Beclin-1 Protein Expression Research Articles

CCND1-Induced Autophagy Contributes to Lymph Node Metastasis in Endometrial Cancer.

Endometrial cancer with lymph node metastasis shows poor prognosis, while the biomarker to predict the metastasis is lacking. The relative mRNA or protein expression of cyclin D1 (CCND1) and autophagy-related molecules were detected in real-time PCR and Western blot. Correlation analysis was applied to identify any significant patterns, and receiver operating characteristics (ROC) was performed to assess the prediction value. CCND1 vector was transfected in Ishikawa (ISK) cells, and the relative expression of autophagy-related molecules was detected with Western blot. CCND1 was overexpressed in endometrial cancer and correlated with lymph node metastasis. ROC analysis found that CCND1 had a predictive value to discriminate tumors from normal tissues (cut off=1.455; sensitivity, 71%; specificity, 84%; area under curve (AUC) 0.82; p<0.001) and had a predictive value to indicate metastasis (cut off=1.871; sensitivity, 54.17%; specificity, 75%; AUC 0.674; p=0.003). Increased BECLIN1 (r=0.39, p<0.001) and ATG5 (r=0.41, p<0.001) expression were positively correlated to CCND1. On the other hand, the relative protein expression of CCND1, BECLIN1, ATG5, ATG7, and LC3 I/II were also increased in tumor tissues. CCND1 overexpressed ISK cells showed upregulated BECLIN1, ATG5, ATG7, and LC3 I/II expression. CCND1 promoted autophagy may contribute to lymph node metastasis in endometrial cancer.

MicroRNA-146a-5p alleviates the pathogenesis of osteoarthritis by inhibiting SDF-1/CXCR4-induced chondrocyte autophagy.

SDF-1/CXCR4 signaling promotes osteoarthritis (OA) development. CXCR4 is a potential target of miR-146a-5p. This study investigated the therapeutic role and the underlying mechanism of miR-146a-5p in OA. Human primary chondrocytes C28/I2 were stimulated with SDF-1. Cell viability and LDH release were examined. Chondrocyte autophagy was assessed using Western blot analysis, ptfLC3 transfection, and transmission electron microscopy. MiR-146a-5p mimics were transfected into C28/I2 cells to investigate the role of miR-146a-5p in SDF-1/CXCR4-induced autophagy of chondrocytes. An SDF-1-induced rabbit OA model was established to investigate the therapeutic role of miR-146a-5p in OA. Histological staining was performed to observe the morphology of osteochondral tissue. SDF-1/CXCR4 signaling promoted autophagy in C28/I2 cells, as demonstrated by increased LC3-II protein expression and autophagic flux induced by SDF-1. SDF-1 treatment significantly inhibited cell proliferation while promoting necrosis and autophagosome formation in C28/I2 cells. In the presence of SDF-1, miR-146a-5p overexpression in C28/I2 cells suppressed CXCR4 mRNA expression, LC3-II and Beclin-1 protein expression, LDH release, and autophagic flux. In addition, SDF-1 increased the autophagy of chondrocytes in rabbits and promoted the development of OA. Compared with the negative control, miR-146a-5p significantly reduced the morphological abnormalities of the rabbit cartilage that were induced by SDF-1, as well as the number of LC3-II-positive cells, protein expression of LC3-II and Beclin 1, and mRNA expression of CXCR4 in osteochondral tissue. These effects were reversed by the autophagy agonist rapamycin. SDF-1/CXCR4 promotes OA development by enhancing chondrocyte autophagy. MicroRNA-146a-5p may alleviate OA by suppressing CXCR4 mRNA expression and SDF-1/CXCR4-induced chondrocyte autophagy.

Phosphate and tension homology-induced kinase 1/Parkin signaling mediates cognitive dysfunction in sepsis-associated encephalopathy through activation of hippocampal mitochondrial autophagy

