Beclin-1 Protein Expression Research Articles

Comparison of biocompatibility of epoxy resin and bioceramic-based root canal sealers.

The purpose of this study is to compare the cytotoxicity of 2 bioceramic-based (Bioserra and AH Plus Bioceramic (Bio)) and 2 epoxy resin-based (Sealart and AH Plus) root canal sealers on the Saos-2 cell line. The cytotoxicity of the root canal sealers was determined using the MTT metabolic activity assay. The mRNA expression levels of Bcl-2, Beclin1, Cas-3, IL-6, LC3, MMP-9 and TNF-α genes were assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and the expression of Beclin1 and LC3 proteins were assessed using western blot (WB) method. The canal sealers' total antioxidant status (TAS), total oxidant status (TOS), and oxidative stress index (OSI) were measured using photometric kits. AH Plus Bio significantly increased metabolic activity compared to the 2 resin-containing sealers, but there was no difference with Bioserra. Sealart was clearly the most cytotoxic group. QRT-PCR analysis revealed that the mRNA expressions of Bcl-2, Beclin1, Cas-3, IL-6, LC3, and MMP-9 in the Bioserra group, Bcl-2, Cas-3, and IL-6 in the Sealart group, Beclin1, Cas-3, IL-6, and MMP-9 in the AH Plus group, and Bcl2 in the AH Plus Bio group were stimulated. The OSI results showed no significant difference between the root canal sealer groups. In the study, the best biocompatibility results in MTT, qRT-PCR, WB and OSI were in the AH Plus Bio group. Although Bioserra, the study's other bioceramic paste, was found to be biocompatible according to MTT and OSI measurements, it increased the expression of all genes in qRT-PCR except for TNF-α, and also increased LC3 protein levels in WB. Since periradicular tissues can be directly affected by the materials used in root canal treatment, the sealers with high biocompatibility are being developed. In vitro studies examining the biocompatibility of newly produced root canal sealers are guiding for clinical success.

Matrine regulates autophagy in ileal epithelial cells in a porcine circovirus type 2-infected murine model.

Porcine circovirus type 2 (PCV2) is an important pathogen that causes diarrhea in nursery and fattening pigs, resulting in huge economic losses for commercial pig farms. Protective efficacy of vaccines is compromised by mutations in pathogens. There is an urgent need to articulate the mechanism by which PCV2 destroys the host's intestinal mucosal barrier and to find effective therapeutic drugs. Increasing attention has been paid to the natural antiviral compounds extracted from traditional Chinese medicines. In the present study, we investigated the role of Matrine in mitigating PCV2-induced intestinal damage and enhancing autophagy as a potential therapeutic strategy in mice. A total of 40 female, specific-pathogen-free-grade Kunming mice were randomly divided into four groups with 10 mice in each group: control, PCV2 infection, Matrine treatment (40 mg/kg Matrine), and Ribavirin treatment (40 mg/kg Ribavirin). Except for the control group, all mice were injected intraperitoneally with 0.5 mL 105.4 50% tissue culture infectious dose (TCID50)/mL PCV2. While attenuating PCV2-induced downregulation of ZO-1 and occludin and restoring intestinal barrier function in a PCV2 Kunming mouse model, treatment with Matrine (40 mg/kg) attenuated ultrastructural damage and improved intestinal morphology. Mechanistically, Matrine reversed PCV2-induced autophagosome accumulation by inhibiting signal transducer and activator of transcription 3 (STAT3) phosphorylation and upregulating Beclin1 protein expression, thus resisting viral hijacking of enterocyte autophagy. Our findings demonstrate that Matrine may be a novel, potential antiviral agent against PCV2 by activating intestine cellular autophagy, which provides a new strategy for host-directed drug discovery.

