Autophagy-related Proteins Beclin-1 Research Articles

Targeting NCAPD2 as a Therapeutic Strategy for Crohn's Disease: Implications for Autophagy and Inflammation.

Our earlier studies identified that non-SMC condensin I complex subunit D2 (NCAPD2) induces inflammation through the IKK/NF-κB pathway in ulcerative colitis. However, its role in the development of Crohn's disease (CD) and the specific molecular mechanism still need to be further studied. NCAPD2 expression in clinical ileal CD mucosa vs normal mucosa was examined, alongside its correlation with CD patients' clinical characteristics via their medical records. The biological function and molecular mechanism of NCAPD2 in CD were explored using a 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced CD mouse model, along with immunofluorescence, western blot, quantitative real-time PCR, immunohistochemistry, hematoxylin and eosin staining, and cell functional analysis. NCAPD2 was overexpressed in CD tissues and significantly correlated with disease activity in CD patients (P = .016). In a TNBS-induced CD mouse model, NCAPD2 knockdown inhibited the development of TNBS-induced intestinal inflammation in mice. In addition, we found that NCAPD2 inhibited autophagy. Mechanistically, NCAPD2 promoted the phosphorylation of mammalian target of the rapamycin (mTOR) and its direct effector S6K and downregulated the expression of autophagy-related proteins Beclin1, LC3II, and Atg5. In addition, NCAPD2 activates the NF-κB signaling pathway, and the downstream inflammatory factors are continuously released, leading to the persistence of inflammation. Our results show that NCAPD2 suppresses autophagy and worsens intestinal inflammation by modulating mTOR signaling and impacting the NF-κB pathway, suggesting a critical role in CD progression. Targeting NCAPD2 could be a promising therapeutic approach to stop CD advancement.

Assessment of Serum Autophagy Related Protein Beclin-1 in Egyptian Adult Beta Thalassemia Patients

Assessment of Serum Autophagy Related Protein Beclin-1 in Egyptian Adult Beta Thalassemia Patients

Stable laminar shear stress induces G1 cell cycle arrest and autophagy in urothelial carcinoma by a torque sensor-coupled cone-and-plate device

The microenvironments of urinary systems play crucial roles in the development and metastasis of cancers due to their generation of complex temporal and spatial fluidic profiles. Because of their versatility in creating desired biomimetic flow, cone-and-plate bioreactors offer great potential for bladder cancer research. In this study, we construct a biocompatible cone-and-plate device coupled with a torque sensor, enabling the application and real-time monitoring of stable shear stress up to 50 dyne/cm². Under a stable shear stress stimulation at 12 dyne/cm2, bladder cancer cell BFTC-905 is arrested at the G1 phase with decreased cell proliferation after 24-hour treatment. This effect is associated with increased cyclin-dependent kinase inhibitors p21 and p27, inhibiting cyclin D1/CDK4 complex with dephosphorylation of serine 608 on the retinoblastoma protein. Consequently, an increase in cyclin D3 and decreases in cyclin A2 and cyclin E2 are observed. Moreover, we demonstrate that the shear stress stimulation upregulates the expression of autophagy-related proteins Beclin-1, LC3B-I and LC3B-II, while caspase cleavages are not activated under the same condition. The design of this system and its application shed new light on flow-induced phenomena in the study of urothelial carcinomas.

Protective effect of irbesartan against hepatic ischemia-reperfusion injury in rats: role of ERK, STAT3, and PPAR-γ inflammatory pathways in rats.

This study aimed to elucidate the possible hepatocellular protective role of irbesartan during hepatic ischemia-reperfusion injury (HIRI) and the probable underlying mechanisms. Wistar rats were allocated into four groups: sham; HIRI (control); irbesartan (50 mg/kg) + HIRI; irbesartan (100 mg/kg) + HIRI; irbesartan + GW9662 (1 mg/kg, i.p.) + HIRI. Rats pretreated orally with irbesartan or vehicle for 14 days underwent 45-min hepatic ischemia followed by 60-min reperfusion. Irbesartan preconditioning diminished alanine transaminase (ALT) and aspartate transaminase (AST) serum levels, and reduced extracellular signal-regulated kinase (ERK) and signal transducer and activator of transcription 3 (STAT3). Irbesartan decreased proapoptotic BAX (bcl-2-like protein 4), increased anti-apoptotic B-cell lymphoma 2 (BCL2) hepatic content, and thereby reduced BAX/BCL2 ratio. Moreover, irbesartan preconditioning reduced autophagy-related proteins Beclin1 and LC3 II, and elevated p62 (protein responsible for autophagosome degradation). It elevated proliferator-activated receptor γ (PPAR-γ), and reduced tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) hepatic gene expression. Also, hepatic protein expressions of nuclear factor kappa-B p65 (NF-κB p65) and caspase-3 were lessoned by irbesartan pretreatment in HIRI rats. However, GW9662 abrogated irbesartan's effect on HIRI. The protective effect of irbesartan on HIRI may be mediated by alleviation of ERK, STAT3, and PPAR-γ inflammatory pathways, exerting anti-apoptotic and anti-autophagic effects in HIRI in rats.

Protective effects of CY-09 and astaxanthin on NaIO3-induced photoreceptor inflammation via the NLRP3/autophagy pathway.