To investigate the effects of gene of phosphate and tension homology (PTEN)-induced putative kinase 1 (PINK1)/Parkin pathway on hippocampal mitophagy and cognitive function in mice with sepsis-associated encephalopathy (SAE) and its possible mechanism. A total of 80 male C57BL/6J mice were randomly divided into Sham group, cecal ligation puncture (CLP) group, PINK1 plasmid transfection pretreatment groups (p-PINK1+Sham group, p-PINK1+CLP group), empty vector plasmid transfection control group (p-vector+CLP group), with 16 mice in each group. The mice in CLP groups were treated with CLP to reproduce SAE models. The mice in the Sham groups were performed laparotomy only. Animals in the p-PINK1+Sham and p-PINK1+CLP groups were transfected with PINK1 plasmid through the lateral ventricle at 24 hours before surgery, while mice in the p-vector+CLP group were transfected with the empty plasmid. Morris water maze experiment was performed 7 days after CLP. The hippocampal tissues were collected, the pathological changes were observed under a light microscope after hematoxylin-eosin (HE) staining, and the mitochondrial autophagy was observed under a transmission electron microscopy after uranyl acetate and lead citrate staining. The expressions of PINK1, Parkin, Beclin1, interleukins (IL-6, IL-1β) and microtubule-associated protein 1 light chain 3 (LC3) were detected by Western blotting. Compared with the Sham group, CLP group mice in Morris water maze experiment had longer escape latency, shorter target quadrant residence time, and fewer times of crossing the platform at 1-4 days. Under the light microscope, the hippocampal structure of the mouse was injured, the neuronal cells were arranged in disorder, and the nuclei were pyknotic. Under the electron microscope, the mitochondria appeared swollen, round, and wrapped by bilayer or multilayer membrane structures. Compared with the Sham group, CLP group had higher expressions of PINK1, Parkin, Beclin1, LC3II/LC3I ratio, IL-6 and IL-1β in hippocampus, indicating that sepsis induced by CLP could activated inflammatory response and caused PINK1/Parkin-mediated mitophagy. Compared with the CLP group, p-PINK1+CLP group had shorter escape latencies, spent more time in the target quadrant and had more number of crossings in the target quadrant at 1-4 days. Under the light microscope, the hippocampal structures of mice was destroyed, the neurons were arranged disorderly, and the nuclei were pyknotic. Under transmission electron microscope, swollen and rounded mitochondria and mitochondrial structure wrapped by double membrane or multilayer membrane structure were observed. Compared with the CLP group, the levels of PINK1, Parkin, Beclin1 and LC3II/LC3 ratio in the p-PINK1+CLP group were significantly increased [PINK1 protein (PINK1/β-actin): 1.95±0.17 vs. 1.74±0.15, Parkin protein (Parkin/β-actin): 2.06±0.11 vs. 1.78±0.12, Beclin1 protein (Beclin1/β-actin): 2.11±0.12 vs. 1.67±0.10, LC3II/LC3I ratio: 3.63±0.12 vs. 2.27±0.10, all P < 0.05], while the levels of IL-6 and IL-1β were significantly decreased [IL-6 protein (IL-6/β-actin): 1.69±0.09 vs. 2.00±0.11, IL-1β protein (IL-1β/β-actin): 1.11±0.12 vs. 1.65±0.12, both P < 0.05], suggesting that overexpression of PINK1 protein could further activate mitophagy and reduce the inflammatory response caused by sepsis. There was no statistically significant difference in the above pathological changes and related indicators between Sham group and p-PINK1+Sham group, CLP group and p-vector+CLP group. PINK1 overexpression can further activate CLP-induced mitophagy by upregulating Parkin, thereby inhibiting inflammation response and alleviate cognitive function impairment in SAE mice.

Silencing SIRT1 reduces 5-fluorouracil resistance of cholangiocarcinoma cells by inhibiting the FOXO1/Rab7 autophagy pathway

To investigate the mechanism by which SIRT1 silencing reduces 5-fluorouracil (5-FU) resistance of cholangiocarcinoma cells and the role of FOXO1/Rab7 autophagy pathway in mediating this effect. Human cholangiocarcinoma HCCC-9810 cells were treated with 50, 100, 150, and 200 μg/mL 5-FU to construct a 5-FU-resistant cell model, whose expressions of SIRT1, FOXO1 and Rab7 were detected with immunofluorescence assay, Western blotting and RTqPCR, and the expression levels of autophagy related proteins (Beclin1, LC3, and p62) were detected with Western blotting. The 5-FU resistant cells were transfected with a SIRT1 siRNA, and the changes in 5-Fu resistance and migration ability of the cells were evaluated using CCK-8 assay and wound healing assay; The changes in FOXO1 and Rab7 mRNA levels and protein expressions of SIRT1, FOXO1, Rab7, Beclin1, LC3 and P62 were detected with RT-qPCR and Western blotting. Treatments with 5-FU at 50, 100, 150, and 200 μg/mL all inhibited the proliferation of HCCC-9810 cells. Immunofluorescence assay revealed significantly enhanced SIRT1 expression in 5-FU-resistant HCC-9810 cells, and Western blotting also showed significantly up-regulated protein expressions of SIRT1, Rab7, P62, FOXO1 and Beclin 1 (P < 0.001) and an increased LC3II/LC3I ratio in the cells (P < 0.001). The mRNA levels of SIRT1, Rab7 and FOXO1 were also up-regulated in 5-Fu-resistant cells (P < 0.05). SIRT1 silencing significantly attenuated 5-FU resistance and migration ability of HCCC-9810 cells, and obviously decreased the protein expressions of SIRT1, Rab7, P62, FOXO1 and Beclin1 and the LC3II/LC3I ratio as well (P < 0.001). FOXO1 and Rab7 mRNA levels were significantly decreased in 5-FU-resistant HCC-9810 cells after SIRT1 silencing (P < 0.05). Silencing SIRT1 attenuates 5-FU resistance in HCC-9810 cells by inhibiting the activation of the FOXO1/Rab7 autophagy pathway.

