodsThe CIRI model was established using middle cerebral artery occlusion (MCAO). Neurological deficit scoring, TTC staining, regional cerebral blood flow (rCBF) measurement, and TUNEL staining were used to assess the neuroprotective effects of ANP pretreatment on CIRI. Acetylomics and proteomics analyses were performed to identify the potential mechanisms by which ANP reduces CIRI. Finally, the role of SIRT1-H4K16ac-mediated autophagy in the neuroprotective effects of ANP was validated by using a SIRT1 inhibitor, EX527. ResultsANP pretreatment markedly lowered neurological deficit scores and cerebral infarct volumes, increased rCBF, and reduced apoptosis. Acetylomics and proteomics results suggested that ANP regulated autophagy at the transcriptional level by modulating H4K16ac. Immunofluorescence and western blot analyses confirmed that ANP promoted the accumulation of sirtuin 1 (SIRT1). Specifically, ANP pretreatment reduced H4K16ac levels, decreased LC3B-II/I ratios, upregulated SQSTM1/p62, and suppressed the expression of ATG5 and ATG7. The ability of EX527 to counteract these effects underscored the importance of the SIRT1-H4K16ac pathway in mediating the protective action of ANP against CIRI. ConclusionsANP provides neuroprotection by modulating the SIRT1-H4K16ac pathway, thereby preventing the excessive autophagy triggered by CIRI.

LINC02987 suppression hepatocellular carcinoma progression by modulating autophagy via the miR-338-3p/ATG12 axis.

Hepatocellular carcinoma (HCC), the most common primary liver cancer, is marked by a high mortality rate, with the misregulation of long non-coding RNAs (LncRNAs) playing a key role in its development. Here, we studied the role of LINC02987 in HCC. We employed bioinformatics tools to identify LncRNAs and miRNAs that exhibit differential expression in HCC. Quantitative real-time reverse transcription PCR (RT-qPCR) and Western blot analysis were utilized to quantify gene and protein expression levels. The interaction between miR-338-3p and LINC02987 or ATG12 was confirmed through dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. We observed that LINC02987 was overexpressed in HCC tumor tissues and cell lines. Silencing of LINC02987 led to a reduction in cell viability, diminished clonogenic potential, and attenuated invasive and migratory capabilities. Also, decreasing protein level and fluorescence intensity of the autophagy-associated LC3 I/II. In HCC, miR-338-3p expression was downregulated, while inversely correlates with the overexpression of the autophagy protein ATG12. Mimicking miR-338-3p suppresses the activity of both LINC02987 and ATG12, as evidenced by reduced luciferase signals in corresponding reporter assays. Mimicking miR-338-3p suppresses the activity of both LINC02987 and ATG12, as evidenced by reduced luciferase signals in reporter assays. Transfection with si-LINC02987 decreased ATG12 expression, an effect that was partially reversed by miR-338-3p knockdown. Inhibition of miR-338-3p or overexpression of ATG12 increased LC3 I/II protein levels. Our results indicate that LINC02987 sequesters miR-338-3p, leading to increased ATG12 and promoting autophagy in HCC cells. These results highlight the potential of LINC02987 as a therapeutic target for the treatment of HCC.

Rapamycin and Niacin combination induces apoptosis and cell cycle arrest through autophagy activation on acute myeloid leukemia cells.

Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy caused by disorders in stem cell differentiation and excessive proliferation resulting in clonal expansion of dysfunctional cells called myeloid blasts. The combination of chemotherapeutic agents with natural product-based molecules is promising in the treatment of AML. In this study, we aim to investigate the anti-cancer effect of Rapamycin and Niacin combination on THP-1 and NB4 AML cell lines. The anti-proliferative effects of Rapamycin and Niacin were determined by MTT cell viability assay in a dose- and time-dependent manner. The combination indexes were calculated by isobologram analysis. Furthermore, apoptosis was investigated by Annexin-V/Propidium Iodide(PI) double staining and cell cycle distribution was measured by PI staining. The expression levels of autophagy-related proteins were detected by western blotting. The combination of Rapamycin and Niacin synergistically decreased cell viability of AML cell lines. The combination treatment induced the apoptotic cell population of THP-1 and NB4 by 4.9-fold and 7.3-fold, respectively. In THP-1 cells, the cell cycle was arrested at the G2/M phase by 10% whereas the NB4 cells were accumulated at the G0/G1 phase. The combination treatment decreased Akt and p-Akt expression. Besides, the ATG7 expression was reduced by combination treatment on THP-1 cells. Similarly, the ATG5 level was downregulated in NB4 cells. The level of LC3B-II/LC3B-I, which is an indicator of autophagy flux, was upregulated in THP-1 and NB4 cells. Although further studies are required, the combination of Rapamycin and Niacin combats cell proliferation by inducing cellular apoptosis, cell cycle arrest and autophagy activation.