Effect of fermentation on the constituents in the branches and leaves of Taxus media and non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is a prominent lung cancer disease worldwide. Currently, commonly used methods, such as surgery and radiotherapy, have significant side effects. Traditional Chinese medicine (TCM) has become a research hotspot because of its safe and effective characteristics. The branches and leaves of Taxus media are abundant in antitumor active compounds, and there has been no research conducted as yet regarding its anti-lung cancer molecular mechanism. The aim of this study is to investigate the antitumor activity of two samples before and after fermentation of T. media, and to research the molecular mechanism of its inhibitory effect on NSCLC. The chemical composition of pre-fermentation T. media (TM) and post-fermentation T. media qu (TMQ) were investigated using UHPLC-Q-Qrbitrap HRMS and high-performance liquid chromatography (HPLC). The anti-lung cancer activities of TM and TMQ were compared using an A549-induced tumor mouse model. An enzyme-linked immunosorbent assay (ELISA), hematoxylin and eosin (H&E) staining, immunohistochemistry, and immunofluorescence were used to determine the of TMQ mechanism of action. The results indicated that TM and TMQ contained 83 compounds, consisting primarily of flavonoids, organic acids, and taxanes. Both taxanes and flavonoids in TMQ were higher than that in TM. Both TM and TMQ effectively inhibited the tumor growth in non-small cell lung cancer (NSCLC), and the inhibition rate was greater in TMQ (57.24%) than in TM (49.62%). TMQ administration downregulated the tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and the glutathione (GSH) level and upregulated interferon-γ (IFN-γ), reactive oxygen species (ROS), and malondialdehyde (MDA) levels in the serum of tumor mice. TMQ treatment also increased the protein expression of Bax, Caspase-3, and Beclin-1 in tumor tissues. In contrast, the bcl-2, PI3K, Ki67, ULK1, and mTOR protein levels were suppressed by TMQ. Protein assay analyses reemphasized the superior antitumor effect of TMQ over TM. These cumulative findings demonstrated that the mechanism of action of TMQ was closely related to the activation of transcriptional misregulation in the cancer pathway that inhibited the cholinergic synaptic, AMPK, and PI3K/Akt/mTOR signaling pathways. This study demonstrated that fermentation increased the active ingredient contents and antitumor effects of T. media. In addition, post-fermentation TMQ was superior to TM as a herbal medicine for NSCLC treatment.

Extracorporeal Shock Wave and Melatonin Alleviate Joint Capsule Fibrosis after Knee Trauma in Rats by Regulating Autophagy.

Joint contracture is a common clinical problem affecting joint function. Capsule fibrosis plays a pivotal role in the progression of Joint contracture. Previous studies have reported that autophagy plays a regulatory role in visceral fibrosis. This study aimed to investigate whether extracorporeal shock wave therapy (ESWT) and melatonin alleviate joint capsule fibrosis in rats with extended knee joint contracture by regulating autophagy. A rat knee joint extension contracture model was made. Then, the rats were treated with ESWT, melatonin, ESWT + melatonin, or ESWT + melatonin + mTOR agonist for 4 weeks. The range of motion (ROM) of the knee joints was measured. Joint capsules were collected and observed for pathological changes by H&E and Masson staining. LC3B protein expression was evaluated by immunofluorescence staining. TGF-β1, MMP-1, Col-Ⅰ, Col-Ⅲ, LC3, ATG7, Beclin1, p-AMPK, p-mTOR and p-ULK1 protein expressions were measured by Western blot assay. The intervention groups had significantly improved ROM of knee joint (P < 0.05), significantly improved pathological changes on HE and Masson staining, significantly decreased protein expressions of TGF-β1, MMP-1, Col-Ⅰ, Col-Ⅲ and pmTOR (P < 0.05), and significantly increased protein expressions of LC3B, LC3II/LC3I ratio, ATG7, Beclin1, p-AMPK, and p-ULK1 (P < 0.05). Among these groups, the effects demonstrated by the ESWT + melatonin group were the best. With the mTOR agonist supplement, the therapeutic effects of extracorporeal shock waves and melatonin were significantly reduced. ESWT plus melatonin alleviated knee joint capsule fibrosis in rats by regulating autophagy.

Scutellarin ameliorates mitochondrial dysfunction and apoptosis in OGD/R-insulted HT22 cells through mitophagy induction

Scutellarin (Scu), a flavonoid from herbal Erigeron breviscapus (Vaniot) Hand-Mazz, exerts neuroprotective effects against cerebral ischemia. However, whether the effects of Scu are related to mitochondrial protection needs further investigation. In this study, we aimed to clarify the mechanisms of Scu against HT22 cells injury caused by oxygen-glucose deprivation and reperfusion (OGD/R). Our results proved that Scu significantly reduced the overload of intracellular reactive oxygen species (cellar ROS) and mitochondria reactive oxygen species (mito-ROS), ameliorating oxidative stress damage. TUNEL positive rate, Caspase-3 activity, and Cytochrome c (Cyto-c) expression remarkably decreased following Scu treatment. Meanwhile, Scu could maintain mitochondrial morphology and reverse ultrastructure changes. And mitochondrial membrane potential (MMP), oxygen consumption rate (OCR), adenosine triphosphate (ATP) production and Na+/K+-ATPase activity were obviously promoted. Additionally, Scu was found to stimulate mitophagy level by increasing the expression of LC3, Beclin1, PINK1 and Parkin proteins, as well as promoting the degradation of p62. More importantly, the regulatory effects of Scu on mito-ROS, MMP, ATP, Na+/K+-ATPase, cell viability and lactate dehydrogenase (LDH) were markedly limited by Mdivi-1 (a mitophagy inhibitor). Of note, the inhibitor also reversed Scu-mediated apoptosis suppression, evidenced by the diminished apoptosis rate, the down-regulated expression activities of Cyto-c, Bax and cleaved Caspase-3, as well as the elevated level of Bcl-2 protein. Collectively, Scu could improve mitochondrial dysfunction and inhibit apoptosis by stimulating mitophagy, thereby attenuating OGD/R-induced HT22 cells injury.