Fuyu Decoction improves ventricular remodeling in rats with heart failure by inhibiting AMPK/mTOR pathway-mediated autophagy

To investigate the effect of Fuyu Decoction on ventricular remodeling and its association with AMPK/mTOR pathway-mediated autophagy in rats with heart failure. Thirty male Wistar rat models of heart failure induced by ligation of the left anterior descending coronary artery were divided into model group, Fuyu Decoction treatment group, Fuyu Decoction treatment +AMPK agonist group (n=10), with another 10 rats receiving sham operation as the Sham group. After 8 weeks of drug intervention, the changes of ventricular function and ventricular remodeling indexs of the rats were assessed. TTC staining was used to detect the myocardial infarction area, and HE and Masson staining were used to observe the pathological changes in the myocardial tissue. Western blotting was performed to detect the protein expressions of p-AMPK, p-mTOR, LC3-II, Beclin1 and p62 in the myocardial tissue. Compared with the sham-operated rats, the rat models of heart failure showed significantly increased left ventricular end-diastolic volume (LVEDV), left ventricular endsystolic volume (LVESV), and left ventricular mass index (LVMI) (P < 0.01), reduced left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), and decreased spherical index (SI) were (P < 0.01). The rat models also showed increased myocardial infarction area, obvious myocardial pathologies and fibrosis, increased apoptosis rate of the cardiomyocytes, enhanced myocardial expressions of p-AMPK, LC3-II/LC3-I and Beclin1 (P < 0.01), and reduced expressions of p-mTOR and p62 (P < 0.01). Fuyu Decoction treatment significantly ameliorated these changes in the rat models (all P < 0.01), but its effects were obviously blocked by treatment with EX229. Fuyu Decoction can improve ventricular remodeling in rats with heart failure by inhibiting AMPK/mTOR signaling-mediated autophagy in the cardiomyocytes.

Mechanism of Cistanches Herba in treatment of cancer-related fatigue based on network pharmacology and experimental verification

This study aimed to explore the mechanism of Cistanches Herba in the treatment of cancer-induced fatigue(CRF) by network pharmacology combined with in vivo and in vitro experiments to provide a theoretical basis for the clinical medication. The chemical constituents and targets of Cistanches Herba were searched from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP). The targets of CRF were screened out by GeneCards and NCBI. The common targets of traditional Chinese medicine and disease were selected to construct a protein-protein interaction(PPI) network, followed by Gene Ontology(GO) functional and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analyses. A visual signal pathway rela-ted to Chinese medicine and disease targets was constructed. The CRF model was induced by paclitaxel(PTX) in mice. Mice were divided into a control group, a PTX model group, and low-and high-dose Cistanches Herba extract groups(250 and 500 mg·kg~(-1)). The anti-CRF effect in mice was evaluated by open field test, tail suspension test, and exhaustive swimming time, and the pathological morphology of skeletal muscle was evaluated by hematoxylin-eosin(HE) staining. The cancer cachexia model in C2C12 muscle cells was induced by C26 co-culture, and the cells were divided into a control group, a conditioned medium model group, and low-, medium-, and high-dose Cistanches Herba extract groups(62.5, 125, and 250 μg·mL~(-1)). The reactive oxygen species(ROS) content in each group was detected by flow cytometry, and the intracellular mitochondrial status was evaluated by transmission electron microscopy. The protein expression levels of hypoxia-inducible factor-1α(HIF-1α), BNIP3L, and Beclin-1 were detected by Western blot. Six effective constituents were screened out from Cistanches Herba. The core genes of Cistanches Herba in treating CRF were AKT1, IL-6, VEGFA, CASP3, JUN, EGFR, MYC, EGF, MAPK1, PTGS2, MMP9, IL-1B, FOS, and IL10, and the pathways related to CRF were AGE-RAGE and HIF-1α. Through GO enrichment analysis, it was found that the main biological functions involved were lipid peroxidation, nutrient deficiency, chemical stress, oxidative stress, oxygen content, and other biological processes. The results of the in vivo experiment showed that Cistanches Herba extract could significantly improve skeletal muscle atrophy in mice to relieve CRF. The in vitro experiment showed that Cistanches Herba extract could significantly reduce the content of intracellular ROS, the percentage of mitochondrial fragmentation, and the protein expression of Beclin-1 and increase the number of autophagosomes and the protein expression of HIF-1α and BNIP3L. Cistanches Herba showed a good anti-CRF effect, and its mechanism may be related to the key target proteins in the HIF-1α signaling pathway.