Scutellaria barbata D.Don and Hedyotis diffusa Willd herb pair combined with cisplatin synergistically inhibits ovarian cancer progression through modulating oxidative stress via NRF2-FTH1 autophagic degradation pathway

BackgroundCisplatin (DDP) is one of the most effective anticancer drugs, commonly used to treat advanced ovarian cancer (OC). However, DDP has significant limitations of platinum-based drugs, including chemical resistance and high-dose toxic side effects. Traditional Chinese medicines (TCMs) often presented in the form of formula, in which the herb pair was the basic unit. Scutellaria barbata D.Don and Hedyotis diffusa Willd (SB-HD) are famous TCMs herb pair that have been shown to help treat multiple types of cancers. However, the synergistic effects and mechanism of combination of SB-HD and DDP to enhance DDP chemosensitivity in OC are still unknown.ResultsIn vitro, we found that the optimal proportion of SB-HD to inhibit the proliferation of OC cells was 2:1, SB-HD and DDP were shown to synergistically reduce the viability of OC cells, inhibit the colony formation, promote cell cycle arrest and apoptosis, as well as inhibit cell migration and invasion. In vivo, combination treatment significantly inhibited the growth of subcutaneous tumors in BALB/c nude mice and reduced the toxic side effects of DDP. Mechanistically, SB-HD and DDP combination treatment significantly promoted oxidative stress response, decreased MMP, inhibited ATP production, decreased ROS levels and increased SOD activity, increased the expression of NRF2, HO-1, ATG5 and LC3, decreased the expression of p62 and FTH1 both in OC cells and tumor tissue of mice. Inhibitor 3-MA (Methyladenine, autophagy inhibitor) and Fer-1 (Ferrostatin-1, iron ion inhibitor) can effectively reverse the expression changes of the key target proteins, but not ZnPP (Zinc protoporphyrin, HO-1 inhibitor). Through bioinformatics analysis, it was found that the abnormal expression level of NRF2 and FTH1 mRNA has a high prognostic value, at the same time, the other four key proteins respectively or interacting with NRF2 and FTH1, also play important roles in the occurrence and development of OC.ConclusionOur findings uncover a synergistic effect of SB-HD and DDP against OC through modulating oxidative stress via NRF2-FTH1 autophagic degradation pathway, which may provide an important theoretical foundation for the use of SB-HD and a new strategy for enhancing DDP chemosensitivity as well as reducing toxic side effects.

Dipeptidyl peptidase-4 (DPP-4) inhibitor sitagliptin ameliorates inflammation and autophagy in mice hindlimb ischemia–reperfusion injury

BackgroundIschemia-reperfusion injury (I/R) for skeletal muscle usually results from vascular injuries or trauma. Sitagliptin (STG) is an effective member of the dipeptidyl peptidase-4 (DPP-4) inhibitors drug family that plays roles in oxidative stress regulation, inflammation, and autophagy control. In this study, we evaluated the protective roles of STG against I/R of gastrocnemius muscle and the underlying mechanisms.Materials and methodsForty-eight mice were randomly allocated into three groups: Group I (n = 24): control group which was subdivided equally into subgroup IA; negative control, subgroup IB; sitagliptin (STG), Group II (n = 12): ischemia–reperfusion injury (I/R), and Group III (n = 12): sitagliptin pretreatment (300 mg/kg/ day; p.o.) for two weeks followed by ischemia–reperfusion injury (STG + I/R). We measured SOD activity and MDA level to assess oxidative stress. Moreover, GLP-1/p-PI3K/ p-AKT expression levels were investigated. Autophagy was estimated by assessing lncRNA H19, Beclin-1 and ATG7 expression by RT-qPCR analysis. Inflammatory markers were assessed by iNOS and NF‐κB expression using immunohistochemistry.ResultsOur results revealed that STG pretreatment significantly attenuated oxidative stress and inflammation and upregulated GLP-1, p-PI3K, and p-AKT protein levels. Also, LnRNA H19, Becline-1, and ATG7 mRNA expression were downregulated as well as decrease the expression of the inflammatory markers iNOS and NF‐κB with STG pretreatment.ConclusionOur results highlighted the useful effects of Sitagliptin during hind limb I/R that could be mediated by antioxidant, anti-inflammatory effects, and attenuation of excessive autophagy.