Effect of electroacupuncture on expression of autophagy-related proteins in nucleus pulposus of cervical spondylosis rabbits.

To observe the effects of electroacupuncture (EA) on the morphological changes of intervertebral disc tissues, apoptosis of nucleus pulposus cells, and the protein expression of Unc-51 like autophagy-activated kinase 1 (ULK1), homologous series of yeast Atg6 (Beclin1), and light chain protease complication 3 type (LC3) in nucleus pulposus tissue of cervical spondylosis rabbits, so as to explore the role of cellular autophagy in EA treatment of cervical spondylosis. A total of 24 New Zealand white rabbits were randomly divided into blank, model and EA groups, with 8 rabbits in each group. In the EA group, both sides of the cervical (C)3-C6 "Jiaji" (EX-B2) were stimulated by EA (2 Hz/100 Hz, 1 mA) for 25 min, once daily for 5 days in a course, with a 2-day interval between courses, totaling 4 treatment courses. X-ray was used to assess cervical spine radiographic changes and evaluate radiographic scores；transmission electron microscopy was used to observe ultrastructural changes in nucleus pulposus cells；HE staining was used to observe morphological changes of intervertebral disc tissues and conduct pathological scoring；TUNEL staining was used to observe apoptosis rate of nucleus pulposus cells；Western blot was performed to detect protein expression levels of ULK1, Beclin1, and LC3 in nucleus pulposus tissue. Compared with the blank group, rabbits in the model group showed significantly higher cervical spine radiographic scores (P<0.01), higher pathological scores of intervertebral disc tissues (P<0.05), increased apoptosis rate of nucleus pulposus cells (P<0.01), and decreased expression levels of ULK1, Beclin1, and LC3Ⅱ proteins in nucleus pulposus tissue (P<0.05). Compared with the model group, the EA group showed significantly lower pathological scores of intervertebral discs (P<0.05), lower apoptosis rate of nucleus pulposus cells (P<0.01), and higher protein expression levels of ULK1, Beclin1, and LC3Ⅱ in nucleus pulposus tissue (P<0.01). Rabbits in the blank control group exhibited generally normal organelle structures in nucleus pulposus tissues with few autophagic vacuoles, indicative of early stages of autophagy；while those in the model group showed disrupted organelle structures with cytoplasmic condensation and those in the EA group exhibited autophagosomes with double-membrane structures in nucleus pulposus tissues. EA promotes the expression of ULK1, Beclin1, and LC3Ⅱ proteins in nucleus pulposus tissues, reduces apoptosis of nucleus pulposus cells, and improves intervertebral disc degeneration.

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.

Tissue factor regulates autophagy in pulmonary artery endothelial cells from chronic thromboembolic pulmonary hypertension rats via the p38 MAPK-FoxO1 pathway

AimsTo detect the expression of autophagy components, p38 MAPK (p38) and phosphorylated forkhead box transcription factor O-1 (pFoxO1) in pulmonary vascular endothelial cells of chronic thromboembolic pulmonary hypertension (CTEPH) rats and to investigate the possible mechanism through which tissue factor (TF) regulates autophagy.MethodsPulmonary artery endothelial cells (PAECs) were isolated from CTEPH (CTEPH group) and healthy rats (control group (ctrl group)) which were cocultured with TF at different time points including 12 h, 24 h, 48 h and doses including 0 nM,10 nM, 100 nM, 1µM, 10µM, 100µM and cocultured with TFPI at 48 h including 0 nM, 2.5 nM, 5 nM. The expression of forkhead box transcription factor O-1 (FoxO1), pFoxO1, p38, Beclin-1 and LC3B in PAECs was measured. Coimmunoprecipitation (co-IP) assays were used to detect the interaction between FoxO1 and LC3.ResultsThe protein expression of p-FoxO1/FoxO1 was significantly lower in the CTEPH groups (cocultured with TF from 0 nM to 100 µM) than in the ctrl group at 12 h, 24 h, and 48 h (P < 0.05) and was significantly lower in the CTEPH groups (cocultured with TFPI from 0 nM to 5 nM) than in the ctrl group at 48 h (P < 0.05). The protein expression of p38 in the CTEPH groups treated with 0 nM, 10 nM, 100 nM or 1 µM TF for 48 h significantly increased than ctrl groups (P < 0.05) and was significantly increased in the CTEPH groups (cocultured with TFPI concentration from 0 nM to 5 nM) than in the ctrl group at 48 h (P < 0.05). The protein expression of Beclin1 at the same concentration (cocultured with TF from 0 nM to 100 µM) was significantly lower in the CTEPH groups than ctrl groups after 24 h and 48 h (P < 0.05) and was significantly decreased in the CTEPH groups (cocultured with TFPI concentration from 2.5 nM to 5 nM) than in the ctrl group at 48 h (P < 0.05). The protein expression of LC3-II/LC3-I at the same concentration (cocultured with TF 0 nM, 1 µM, 10 µM, and 100 µM) was significantly lower in the CTEPH than in the ctrl groups after 12 h (P < 0.05) and was significantly lower in the CTEPH groups (cocultured with TFPI concentration from 0 nM to 5 nM) than in the ctrl group at 48 h (P < 0.05). There were close interactions between FoxO1 and LC3 in the control and CTEPH groups at different doses and time points.ConclusionThe autophagic activity of PAECs from CTEPH rats was disrupted. TF, FoxO1 and p38 MAPK play key roles in the autophagic activity of PAECs. TF may regulate autophagic activity through the p38 MAPK-FoxO1 pathway.