To study the effect of the NLRP3/autophagy pathway on the photoreceptor inflammatory response and the protective mechanism of CY-09 and astaxanthin (AST). ICR mice were intraperitoneally injected NaIO3, CY-09, AST successively and divided into 5 groups, including the control, NaIO3, NaIO3+CY-09, NaIO3+AST, and NaIO3+CY-09+AST groups. Spectral domain optical coherence tomography and flash electroretinogram were examined and the retina tissues were harvested for immunohistochemistry, enzyme linked immunosorbent assay (ELISA), and Western blotting. Retinal pigment epithelium cell line (ARPE-19 cells) and mouse photoreceptor cells line (661W cells) were also treated with NaIO3, CY-09, and AST successively. Cell proliferation was assessed by cell counting kit-8 (CCK-8) assay. Apoptosis was analyzed by flow cytometry. Changes in autophagosome morphology were observed by transmission electron microscopy. Quantitative polymerase chain reaction (qPCR) was used to detect NLRP3 and caspase-1. NLRP3, caspase-1, cleaved caspase-1, p62, Beclin-1, and LC3 protein levels were measured by Western blotting. IL-1β and IL-18 were measured by ELISA. Compared with the control group, the activity of NaIO3-treated 661W cells decreased within 24 and 48h, apoptosis increased, NLRP3, caspase-1, IL-1β and IL-18 levels increased, and autophagy-related protein levels increased (P<0.05). Compared with NaIO3 group, CY-09 and AST inhibited apoptosis (P<0.05), reduced NLRP3, caspase-1, IL-1β and IL-18 expression (P<0.05), and inhibited autophagy. Compared with the other groups, CY-09 combined with AST significantly decreased NLRP3 expression and inhibited the expression of the autophagy-related proteins p62, Beclin-1, and LC3 in vitro and in vivo (P<0.05). CY-09 and AST inhibit NaIO3-induced inflammatory damage through the NLRP3/autophagy pathway in vitro and in vivo. CY-09 and AST may protect retina from inflammatory injury.

Kidney Injury in a Murine Hemorrhagic Shock/Resuscitation Model Is Alleviated by sulforaphane's Anti-Inflammatory and Antioxidant Action.

Hemorrhagic shock/resuscitation (HS/R) can lead to acute kidney injury, mainly manifested as oxidative stress and inflammatory injury in the renal tubular epithelial cells, as well as abnormal autophagy and apoptosis. Sulforaphane (SFN), an agonist of the nuclear factor-erythroid factor 2-related factor 2 (Nrf2) signaling pathway, is involved in multiple biological activities, such as anti-inflammatory, antioxidant, autophagy, and apoptosis regulation. This study investigated the effect of SFN on acute kidney injury after HS/R in mice. Hemorrhagic shock was induced in mice by controlling the arterial blood pressure at a range of 35-45mmHg for 90min within arterial blood withdrawal. Fluid resuscitation was carried out by reintroducing withdrawn blood and 0.9% NaCl. We found that SFN suppressed the elevation of urea nitrogen and serum creatinine levels in the blood induced by HS/R. SFN mitigated pathological alterations including swollen renal tubules and renal casts in kidney tissue of HS/R mice. Inflammation levels and oxidative stress were significantly downregulated in mouse kidney tissue after SFN administration. In addition, the kidney tissue of HS/R mice showed high levels of autophagosomes as observed by electron microscopy. However, SFN can further enhance the formation of autophagosomes in the HS/R + SFN group. SFN also increased autophagy-related proteins Beclin1 expression and suppressed P62 expression, while increasing the ratio of microtubule-associated protein 1 light chain 3 (LC3)-II and LC3-I (LC3-II/LC3-I). SFN also effectively decreased cleaved caspase-3 level and enhanced the ratio of anti-apoptotic protein B cell lymphoma 2 and Bcl2-associated X protein (Bcl2/Bax). Collectively, SFN effectively inhibited inflammation and oxidative stress, enhanced autophagy, thereby reducing HS/R-induced kidney injury and apoptosis levels in mouse kidneys.

Extracellular Vesicles from hiPSC-derived NSCs Protect Human Neurons against Aβ-42 Oligomers Induced Neurodegeneration, Mitochondrial Dysfunction and Tau Phosphorylation.

One of the hallmarks of Alzheimer's disease (AD) is the buildup of amyloid beta-42 (Aβ-42) in the brain, which leads to various adverse effects. Therefore, therapeutic interventions proficient in reducing Aβ-42-induced toxicity in AD are of great interest. One promising approach is to use extracellular vesicles from human induced pluripotent stem cell-derived neural stem cells (hiPSC-NSC-EVs) because they carry multiple therapeutic miRNAs and proteins capable of protecting neurons against Aβ-42-induced pathological changes. Therefore, this in vitro study investigated the proficiency of hiPSC-NSC-EVs to protect human neurons derived from two distinct hiPSC lines from Aβ-42o-induced neurodegeneration. We isolated hiPSC-NSC-EVs using chromatographic methods and characterized their size, ultrastructure, expression of EV-specific markers and proficiency in getting incorporated into mature human neurons. Next, mature human neurons differentiated from two different hiPSC lines were exposed to 1 µM Aβ-42 oligomers (Aβ-42o) alone or with varying concentrations of hiPSC-NSC-EVs. The protective effects of hiPSC-NSC-EVs against Aβ-42o-induced neurodegeneration, increased oxidative stress, mitochondrial dysfunction, impaired autophagy, and tau phosphorylation were ascertained using multiple measures and one-way ANOVA with Newman-Keuls multiple comparisons post hoc tests. Significant neurodegeneration was observed when human neurons were exposed to Aβ-42o alone. Notably, neurodegeneration was associated with elevated levels of oxidative stress markers malondialdehyde (MDA) and protein carbonyls (PCs), increased expression of proapoptotic Bax and Bad genes and proteins, reduced expression of the antiapoptotic gene and protein Bcl-2, increased expression of genes encoding mitochondrial complex proteins, decreased expression of autophagy-related proteins Beclin-1 and microtubule-associated protein 1 light chain 3B, and increased phosphorylation of tau. However, the addition of an optimal dose of hiPSC-NSC-EVs (6 x 10 9 EVs) to human neuronal cultures exposed to Aβ-42o significantly reduced the extent of neurodegeneration, along with diminished levels of MDA and PCs, normalized expressions of Bax, Bad, and Bcl-2, and genes linked to mitochondrial complex proteins, and reduced tau phosphorylation. The findings demonstrate that an optimal dose of hiPSC-NSC-EVs could significantly decrease the degeneration of human neurons induced by Aβ-42o. The results also support further research into the effectiveness of hiPSC-NSC-EVs in AD, particularly their proficiency in preserving neurons and slowing disease progression.