MicroRNA-122 regulates inflammatory and autophagic proteins by downregulating pyruvate kinase M2 in non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is one of the serious global health concerns, leading to non-alcoholic steatohepatitis (NASH), and to hepatocellular carcinoma (HCC). Despite its prevalence, the molecular mechanisms regulating NAFLD progression remain elusive. The present study aims to determine role of microRNA-122-mediated regulation of pyruvate kinase M2 (PKM2) on regulating inflammatory and autophagic proteins during the pathogenesis of NAFLD. Huh7 cells were incubated with free fatty acids (FFAs) or transfected with single guide RNA to PKM2 containing CRISPR-Cas9 system or miR-122 for up to 72h. C57BL/6 mice were fed with sham-operated control, choline sufficient L-amino acid defined (CSAA) or choline-deficient L-amino acid defined (CDAA) diet for 6, 18, 32 and 54weeks. The RNA or protein was isolated from the Huh7 cells and the liver tissue of the mice. RT-PCR was performed for miR-122 expression and Western blots were performed for PKM2, iNOS, COX2, Beclin-1, Atg7 and LC3-II. FFAs induced the expression of PKM2, iNOS and COX2, while decreased the expression of miR-122, Beclin-1, Atg7 and LC3-II. Overexpression of miR-122 resulted in decreased PKM2, iNOS and COX2 and increased Beclin-1, Atg7 and LC3-II. Silencing of PKM2 led to decreased iNOS and COX2 and increased Beclin-1, Atg7 and LC3-II. In CDAA fed-mice, there was a significant increase in PKM2, iNOS and COX2 and decreased miR-122, Beclin-1, Atg7 and LC3-II. The data showed that FFAs downregulated miR-122 expression, which resulted in the upregulation of PKM2, which in turn upregulated inflammatory proteins and downregulated autophagic proteins during the pathogenesis of NAFLD.

Cinnamon powder intake enhances the effect of caloric restriction on white adipose tissue in male rats.

Caloric Restriction (CR) and cinnamon promote several benefits, including the modulation of lipid metabolism and body fat mass. We hypothesize that cinnamon may act as a mimetic of restriction or enhance the effects of caloric restriction on adipose tissue. Adult male Wistar rats were divided into Control (CT, n = 8) and Cinnamon (CIN, n = 7), with free access to standard chow; Calorie Restriction (CR, n = 8) and Calorie Restriction with Cinnamon (CIN-CR, n = 7), subjected to a 30% reduction in food intake compared to the average consumption of CT rats. Both CIN groups received 50mg cinnamon powder (Cinnamomun verum) per kg body mass, by gavage, over 6 weeks. Cinnamon treatment did not alter food intake under either ad libitum or caloric restriction conditions. The CR and CIN-CR groups exhibit lower body mass. Basal glycemia, lipid profile, and triglyceride-glycemic index were similar between groups. The combination of both interventions induced lower visceral white adipose tissue (WAT) mass, and smaller adipocyte diameter in the visceral and subcutaneous WAT compartments, accompanied by reduced expression of genes related to lipid metabolism (Acaca, Fasn, Cd36, Srebf1c), suggesting decreased lipid synthesis. Histological analyses identified a browning phenotype in the CR, CIN, and CIN-CR groups, positive for UCP1 immunostaining. The CR and CIN-CR groups showed lower Atg7 expression, and CIN-CR animals expressed increased levels of Lamp2, suggesting modulation of autophagy. Brown adipose tissue mass and lipid content were not influenced by any intervention. These findings suggest that cinnamon may enhance the effects of caloric restriction in promoting adipocyte metabolic health.

Oxyresveratrol and/or Dapagliflozin Attenuate Doxorubicin-Induced Nephrotoxicity via Modulation of PPAR-γ/Nrf-2/HO-1, NF-κB/TNF-α/Keap-1, and Bcl-2/Caspase-3/ATG-5 signaling pathways in rats.