Melatonin alleviates autophagy in cortical neurons of neonatal rats with hypoxic- ischemic brain damage via the PI3K/AKT pathway

To observe the effects of melatonin on autophagy in cortical neurons of neonatal rats with hypoxic-ischemic brain damage (HIBD) and to explore its mechanisms via the PI3K/AKT signaling pathway, aiming to provide a basis for the clinical application of melatonin. Seven-day-old Sprague-Dawley neonatal rats were randomly divided into a sham operation group, an HIBD group, and a melatonin group (n=9 each). The neonatal rat HIBD model was established using the classic Rice-Vannucci method. Neuronal morphology in the neonatal rat cerebral cortex was observed with hematoxylin-eosin staining and Nissl staining. Autophagy-related protein levels of microtubule-associated protein 1 light chain 3 (LC3) and Beclin-1 were detected by immunofluorescence staining and Western blot analysis. Phosphorylated phosphoinositide 3-kinase (p-PI3K) and phosphorylated protein kinase B (p-AKT) protein expression levels were measured by immunohistochemistry and Western blot. The correlation between autophagy and the PI3K pathway in the melatonin group and the HIBD group was analyzed using Pearson correlation analysis. Twenty-four hours post-modeling, neurons in the sham operation group displayed normal size and orderly arrangement. In contrast, neurons in the HIBD group showed swelling and disorderly arrangement, while those in the melatonin group had relatively normal morphology and more orderly arrangement. Nissl bodies were normal in the sham operation group but distorted in the HIBD group; however, they remained relatively intact in the melatonin group. The average fluorescence intensity of LC3 and Beclin-1 was higher in the HIBD group compared to the sham operation group, but was reduced in the melatonin group compared to the HIBD group (P<0.05). The number of p-PI3K+ and p-AKT+ cells decreased in the HIBD group compared to the sham operation group but increased in the melatonin group compared to the HIBD group (P<0.05). LC3 and Beclin-1 protein expression levels were higher, and p-PI3K and p-AKT levels were lower in the HIBD group compared to the sham operation group (P<0.05); however, in the melatonin group, LC3 and Beclin-1 levels decreased, and p-PI3K and p-AKT increased compared to the HIBD group (P<0.05). The correlation analysis results showed that the difference of the mean fluorescence intensity of LC3 and Beclin-1 protein in the injured cerebral cortex between the melatonin and HIBD groups was negatively correlated with the difference of the number of p-PI3K+ and p-AKT+ cells between the two groups (P<0.05). Melatonin can inhibit excessive autophagy in cortical neurons of neonatal rats with HIBD, thereby alleviating HIBD. This mechanism is associated with the PI3K/AKT pathway.

Effect of electroacupuncture on autophagy of ovarian granulosa cells in rats with premature ovarian insufficiency