Assessment of Serum Autophagy Related Protein Beclin1 in Egyptian Adult Beta Thalathemic Patients

Abstract Background Beta-thalassemia is a chronic hemolytic anemia that is inherited in an autosomal recessive manner. It is characterized by reduced hemoglobin levels and red blood cell production. Aim of the Work Assessment of serum autophagy related protein beclin1 in Egyptian adult beta thalathemic patients and Its relation to ineffective erythropoiesis, transfusion requirements, iron overload. Subjects and Methods This is a case control study which was carried out at Ain Shams university hospitals. Internal medicine department, Clinical hematology and stem cell transplantation unit, outpatient clinic and inpatient department, during the period between January 2022 and August 2022. Results Our study revealed significant decreased fertility between patients group and control group (P = 0.011). Transfusion-dependent beta-thalassemia males have a high proportion of fertility impairment and iron overload might contribute to disturbed sperm quality and testicular tissue injury. Such findings might explain the high prevalence of impaired fertility in transfusion-dependent beta- thalassemia patients which agrees with our results. Also our study revealed significant differences between non-transfusion dependent group and transfusion-dependent group regarding spleen size (cm) below costal margin which was increased in (TD group), transfusion per year (ml/yr)(which was increased in (TD group), virology (HIV, HCV,HBV) revealed that percentage of HCV+ve higher in (TD group) with (P &amp;lt; 0.001): Our study revealed significant difference between non-transfusion- dependent group and Transfusion-dependent group regarding percentage of patients of with positive HCV which was increased in Transfusion-dependent group (P = 0.021): In our study Iron chelation therapy was found to be given in a higher percentage of TD patients compared to non TD patients (P &amp;lt; 0.001). Also our study revealed significant difference regarding cardic function(EF%) between patients group and control group (EF found to be lower in patients group than controls) (P &amp;lt; 0.001): Also our study showed that Beclin-1 level is significantly higher in patients group compared to control group (P &amp;lt; 0.001). We couldn’t find previous studies of Beclin-1 level in thalassemia patients. We postulate that Beclin 1 could be used as a marker of worsening cardiac functions in thalassemia patients, but further studies should measure serial levels of Beclin 1 and correlate it with cardiac parameters over longer periods of follow up. Conclusion Beclin 1 level increased in Adult Beta Thalasemia patients and it can be used as a prognostic marker for Adult Beta Thalasemia patients.

Effect of total saponins from Panacis Majoris Rhizoma on proliferation, apoptosis and autophagy of human cervical carcinoma HeLa cells

This paper investigated the effect of total saponins from Rhizoma Panacis Majoris on the proliferation, apoptosis, and autophagy of human cervical carcinoma HeLa cells. The saponin content was detected by ultraviolet-visible spectrophotometry. Cell coun-ting kit-8(CCK-8) assay, 4,6-diamidino-2-phenylindole(DAPI) staining, and flow cytometry were used to detect the effects of total saponins of Panacis Majoris Rhizoma on cell viability, morphology, cell cycle and apoptosis of HeLa cells. Western blot was used to detect the expression of apoptosis-related proteins B cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), cleaved caspase-9, and cleaved caspase-3, autophagy-related proteins Beclin-1 and SQSTM1(p62), and the proteins related to the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR) and mitogen-activated protein kinase(MAPK) signaling pathways. It was found that the yield and saponin content of total saponins from Rhizoma Panacis Majoris were 6.3% and 78.3%, respectively. Total saponins from Rhizoma Panacis Majoris could significantly inhibit the proliferation(P&lt;0.001), effect the nuclear morphology, block the G_0/G_1 cycle, and induce cell apoptosis in HeLa cells with a concentration-dependent manner. In addition, total saponins from Rhizoma Panacis Majoris up-regulated the expression of pro-apoptotic proteins Bax, cleaved caspase-9, and cleaved caspase-3, and autophagy-related protein p62(P&lt;0.05), while down-regulated the expression of anti-apoptotic protein Bcl-2 and autophagy-related protein Beclin-1(P&lt;0.01). Total saponins from Rhizoma Panacis Majoris could promote the expression of p-p38/p38, p-Jun N-terminal kinase(JNK)/JNK, p-PI3K/PI3K, p-Akt/Akt, p-mTOR/mTOR proteins in PI3K/Akt/mTOR and MAPK signaling pathways(P&lt;0.05). In contrast, the effect on p-ERK/ERK expression was not obvious. Therefore, total saponins from Rhizoma Panacis Majoris may inhibit autophagy and promote apoptosis of HeLa cells through the activation of the PI3K/Akt/mTOR, c-JNK, and p38 MAPK signaling pathways, which indicates that total saponins from Rhizoma Panacis Majoris may have a potential role in cervical cancer treatment.