Among the most undesirable effects that lead to the restriction of doxorubicin (DOX) use in chemotherapy is kidney damage. This research aimed to assess the possible defenses against DOX-induced nephrotoxicity offered by oxyresveratrol (ORES) and/or dapagliflozin (DAPA). Five groups of eight male Swiss albino rats each were created from a total of sixty-four. One intravenous injection of DOX (10mg/kg) was given into the tail vein on the fourteenth day of the experiment; in the meantime, ORES (80mg/kg) and DAPA (10mg/kg) were given orally 14 days prior to the DOX injection and 2 days following the DOX injection. In rats given DOX, ORES and/or DAPA both successfully reduced the kidney weight, kidney/bodyweight ratio, and blood levels of creatinine, uric acid, and urea. They also increased final body weight and albumin serum levels. Additionally, lower serum concentrations of TNF-α and IL-6 were noted, along with a lower kidney content of caspase-3. Furthermore, the expression of the Bcl-2 gene was upregulated, as were the Nrf-2, PPAR-γ, and HO-1 proteins, and there was a downregulation of the ATG-5, Keap-1, and NF-κB renal gene expression. These findings support a decrease in oxidative stress and relief of histopathological alterations. The current study's findings suggest that ORES and/or DAPA pretreatment could be a viable therapeutic approach to ameliorate DOX-induced nephrotoxicity.

Atg5-Mediated Lipophagy Induces Ferroptosis in Corneal Epithelial Cells in Dry Eye Disease.

Ferroptosis occurred in corneal epithelial cells has been implicated in the inflammation in dry eye disease (DED). Given the proposed link between ferroptosis and autophagy, this study aims to investigate the role of autophagy in driving ferroptosis in corneal epithelial cell and enrich the pathogenesis underlying DED. DED models were established in C57BL/6 mice via scopolamine injection and in human corneal epithelial cell line (HCEC) using hyperosmotic medium. Lipidomic and transcriptomic analysis were conducted to assess lipid metabolism and regulatory pathways. Atg5 expression was manipulated in vivo using cholesterol-modified small interfering RNA. Lipid droplets (LDs) and lysosomes were labeled with BODIPY 493/503 and Lysotracker Red DND-99, respectively. Western blot, immunofluorescence (IF) staining, co-immunoprecipitation (CO-IP), transmission electron microscopy and microplate reader were used to explore protein expressions and interactions, cellular structures, and free fatty acid (FFA) content. Our results revealed that autophagy was activated in DED, as evidenced by lipidomic and transcriptomic analyses. Enhanced lipophagy was observed in HCECs exposed to hyperosmolarity, manifested by lysosome-LD co-localization and autophagic vacuoles containing LDs. Upregulation of Atg5 promoted lipophagy, leading to elevated cellular FFA levels, lipid peroxidation, and expression of ferroptosis markers. Interaction between Atg5 and perilipin3 was confirmed through CO-IP and IF. In the DED mouse model, Atg5 inhibition effectively ameliorated corneal damage, suppressed ferroptosis and ocular surface inflammation. Our findings highlight the pivotal role of Atg5-mediated lipophagy in driving ferroptosis in corneal epithelial cells in DED, proposing Atg5 as a promising therapeutic target for mitigating ferroptosis-induced cell damage and inflammation in DED.

Inhibition of miR-30d-5p promotes mitochondrial autophagy and alleviates high glucose-induced injury in podocytes

To study the role of microRNA (miR)-30d-5p in high glucose-induced podocyte injury. Podocytes were hyperglycated with 30 mmol/L glucose, transfected with miR-30d-5p inhibitor and mimic, and then treated with 1 mg/mL 3-methyladenine (3-MA). The transfection efficiency of miR-30d-5p was quantified by reverse transcription PCR. Apoptosis was detected by flow cytometry. The expressions of nephrin, microtubule-associated protein light chain (LC) 3Ⅱ/LC3Ⅰ, P62, autophagy-related gene (ATG) 5, PTEN induced putative kinase (PINK) 1 and Parkin gene (PARK2) were detected by Western blotting. The mito-chondrial membrane potential was detected by JC-1 fluorescent probe, and adenosine triphosphate (ATP) content in cells was detected by relevant kits. Under high glucose induction, podocyte apoptosis increased, miR-30d-5p and P62 expressions were upregulated, while nephrin, ATG5, PINK1, PARK2 and LC3Ⅱ/LC3Ⅰ expressions decreased (all P<0.01). MiR-30d-5p inhibitor reversed the effect of high glucose on apoptosis, and the expression of ATG5, PINK1, PARK2, nephrin, LC3Ⅱ/LC3Ⅰ and P62 (all P<0.01). High glucose induced loss of mitochondrial membrane potential and ATP content in podocytes, while inhibition of miR-30d-5p increased them. Autophagy inhibitors 3-MA and miR-30d-5p mimics reversed the effects of miR-30d-5p inhibition on apoptosis, autophagy and mitochondrial function of podocytes induced by high glucose (all P<0.05). Inhibition of miR-30d-5p may promote mitochondrial autophagy (mitophagy) by promoting the expression of ATG5, PINK1, PARK2 and alleviating high glucose-induced podocyte damage.