To observe the effects of electroacupuncture (EA) on the autophagy of ovarian granulosa cells in rats with premature ovarian insufficiency (POI), and explore the mechanism of EA in improving POI. Thirty-two female SD rats were randomly divided into a blank group (n=8) and a model making group (n=24). The rats in the model making group were injected intraperitoneally with cyclophosphamide for 15 days to establish the POI model (the dosage on the 1st day was 50 mg/kg, and 8 mg/kg from the 2nd day to 15th day). The successfully modeled rats were then randomly divided into a model group, an EA group, and an estradiol (E2) group, with 8 rats in each group. Rats in the EA group received EA at bilateral "Gongsun" (SP 4) with continuous wave, frequency of 2 Hz, and current intensity of 0.1 to 1 mA, 20 min per treatment, once daily for 14 days. Rats in the E2 group were administered with E2 (0.01 mg/mL) by gavage (10 mL/kg), once daily for 14 days. The changes in estrous cycle were observed by rapid Giemsa staining before and after modeling. After intervention, ovarian tissue morphology was observed by HE staining; serum levels of follicle-stimulating hormone (FSH), E2, anti-Mullerian hormone (AMH), and inhibin B (INHB) were detected by ELISA; immunofluorescence staining was used to observe the expression of p62 in ovarian granulosa cells; the ultrastructure of ovarian granulosa cells was observed by transmission electron microscopy, and the number of autophagosomes and autolysosomes was compared; Western blot and real-time fluorescence quantitative PCR were used to detect the protein and mRNA expression of p62, Beclin-1, and microtubule-associated protein 1A/1B-light chain 3 (LC3) in ovarian tissue. The results of vaginal smears in the blank group showed regular cyclical changes; the rats in the model group showed prolonged estrous cycle or cycle arrest, mostly in proestrus or metestrus, with overall ovarian atrophy, disordered structure, and decreased granulosa cells. Compared with the blank group, rats in the model group showed increased serum FSH level (P<0.01), decreased serum levels of E2, AMH, and INHB (P<0.01), decreased positive expression of p62 in ovarian granulosa cells (P<0.01), with obvious swelling of ovarian granulosa cells, mild to moderate swelling of mitochondria, slight expansion of rough endoplasmic reticulum, and hypertrophy of Golgi apparatus; the number of autophagosomes and autolysosomes in the ovaries was increased (P<0.01), the expression of p62 protein and mRNA was decreased (P<0.01), and the expression of Beclin-1 and LC3 protein and mRNA in ovarian tissue was increased (P<0.01). Compared with the model group, rats in the EA group and the E2 group showed decreased serum FSH levels (P<0.01), increased levels of E2, AMH, and INHB (P<0.01), increased positive expression of p62 in ovarian granulosa cells (P<0.01), alleviated degree of ovarian granulosa cell damage, with relatively intact organelle morphology, and decreased number of autophagosomes and autolysosomes in the ovaries (P<0.01); the rats also showed increased expression of p62 protein and mRNA (P<0.01), and decreased expression of Beclin-1 and LC3 protein and mRNA (P<0.01) in ovarian tissue. EA at "Gongsun" (SP 4) could improve ovarian reserve function in POI rats by reducing the number of autophagosomes and autolysosomes, up-regulating p62 expression, and down-regulating Beclin-1 and LC3 expression, thus inhibiting autophagy of ovarian granulosa cells, and regulating the serum levels of FSH, E2, AMH, and INHB.

Baicalin reduced injury of and autophagy-related gene expression in RAW264.7 cells infected with H6N6 avian influenza virus

In the present study, we investigated whether baicalin could reduce the damage caused to RAW264.7 cells following infection with H6N6 avian influenza virus. In addition, we studied the expression of autophagy-related genes. The morphological changes in cells were observed by hematoxylin and eosin (H&E) staining, and the inflammatory factors in the cell supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Transmission electron microscopy (TEM) was used to detect the levels of RAW264.7 autophagosomes, and western blotting and immunofluorescence were used to detect the protein expression of autophagy marker LC3. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was used to detect the mRNA transcription levels of autophagy key factors. The results showed that different doses of baicalin significantly reduced the H6N6 virus-induced damage of RAW264.7 cells. The contents of interleukin (IL)-1β, IL-2, IL-6, and tumor necrosis factor (TNF)-α in the cell supernatant significantly decreased. In addition, the protein expression of LC3 and Beclin-1, ATG12, ATG5 the mRNA levels were significantly decreased. This study showed that baicalin can reduce cell damage and affect the H6N6-induced autophagy level of RAW264.7 cells.

Galectin-receptor interaction: a key player in liver fibrosis induced by Schistosoma japonicum infection