NAcM-OPT protects keratinocytes from H2O2-induced cell damage by promoting autophagy.

This study aimed to investigate the protective effect of NAcM-OPT, a small molecule inhibitor of defective in cullin neddylation 1 (DCN1), on H2O2-induced oxidative damage in keratinocytes. Immortalized human keratinocytes (HaCaT cells) were treated with NAcM-OPT and exposed to oxidative stress. CCK-8 assays were used to measure cell viability. The mGFP-RFP-LC3 dual fluorescent autophagy indicator system was utilized to evaluate changes in autophagic flux. Western blotting was used to measure the expression of the autophagy-related proteins LC3 and Beclin 1. Keratinocytes were treated with the autophagy activator rapamycin, and HaCaT cell supernatant was added to PIG1 cells (immortalized human melanocytes), followed by evaluation of tyrosinase (TYR) expression via qRT-PCR. NAcM-OPT increased cell viability and cell proliferation. Furthermore, this molecule promoted autophagic flux through increased expression of autophagy-related proteins under H2O2-induced oxidative stress. Additionally, rapamycin increased the mRNA levels of TYR in PIG1 cells. Moreover, NAcM-OPT alleviated mitochondrial damage, restored mitochondrial function, and upregulated the expression of NFE2L2, HO1, NQO1, and GCLM. Importantly, NAcM-OPT also increased epidermal thickness, follicle length, and melanin synthesis under oxidative stress in vivo. These findings suggest that NAcM-OPT may be a promising small molecule antioxidant drug for the treatment of vitiligo.

Progranulin Protects against Aspergillus fumigatus Keratitis by Attenuating the Inflammatory Response through Enhancing Autophagy.

Fungal keratitis (FK) is a severe corneal condition caused by pathogenic fungi and is associated with the virulence of fungi and an excessive tissue inflammatory response. Progranulin (PGRN), functioning as a multifunctional growth factor, exerts a pivotal influence on the regulation of inflammation and autophagy. The aim of our research was to analyze the role of PGRN in Aspergillus fumigatus (A. fumigatus) keratitis. We found that PGRN expression was increased in the mouse cornea with A. fumigatus keratitis. In our experiments, corneas of mice with FK were treated with 100 ng/mL of PGRN. In vitro, RAW 264.7 cells were treated with 10 ng/mL of PGRN before A. fumigatus stimulation. The findings suggested that PGRN effectively alleviated corneal edema and decreased the expression of pro-inflammatory cytokines in mice. In stimulated RAW 264.7 cells, PGRN treatment suppressed the expression of pro-inflammatory cytokines IL-6 and TNF-α but promoted the expression of the anti-inflammatory cytokines IL-10. PGRN treatment significantly upregulated the expression of autophagy-related proteins LC3, Beclin-1, and Atg-7. 3-Methyladenine (3-MA, autophagy inhibitor) reversed the regulation of inflammatory cytokines by PGRN. In addition, our study demonstrated that PGRN also enhanced phagocytosis in RAW 264.7 cells. In summary, PGRN attenuated the inflammatory response of A. fumigatus keratitis by increasing autophagy and enhanced the phagocytic activity of RAW 264.7 cells. This showed that PGRN had a protective effect on A. fumigatus keratitis.

Immunohistochemistry analysis of autophagy-related proteins Beclin-1, p62/SQSTM1, and LC3B in breast carcinoma progression to bone metastasis

Autophagy is a catabolic pathway involved both in tissue homeostasis and in cellular response to stress. The precise role of autophagy in cancer is still undefined and seems to depend on the tumor stage, appearing tumor-suppressive in physiological conditions and helpful to tumor progression in the established tumor. Here we analyzed by immunohistochemistry Beclin-1, p62, and LC3B, autophagic markers, in human specimens of normal breast, bone metastasis together with pair-matched invasive breast carcinoma of no special type (IBC-NST) as well as non-metastatic breast carcinoma, to disclose the possibility that they could be early prognostic indicators of the evolution of the disease toward the worst outcome. Different regions of metastatic carcinomas, i.e., areas adjacent to the tumor without signs of neoplastic growth, dysplastic lesions, and areas with invasive growth were considered. The pattern of autophagic parameters showed differences among the stages of breast carcinoma progression with a trend that indicated the activation of autophagic process in normal breast (Beclin-1 more elevated than p62), a pattern that was maintained in non-metastatic carcinoma. As the neoplasia proceeds with malignancy, the modification of the pattern of expression of autophagic markers (low ratio between Beclin-1 and p62) in areas of invasive growth of carcinomas suggested inhibition of the process. Of note, the parameters showed a different pattern in bone metastasis with respect to bone metastatic (bm)-IBC-NST, suggesting the reactivation of the autophagic process in the new growth site, helpful to the colonization. The course of autophagy markers during tumor progression could have a prognostic value towards bone metastasis and reveal different roles of the process in different phases of neoplastic growth. The understanding of the role of autophagy in bone metastasis could disclose new therapeutic targets to improve the conditions of patients.