BackgroundSchistosoma japonicum eggs lodge in the liver and induce a fibrotic granulomatous immune response in the liver of host. Galectin 3 (Gal-3) is a protein implicated in fibrosis in multiple organs. However, the pathology and molecular mechanisms promoting hepatic granuloma formation remain poorly understood.MethodsTo investigate the effect of blocking galectin-receptor interactions by α-lactose on liver immunopathology in mice with S. japonicum infection, C57BL/6 mice were infected with S. japonicum and alpha (α)-lactose was intraperitoneally injected to block the interactions of galectins and their receptors.ResultsCompared with S. japonicum-infected mice, there were significantly decreased Gal-3 mRNA and protein expression levels, decreased intensity of Gal-3 fluorescence in the liver, decreased serum ALT and AST levels, decreased egg numbers of S. japonicum in the liver section, attenuated hepatic and spleen pathology, and alleviated liver fibrosis accompanied with decreased protein expression levels of fibrosis markers [α-smooth muscle actin (α-SMA), collagen I, and collagen IV] in the liver of S. japonicum-infected mice blocked galectin-receptor interactions with hematoxylin-eosin staining, Masson’s trichrome staining, immunohistochemistry, or Western blot analysis. Compared with S. japonicum-infected mice, blocking galectin-receptor interactions led to increased eosinophil infiltration and higher eosinophil cationic protein (ECP) expression in the liver, accompanied by increased mRNA levels of eosinophil granule proteins [ECP and eosinophil peroxidase (EPO)], IL-5, CCL11, and CCR3 in the liver and decreased mRNA levels of Gal-3 and M2 macrophage cytokines (TGF-β, IL-10, and IL-4) in the liver and spleen by using quantitative real-time reverse transcription-polymerase chain reaction. In addition, there were increased Beclin1 protein expression and protein expression ratio of LC3B-II/LC3B-I and decreased p62 protein expression and protein expression ratios of phospho-mTOR/mTOR and phospho-AKT/AKT by Western blot; increased double-labeled F4/80+/LC3B+ cells by immunofluorescence staining; increased M1 macrophage polarization in the liver of S. japonicum-infected mice blocked galectin-receptor interactions by flow cytometric analysis and immunofluorescence staining.ConclusionsOur data found that blockage of galectin-receptor interactions downregulated Gal-3, which in turn led to reduced liver functional damage, elevated liver eosinophil recruitment, promoted macrophage autophagy through the Akt/mTOR signaling pathway, and alleviated liver pathology and fibrosis. Therefore, Gal-3 plays a pivotal role during S. japonicum infection and could be a target of pharmacologic potential for liver fibrosis induced by S. japonicum infection.Graphical

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.

Pesticide-Induced Alterations in Locomotor Activity, Anxiety, and Depression-like Behavior Are Mediated through Oxidative Stress-Related Autophagy: A Persistent Developmental Study in Mice.

Chlorpyrifos (CPF), dichlorvos (DDV), and cypermethrin (CP), as commonly used pesticides, have been implicated in inducing neuropsychiatric disorders, such as anxiety, depression-like behaviors, and locomotor activity impairment. However, the exact molecular mechanisms of these adverse effects, particularly in both sexes and their next-generation effects, remain unclear. In this study, we conducted behavioral analysis, along with cellular assays (monodansylcadaverine staining) and molecular investigations (qRT-PCR and western blotting of mTOR, P62, and Beclin-1) to clear the potential role of autophagy in pesticide-induced behavioral alterations. For this purpose, 42 adult female and 21 male inbred ICR mice (F0) were distributed into seven groups. Maternal mice (F0) and 112 F1 offspring were exposed to 0.5 and 1 ppm of CPF, DDV, and CP through drinking water. F1 male and female animals were studied to assess the sex-specific effects of pesticides on brain tissue. Our findings revealed pronounced anxiogenic effects and impaired locomotor activity in mice. F1 males exposed to CPF (1 ppm) exhibited significantly elevated depression-like behaviors compared to other groups. Moreover, pesticide exposure reduced mTOR and P62 levels, while enhancing the Beclin-1 gene and protein expression. These changes in autophagy signaling pathways, coupled with oxidative and neurogenic damage in the cerebral cortex and hippocampus, potentially contribute to heightened locomotor activity, anxiety, and depression-like behaviors following pesticide exposure. This study underscores the substantial impact of pesticides on both physiological and behavioral aspects, emphasizing the necessity for comprehensive assessments and regulatory considerations for pesticide use. Additionally, the identification of sex-specific responses presents a crucial dimension for pharmaceutical sciences, highlighting the need for tailored therapeutic interventions and further research in this field.

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.

Gender and age peculiarities of autophagy in men and women induced by a comprehensive wellness program: a prospective observational study

Background. Autophagy refers to an evolutionary physiological mechanism of cell self-purification. The use of its positive effects in regenerative and preventive medicine implies this process to be investigated under the conditions of various therapeutic factors.Objective. To investigate the influence of gender, age and somatic-morphological factors on the autophagy processes induced by a comprehensive wellness program as expressed by the marker protein Beclin-1.Methods. A prospective observational study involved 107 patients to be monitored before and after comprehensive wellness procedures. All study participants underwent a wellness program in the clinic of OOO Centr Zdorovie (Maykop, Russia). The study was conducted in the periods of September‒December 2020 and January 2021. The study involved measuring the concentration of Beclin-1 protein in the blood before and 12 days after the wellness procedures. In order to characterize the degree of activation of the autophagy process, the index “delta-Beclin-1” was introduced (difference between the initial level of Beclin-1 protein concentration and the level registered after 12 days). The autophagy response was evaluated in terms of its upward or downward direction (positive/negative delta- Beclin-1 value, respectively). The effect of the comprehensive wellness procedures on the functional systems of the organism was determined in accordance with the laboratory tests (complete blood count, lipid profile), morphometric evaluation, and the assessment of body composition by impedance monitoring. The obtained data were processed using SPSS Statistics 26.0 (IBM, USA).Results. Monitoring of Beclin-1 protein concentration revealed that basic autophagy activity is age-specific and significantly lower in individuals over 60 years as compared to both individuals under 60 years and healthy persons. This correlation with age appears more pronounced in obese individuals (r = -0.59). The basic activity of autophagy in men was 30% lower than in women, however, after a set of wellness procedures the mentioned response in men increased by 7%. The study found that not all patients responded unidirectionally to the comprehensive wellness procedures: 49 out of 77 patients showed an increase in Beclin-1 protein concentration, while 28 — a decrease. Patients who participated in the wellness program demonstrated negative correlation between the level of Beclin-1 protein expression and age, while those responding with an increase in Beclin-1 protein level — with body mass index, weight and fat mass. The source of autophagy activity in men under 60 years can refer to fat mass, and in women — lean body mass, especially in the mature age of period II (36‒60 years).Conclusion. The factors of basic autophagy and autophagy induced by the complex of wellness procedures include age, gender and body weight. These factors obtain different significance at different age periods. A comprehensive wellness program can serve as an alternative to existing pharmacological methods for activating autophagy in humans.

Exercise promotes irisin expression to ameliorate renal injury in type 2 diabetic rats

To investigate the role of irisin in exercise-induced improvement of renal function in type 2 diabetic rats. Forty male SD rats aged 4-6 weeks were randomized into normal control group, type 2 diabetes mellitus model group, diabetic exercise (DE) group and diabetic irisin (DI) group (n=8). The rats in DE group were trained with treadmill running for 8 weeks, and those in DI group were given scheduled irisin injections for 8 weeks. After the treatments, blood biochemical parameters of the rats were examined, and renal histopathology was observed with HE, Masson and PAS staining. Western blotting was used to detect the protein expression levels in the rats'kidneys. The diabetic rats showed significantly increased levels of fasting insulin, total cholesterol, triglyceride, serum creatinine and blood urea nitrogen with lowered serum irisin level (all P < 0.05). Compared with those in DM group, total cholesterol, triglyceride, serum creatinine and blood urea nitrogen levels were decreased and serum irisin levels were increased in both DE and DI groups (all P < 0.05). The rats in DM group showed obvious structural disorders and collagen fiber deposition in the kidneys, which were significantly improved in DE group and DI group. Both regular exercises and irisin injections significantly ameliorated the reduction of FNDC5, LC3-II/I, Atg7, Beclin-1, p-AMPK, AMPK and SIRT1 protein expressions and lowered of p62 protein expression in the kidneys of the diabetic rats (all P < 0.05). Both exercise and exogenous irisin treatment improve nephropathy in type 2 diabetic rats possibly due to irisin-mediated activation of the AMPK/SIRT1 pathway in the kidneys to promote renal autophagy.

Exploring the Mechanism of KLF15 on the Biological Activity and Autophagy of Gastric Cancer Cells based on PI3K/Akt/Mtor Signaling Pathway.

To explore the mechanism of KLF15 on the biological activity and autophagy of gastric cancer cells based on the PI3K/Akt/mTOR signaling pathway. The gastric cancer AGS cells were divided into the Con group, pcDNANC group, pcDNA-KLF15 group, LY294002 group and IGF-1 group. RT-PCR was used to detect the expression of KLF15 in human gastric mucosal cells and gastric cancer cells; MTT method to detect cell proliferation; Transwell method to detect cell invasion; flow cytometry to detect cell apoptosis; Western blotting to detect PI3K, Akt, mTOR in cells, LC3, Beclin1, p62 protein expression.P<0.05 was used to indicate statistical significance. Compared with the human gastric mucosal cell line GES-1 cells, the expression of KLF15 in human gastric cancer cell lines MKN-28, MFC, SCG-7901 and AGS cells was significantly decreased, And the expression of KLF15 in AGS cells, was the lowest (P=0.006). Compared with the Con group, The expression of KLF15 in the cells of the PCDNA-KLF15 group was significantly increased (P=0.018); There was no significant difference in the expression of KLF15 between the Con group and the PCDNA-NC group (P=0.225). Compared with the Con group, the proliferation and invasion abilities of the cells in the pcDNA-KLF15 group were significantly reduced, And the apoptosis ability was significantly increased (P=0.019). The ratio of LC3II/LC31 and the expression of Beclin1 Protein in the control group were significantly higher than those in the Con group (P=0.017). Overexpression of KLF15 can inhibit the proliferation and invasion of Gastric cancer cells and promote cell apoptosis and autophagy, and its mechanism may be related to the regulation of the PI3K/Akt/mTOR signaling pathway.