Mechanism study of 6-shogaol alleviating cerebral ischemia/reperfusion injury by regulating microRNA-26a-5p/death-associated protein kinase 1

To investigate whether 6-shogaol (6-SH) alleviates oxygen-glucose deprivation/reoxygenation (OGD/R)-induced neuronal autophagy and calcium overload by promoting the expression of microRNA-26a-5p (miR-26a-5p) and inhibiting death-associated protein kinase 1 (DAPK1), and to explore its potential mechanisms. Primary cultured logarithmic growth phase mouse hippocampal neurons HT22 cells were taken and cell counting kit-8 (CCK-8) was used to detect cell viability, searching for the optimal concentration of Na2S2O4. HT22 cells were divided into blank control group (NC group), OGD/R group (sugar-free culture medium + 10 mmol/L Na2S2O4 treatment for 1.5 hours followed by normal culture medium for 4 hours), 6-SH intervention group (cultured with 10 μmol/L 6-SH for 4 hours after OGD), negative control inhibitor pretreatment group (transfected with negative control inhibitor for 48 hours followed by OGD, then cultured with 6-SH for 4 hours), and miR-26a-5p inhibitor pretreatment group (transfected with miR-26a-5p inhibitor for 48 hours followed by OGD, then cultured with 6-SH for 4 hours). Cell viability of each group was detected by CCK-8 method; cell ultrastructure was observed under transmission electron microscopy; real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the gene expressions of DAPK1 and miR-26a-5p; molecular docking were used to verify the interaction between 6-SH and miR-26a-5p; dual-luciferase assay was used to verify the targeting relationship between DAPK1 and miR-26a-5p; flow cytometry was used to determine the levels of intracellular Ca2+; Western blotting was used to detect the protein expressions of phosphorylated-glutamate receptor 2B (p-NMDAR2B) Ser1303, DAPK1, autophagy related protein Beclin1, light chain 3 (LC3), and p-DAPK1 Ser308; immunofluorescence was used to detect the expression of LC3 and Beclin1. The results of the CCK-8 assay showed that the cell viability of the 6-SH intervention group was significantly increased compared to the OGD/R group, while the cell viability of the miR-26a-5p inhibitor pretreatment group was significantly decreased compared to the 6-SH intervention group. Transmission electron microscopy revealed that the number of autophagosomes in the 6-SH intervention group was significantly reduced compared to the OGD/R group, while the number of autophagosomes in the miR-26a-5p inhibitor pretreatment group was significantly increased compared to the 6-SH intervention group. RT-qPCR results showed that compared with the OGD/R group, the expression of miR-26a-5p was significantly upregulated and the expression of DAPK1 mRNA was significantly downregulated in the 6-SH intervention group; compared with the 6-SH intervention group, the expression of miR-26a-5p was significantly downregulated and the expression of DAPK1 mRNA was significantly upregulated in the miR-26a-5p inhibitor pretreatment group. Molecular docking verified the interaction between 6-SH and miR-26a-5p. Dual-luciferase reporter gene assay showed that compared with the negative control group, mmu-miR-26a-5p significantly downregulated the luciferase expression of m-DAPK1-3UTR-WT, indicating a binding interaction between them. Flow cytometry results showed that compared with the OGD/R group, the level of intracellular Ca2+; was significantly decreased in the 6-SH intervention group; compared with the 6-SH intervention group, the level of Ca2+ was significantly increased in the miR-26a-5p inhibitor pretreatment group. Western blotting results showed that compared with the OGD/R group, the protein expressions of p-NMDAR2B Ser1303, DAPK1, Beclin1, and LC3 were significantly decreased in the 6-SH intervention group (p-NMDAR2B Ser1303/β-actin: 2.34±0.27 vs. 4.78±0.39, DAPK1/β-actin: 1.40±0.13 vs. 2.37±0.21, Beclin1/β-actin: 2.61±0.32 vs. 4.32±0.29, LC3/β-actin: 2.52±0.45 vs. 5.09±0.18, all P < 0.05), while the protein expression of p-DAPK1 Ser308 was significantly increased (p-DAPK1 Ser308/β-actin: 0.66±0.09 vs. 0.40±0.02, P < 0.05); compared with the 6-SH intervention group, the protein expressions of p-NMDAR2B Ser1303, DAPK1, Beclin1, and LC3 were significantly increased in the miR-26a-5p inhibitor pretreatment group (p-NMDAR2B Ser1303/β-actin: 4.08±0.14 vs. 2.34±0.27, DAPK1/β-actin: 1.96±0.15 vs. 1.40±0.13, Beclin1/β-actin: 3.92±0.31 vs. 2.61±0.32, LC3/β-actin: 4.33±0.33 vs. 2.52±0.45, all P < 0.05), while the expression of p-DAPK1 Ser308 protein was significantly decreased (p-DAPK1 Ser308/β-actin: 0.33±0.12 vs. 0.66±0.09, P < 0.05); immunofluorescence staining showed that compared with the OGD/R group, the fluorescence intensity of LC3 and Beclin1 was significantly decreased in the 6-SH intervention group; compared with the 6-SH intervention group, the fluorescence intensity of LC3 and Beclin1 was significantly increased in the miR-26a-5p inhibitor pretreatment group. 6-SH can alleviate neuronal damage by regulating miR-26a-5p/DAPK1 to reduce autophagy and calcium overload in cells.