The potential anticancer effect of bee venom in combination with sorafenib against HepG2 cell lines via induction of apoptosis and autophagy candidate genes

Abstract Background Hepatocellular carcinoma (HCC) is a severe threat and a main reason for cancer-related deaths around the world. Drug resistance to sorafenib (Sorf), the effective HCC first-line therapy, is very common. A number of natural compounds, notably bee venom (BV), have been claimed to show a great impact against cancer when administered on its own or in conjunction with chemotherapy. Thus, this study aimed to investigate the anticancer effect of BV alone and/or combined with Sorf on HepG2 liver cancer cell lines. Methods Both mRNA and protein expressions of Bax, Bcl-2 and Beclin-1 were investigated by quantitative real-time PCR (qPCR) and western blot respectively, to examine the apoptotic and autophagic regulatory effects of BV and Sorf single treatments plus BV/Sorf combination on HepG2 cell lines. Results Our findings showed that BV and Sorf had considerable dose-dependent anti-proliferative effects on HepG2 cells whether administered alone or in combination, with the greatest impact for the combined therapies. Single BV and Sorf treatments showed IC50 of 93.21 and 7.28 μg/ml respectively, while combined treatment showed IC50 of 6.73 μg/ml BV + 6.73 μg/ml Sorf. Moreover, both the pro-apoptotic gene Bax and the autophagy-related gene Beclin-1 showed significant up-regulation in their mRNA expression, while the anti-apoptotic Bcl-2 mRNA gene expression showed significant down-regulation after BV/Sorf treatment as compared to either BV or Sorf single treatment. These qPCR results were further confirmed by western blot. Conclusions These findings indicate that BV synergistically potentiates the anticancer effect of Sorf on HepG2 cells through induction of apoptotic and autophagic machineries.

Effect of Shikonin on Autophagy and Apoptosis of Human Promyelocytic Leukemia Cells

To explore the effect of shikonin on autophagy and apoptosis of human promyelocytic leukemia cells and its possible mechanism. Human promyelocytic leukemia cells NB4 in the logarithmic growth phase were divided into control group (untreated NB4 cells), shikonin group (0.3 µmol/L shikonin treatment), 740Y-P group (15 µmol/L PI3K/Akt/mTOR pathway activator 740Y-P treatment), shikonin+740Y-P group (0.3 µmol/L shikonin and 15 µmol/L 740Y-P co-treatment), after 24 hours of treatment, the cells were used for subsequent experiments. CCK-8 method was used to detect cell viability, monodansylcadaverine (MDC) staining to detect the aggregation of autophagic vesicles, flow cytometry to detect cell apoptosis, and Western blot to detect the expression of Beclin1, LC3, p62, Bax, cleaved caspase-3, Bcl-2 and PI3K/Akt/mTOR pathway related proteins. Compared with the control group, the purple punctate fluorescence intensity, apoptosis rate, Beclin1, LC3-Ⅱ/LC3-Ⅰ, cleaved caspase-3, and Bax protein expression in NB4 cells were increased in the shikonin group, while OD450 value (24, 48 h) and the expressions of Bcl-2 and p62 proteins were decreased (all P < 0.05). Compared with the control group, the purple punctate fluorescence intensity, apoptosis rate, Beclin1, LC3-Ⅱ/LC3-Ⅰ, cleaved caspase-3, and Bax protein expression in NB4 cells were decreased, while OD450 value (24, 48 h) and the expressions of Bcl-2 and p62 proteins were increased in the 740Y-P group (all P < 0.05). Compared with the shikonin group, the purple punctate fluorescence intensity, apoptosis rate, Beclin1, LC3-Ⅱ/LC3-Ⅰ, cleaved caspase-3, and Bax protein expression in NB4 cells were decreased, while OD450 value (24, 48 h) and the expressions of Bcl-2 and p62 proteins were increased in the shikonin+740Y-P group (all P < 0.05). Compared with the control group, the expression of PI3K/Akt/mTOR pathway related proteins p-PI3K, p-Akt, and p-mTOR in NB4 cells were significantly decreased in the shikonin group, while those in the 740Y-P group were increased (all P < 0.05). Compared with the shikonin group, the expressions of p-PI3K, p-Akt, and p-mTOR proteins in NB4 cells were significantly increased in the shikonin+740Y-P group (all P < 0.05). Shikonin may promote autophagy and apoptosis of NB4 cells by inhibiting PI3K/Akt/mTOR pathway.