Keratinocyte-derived small extracellular vesicles delay diabetic wound healing by triggering fibroblasts autophagy

Keratinocyte and fibroblast dysfunctions contribute to delayed healing of diabetic wounds. Small extracellular vesicles (sEV) are key mediators of intercellular communication and are involved in the pathogenesis of several diseases. Recent findings suggest that sEV derived from high-glucose-treated keratinocyte (HaCaT-HG-sEV) can transport LINC01435 to inhibit tube formation and migration of HUVECs, thereby delaying wound healing. This study aimed to elucidate sEV-related communication mechanisms between keratinocytes and fibroblasts during diabetic wound healing. HaCaT-HG-sEV treatment and LINC01435 overexpression significantly decreased fibroblast collagen level and migration ability but significantly increased fibroblast autophagy. However, treatment with an autophagy inhibitor suppressed LINC01435 overexpression-induced decrease in collagen levels in fibroblasts. In diabetic mice, HaCaT-HG-sEV treatment decreased collagen levels and increased the expression of the autophagy-related proteins Beclin-1 and LC3 at the wound site, thereby delaying wound healing. Conclusively, LINC01435 in keratinocyte-derived sEV activates fibroblast autophagy and reduces fibroblast collagen synthesis, leading to impaired diabetic wound healing.

Induction of Peroxiredoxin 1 by Hypoxia Promotes Cellular Autophagy and Cell Proliferation in Oral Leukoplakia via HIF-1α/BNIP3 Pathway.

Hypoxia is a key trigger in the transformation of oral leukoplakia into oral cancer. However, it is still too early to determine the role of hypoxia in the development of oral leukoplakia. Prx1, an antioxidant protein, upregulated by hypoxia, regulates cellular autophagy in leukoplakia. This study aimed to understand the mechanisms by which hypoxia induces Prx1 expression during autophagy in oral leukoplakia. We used an experimental model of tongue epithelial hyperplasia induced by 4-nitroquinoline-1-oxide (4NQO) and dysplastic oral keratinocytes. Prx1 knockdown DOK cells, Leuk-1 cells and control cells were harvested, and cell proliferation was assayed using the Cell Counting Kit-8. Several hypoxia and autophagy-related proteins were examined using quantitative real-time polymerase chain reaction, immunohistochemistry, immunofluorescence, and western blotting in cells and mouse tongue tissues. In addition, the ultrastructure of the cells was observed by transmission electron microscopy. Hypoxia induces cell proliferation, autophagic vesicles and the expression of Prx1, BNIP3, LC3II/I and Beclin-1 in DOK and Leuk-1 cells. However, these effects were all attenuated by Prx1 knockdown. Histologically, 4NQO induced epithelial hyperplasia in the tongue mucosa. The expression of proliferation marker PCNA, autophagy-related proteins LC3B and Beclin-1, as well as HIF-1α/BNIP3 was significantly lower in the tongue tissues of Prx1flox/flox:Cre+ mice compared with Prx1flox/flox mice. In Prx1flox/flox:Cre+ mice, an increased expression of HIF-1α/BNIP3, LC3B and Beclin-1 was detected in epithelial hyperplasia tongue tissues compared to normal tissues. The current study suggests that Prx1 may promotes cell proliferation and autophagy in oral leukoplakia cells via the HIF-1α/BNIP3 pathway.

Study of proanthocyanidin promotes osteogenic differentiation of human periodontal ligament stem cells through the transcription factor EB-induced autophagy-lysosome pathway

Objective: To investigate the mechanism of proanthocyanidin (PA) in regulating the osteogenic differentiation of human periodontal ligament stem cells (PDLSCs), and to explore the effects of PA on the expression and nuclear translocation of transcription factor EB (TFEB) and on the autophagy-lysosome pathway. Methods: PDLSCs were divided into control group and PA group, which were subjected to RNA sequencing analysis (RNA Seq) to detect differentially expressed genes. The osteogenic differentiation ability and autophagy level were observed by real-time fluorescence quantitative PCR (RT-qPCR) analysis, alkaline phosphatase (ALP) staining and transmission electron microscope (TEM), respectively. Scratch assay and Transwell assay were used to detect the migration ability of PDLSCs. Lysotracker and immunofluorescence staining were used to detect the biogenesis of lysosomes. The total protein expression of transcription factor EB (TFEB) as well as that in cytoplasm and nucleus were detected by Western blotting. Confocal laser scanning microscope (CLSM) was used to observe the nuclear translocation of TFEB. The PDLSCs were treated with small interfering RNA (siRNA) technology to knock down the expression levels of TFEB gene with or without PA treatment. Western blotting was used to analyze the expressions of autophagy-related proteins Beclin1 and microtubule-associated protein 1 light chain 3 (LC3B), as well as osteogenic-related proteins runt-related transcription factor 2 (RUNX2), ALP, and osteocalcin in PDLSCs. Results: Compared with the control group, the osteogenic-related and autophagy-related genes showed differential expression in PDLSCs after PA treatment (P<0.05). The mRNA expression levels of osteogenic-related genes RUNX2 (2.32±0.15) and collagen type Ⅰ alpha 1 (COL1α1) (1.80±0.18), as well as the autophagy related genes LC3B (1.87±0.08) and Beclin1 (1.63±0.08) were significantly increased in the PA group, compared with the control group (1.01±0.16, 1.00±0.10, 1.00±0.07, 1.00±0.06, respectively, all P<0.01). Compared with the control group, the PA group had higher ALP activity, and more autophagosomes and autophagolysosomes observed by TEM. PA promoted the migration of PDLSCs (P<0.05) and the increased number of lysosomes and the expression of lysosomal associated membrane protein 1 (LAMP1). In the PA group, the relative expression level of total TFEB protein (1.49±0.07) and the nuclear/cytoplasmic expression of TFEB protein (1.52±0.12) were significantly higher than the control group (1.00±0.11, 1.00±0.13, respectively) (t=6.43, P<0.01; t=5.07, P<0.01). The relative nuclear/cytoplasmic fluorescence intensity of TFEB in the PA group (0.79±0.09) was increased compared with the control group (0.11±0.08) (t=8.32, P<0.01). Knocking down TFEB significantly reduced the expression of TFEB (1.00±0.15 vs 0.64±0.04), LAMP1 (1.00±0.10 vs 0.69±0.09), Beclin1 (1.00±0.05 vs 0.60±0.05), and LC3B Ⅱ/Ⅰ (1.00±0.06 vs 0.73±0.07) in PDLSCs (P<0.05, P<0.05, P<0.01, P<0.01). When TFEB gene was knocked down, the expression levels of Beclin1 (1.05±0.11), LC3B Ⅱ/Ⅰ (1.02±0.09), RUNX2 (1.04±0.10), ALP (1.04±0.16), and osteocalcin (1.03±0.15) proteins were significantly decreased in the PA group compared with the pre-knockdown period (1.28±0.03, 1.44±0.11, 1.38±0.11, 1.62±0.11, 1.65±0.17, respectively) (P<0.05, P<0.01, P<0.05, P<0.01, and P<0.01, respectively). Conclusions: PA promotes the osteogenic differentiation of PDLSCs through inducing the expression and nuclear translocation of TFEB and activating the autophagy-lysosome pathway.

Quercetin Ameliorates Myocardial Injury in Diabetic Rats by Regulating Autophagy and Apoptosis through AMPK/mTOR Signaling Pathway.

A high-glucose environment is involved in the progression of diabetes mellitus (DM). This study aims to explore the regulatory effects of quercetin (QUE) on autophagy and apoptosis after myocardial injury in rats with DM. The type 2 DM rat models were constructed using low-dose streptozotocin (STZ) treatment combined with a high-carbohydrate (HC) diet in vivo. Compared with the control group, the body weight was decreased, whereas blood pressure, blood glucose, and the LVW/BW ratio were increased in the diabetic group. The results showed that the myocardial fibers were disordered in the diabetic group. Moreover, we found that the myocardial collagen fibers, PAS-positive cells, and apoptosis were increased, whereas the mitochondrial structure was destroyed and autophagic vacuoles were significantly reduced in the diabetic group compared with the control group. The expression levels of autophagy-related proteins LC3 and Beclin1 were decreased, whereas the expression levels of P62, Caspae-3, and Bax/Bcl-2 were increased in the diabetic group in vitro and in vivo. Moreover, QUE treatment alleviated the cellular oxidative stress reaction under high-glucose environments. The results of immunoprecipitation (IP) showed that the autophagy protein Beclin1 was bound to Bcl-2, and the binding capacity increased in the HG group, whereas it decreased after QUE treatment, suggesting that QUE inhibited the binding capacity between Beclin1 and Bcl-2, thus leading to the preservation of Beclin1-induced autophagy. In addition, the blood pressure, blood glucose, and cardiac function of rats were improved following QUE treatment. In conclusion, QUE suppressed diabetic myocardial injury and ameliorated cardiac function by regulating myocardial autophagy and inhibition of apoptosis in diabetes through the AMPK/mTOR signaling pathway.

Exosomes Derived from Mesenchymal Stem Cells Increase the Viability of Damaged Endometrial Cells via the miR-99b-5p/PCSK9 Axis.

The aim of this article was to investigate whether exosomes derived from bone marrow mesenchymal stem cells repair damaged endometrial stromal cells (EnSCs) through the miR-99b-5p/PCSK9 axis. Exosomes derived from bone marrow mesenchymal stem cells (BMSC-exos) were isolated by ultracentrifugation and characterized using transmission electron microscopy and nanoflow cytometry. A mifepristone-induced EnSC injury model was established in vitro, and the uptake of BMSC-exos was assessed. EnSCs were divided into three groups: the normal group (ctrl), EnSC injury group (model), and BMSC-exo treatment group. The effects of BMSC-exos on EnSC proliferation, apoptosis, and vascular endothelial growth factor (VEGF) expression were assessed by coculturing MSC-exos with endometrial cells. Furthermore, high-throughput sequencing was used to identify differentially expressed genes (DEGs). Through bioinformatics analysis, reverse transcription-quantitative polymerase chain reaction, western blotting, the CCK8 assay, immunohistochemistry, and dual-luciferase experiments, the potential mechanism by which BMSC-exos-derived miRNAs repair EnSC injury was studied. BMSC-exos expressed the marker proteins CD9 and CD63. Laser confocal microscopy showed that BMSC-exos could enter damaged EnSCs. In the BMSC-exos-EnSC coculture group compared with the model group, BMSC-exos significantly increased the proliferation of damaged EnSCs and inhibited cell apoptosis in a dose-dependent manner. The expression levels of Caspase-3, Caspase-9, Bax, and VEGF mRNA were significantly downregulated in the BMSC-exos-EnSC coculture group, whereas Bcl-2 expression was upregulated. We identified 28 overlapping DEGs between the model and ctrl groups and between the BMSC-exo and model groups. Transfection with miR-99b-5p mimics significantly decreased PCSK9 gene expression and inhibited the expression of the autophagy-related proteins Beclin-1 and LC3-II/I and apoptosis, thereby promoting EnSC proliferation. Transfection with a miR-99b-5p inhibitor showed the opposite effects. Beclin-1, LC3-II/I, and PCSK9 expression in the thin endometrium was significantly increased. miR-99b-5p promoted cell proliferation by targeting PCSK9. BMSC-exos promoted endometrial proliferation, and miR-99b-5p inhibited cell apoptosis and promoted EnSC proliferation by targeting PCSK9, providing a new target for the treatment of thin endometrium.

Allicin regulates Sestrin2 ubiquitination to affect macrophage autophagy and senescence, thus inhibiting the growth of hepatoma cells

BackgroundAllicin regulates macrophage autophagy and senescence, and inhibits hepatoma cell growth. This study investigated the mechanism by which allicin inhibits the growth of hepatoma cells. MethodsHepa1–6 mouse hepatoma cells were subcutaneously injected into C57BL/6 J mice to construct a tumor transplantation model. Macrophages were cultured with the supernatant of hepatoma cells to construct a cell model. The levels of mRNA and proteins and the level of Sestrin2 ubiquitination were measured by RTqPCR, immunofluorescence and Western blotting. The levels of autophagy-related factors and the activity of senescence-associated β-galactosidase were determined by kits, and protein stability was detected by cycloheximide (CHX) tracking. ResultsData analysis of clinical samples revealed that RBX1 was highly expressed in tumor tissues, while Sestrin2 was expressed at low levels in tumor tissues. Allicin can promote the expression of the autophagy-related proteins LC3 and Beclin-1 in tumor macrophages and inhibit the expression of the aging-related proteins p16 and p21, thus promoting autophagy in macrophages and inhibiting cell senescence. Moreover, allicin can inhibit the expression of RBX1, thereby reducing the ubiquitination of Sestrin2, enhancing the stability of Sestrin2, activating autophagy in tumor macrophages and inhibiting senescence. In addition, allicin treatment inhibited the proliferation and migration of hepatoma carcinoma cells cocultured with macrophages and significantly improved the development of liver cancer in mice. ConclusionAllicin can affect the autophagy of macrophages and restrain the growth of hepatoma cells by regulating the ubiquitination of Sestrin2.

Oxidative medicine and cellular longevity the role and mechanism of NCOA4 in ferroptosis induced by intestinal ischemia reperfusion

BackgroundFerroptosis is an iron-dependent and cystathione-non-dependent non-apoptotic cell death characterized by elevated intracellular free iron levels and reduced antioxidant capacity, leading to the accumulation of lipid peroxides. Nuclear receptor coactivator 4 (NCOA4) mediates ferritinophagy, increasing labile iron levels, which can result in oxidative damage. However, the specific mechanism of NCOA4-mediated ferritinophagy in intestinal ischemia–reperfusion and the underlying mechanisms have not been reported in detail. Object1. To investigate the role of NCOA4 in ferroptosis of intestinal epithelial cells induced by II/R injury in mouse. 2. To investigate the mechanism of action of NCOA4-induced ferroptosis. Methods1. Construct a mouse II/R injury model and detect ferroptosis related markers such as HE staining, immunohistochemistry, ELISA, and WB methods.2. Detect expression of NCOA4 in the intestine of mouse with II/R injury model and analyze its correlation with intestinal ferroptosis in mouse with II/R injury model.3. Construct an ischemia–reperfusion model at the cellular level through hypoxia and reoxygenation, and overexpress/knockdown NCOA4 to detect markers related to ferroptosis. Based on animal experimental results, analyze the correlation and mechanism of action between NCOA4 and intestinal epithelial ferroptosis induced by II/R injury in mouse. Results1. Ferroptosis occurred in the intestinal epithelial cells of II/R-injured mouse, and the expression of critical factors of ferroptosis, ACSL4, MDA and 15-LOX, was significantly increased, while the levels of GPX4 and GSH were significantly decreased. 2. The expression of NCOA4 in the intestinal epithelium of mouse with II/R injure was significantly increased, the expression of ferritin was significantly decreased, and the level of free ferrous ions was significantly increased; the expression of autophagy-related proteins LC3 and Beclin-1 protein was increased, and the expression of P62 was decreased, and these changes were reversed by autophagy inhibitors. 3. Knockdown of NCOA4 at the cellular level resulted in increased ferritin expression and decreased ferroptosis, and CO-IP experiments suggested that NCOA4 can bind to ferritin, which suggests that NCOA4 most likely mediates ferritinophagy to induce ferroptosis. ConclusionThis thesis explored the role of NCOA4 in II/R injury in mice and the mechanism of action. The research results suggest that NCOA4 can mediate ferritinophagy to induce ferroptosis during II/R injury. This experiment reveals the pathological mechanism of II/R injury and provides some scientific basis for the development of drugs for the treatment of II/R injury based on the purpose of alleviating ferroptosis.