Salidroside attenuates LPS-induced kidney injury through activation of SIRT1/Nrf2 pathway.

Salidroside (SAL) is an anti-inflammatory, antioxidant, anticancer, neuroprotective, and renal protective active ingredient extracted from the Chinese herb. Rhodiola Rosea. However, the role of SAL in kidney injury has not yet been elucidated. The study investigates SAL's protective effect and mechanism in lipopolysaccharide (LPS)-induced kidney injury. Male C57BL/6 wild-type mice (6-8weeks old) were intraperitoneally injected with 10mg/kg LPS for 24h and SAL (50mg/kg) 2h before the LPS injection. Biochemical and TUNNEL staining assay analyses were carried out to assess kidney injury. The Elisa assay analyzed the mRNA expression of NGAL and KIM-1. RT-qPCR and Western blotting measured the mRNA and protein expression of HO-1, NQO1, Beclin1, P62, SIRT1, Nrf2, and PNCA, respectively. Our study found that mice co-treated with SAL had significantly reduced blood urea nitrogen (BUN), serum creatinine (Scr), neutrophil gelatinase-associated lipocalin (NGAL), and kidney injury molecule-1 (KIM-1) levels in serum of LPS-induced mice. SAL cotreatment potentially decreased the apoptosis rate of kidney tissue and podocytes induced by LPS. SAL significantly reduced the content of malondialdehyde (MDA) and increased superoxide dismutase (SOD) in LPS-treated mice. Autophagy-related proteins Beclin-1 increased but decreased P62 protein expression by cotreatment of SAL in LPS-injected mice. SAL enhanced the Sirtuin 1 (SIRT1) and nuclear factor erythroid 2-related factor 2 (Nrf2) protein expression in LPS-induced kidney tissues. Our results speculate that SAL protects against LPS-induced kidney injury through activation of the SIRT1/Nrf2 pathway.

Targeted magnetochemotherapy modified by 5-Fu-loaded thermally on/off switching nanoheaters for the eradication of CT26 murine colon cancer by inducing apoptotic and autophagic cell death

Despite significant breakthroughs in diagnosis and treatment of colorectal cancer (CRC), the extent of morbidity and mortality secondary to CRC is still concerning. In this study, we evaluated the efficacy of our new tumor-selective nanoplatforms at induction of apoptosis and autophagy, which was tested using active 5-fluorouracil (5-Fu)-based targeting of tumor cells in a BALB/c murine model of CRC combined with magnetic thermal therapy. Nanoparticles were synthesized and characterized by zeta sizer, transmission electron microscopy (TEM), and scanning electron microscopy (SEM). The cytotoxicity and tissue uptake of 5-Fu-loaded folic acid (Fa)-modified magnetic nanoparticles (5-Fu/MNPs-Fa) was assessed using MTT, ICP-OES, and HPLC. The rate of apoptosis and autophagy, as two major indicators of antitumor activity, was measured based on protein expression of Bax, Bcl2, Caspase 3, mTOR, P-mTOR, Beclin-1, and LC3B in CT-26 murine CRC, along with tumor volume and survival time. The spherical 5-Fu/MNPs-Fa exhibited sustained thermal on/off switching drug release and higher therapeutic index compared to free 5-Fu. Our de novo synthetized magnetic nanoheaters successfully delivered the therapeutic agent to the tumor site, enhanced the conversion of radio frequency energy to heat in tumor cells, exhibited higher antitumor efficiency based on Bax/Bcl2 ratio and overexpression of Beclin-1 and LC3B, increased the survival time, and decreased the tumor volume (P < 0.05). Our findings indicated that magnetochemotherapy (MHC) was substantially more effective than hyperthermia and/or chemotherapy alone. From a translational standpoint, the 5-Fu/MNPs-Fa would be a promising candidate sustained drug targeting system that could improve cancer cell therapy via inducing apoptosis and autophagy.Graphical

Effects of Emodin on Protein Expression Related to Autophagy of Interstitial Cells of Cajal in Diabetic Rats.

This work aims to investigate the effects and mechanism of emodin in treating diabetic gastroenteropathy and colonic dysmotility in STZ + HS/HF diet induced diabetic gastroenteropathy rats. Diabetic colonic dysmotility model was established by high-fat/high-glucose (HS/HF) feeding combined with streptozotocin (STZ). Emodin was divided into high, medium and low dose groups. After eight weeks of intervention, fasting blood glucose (FBG) and body weight were measured. Gastrointestinal transmission time was evaluated. Serum vasoactive intestinal peptide (VIP) and substance P (SP) were detected. Colonic protein expression of selective autophagy adaptor proteins p62 and beclin1 were detected by immunohistochemistry. Colonic protein expression of beclin1, autophagy related gene 5 (Atg5), C-kit and p62 were detected by Western blot. After treating with emodin, gastrointestinal transmission rate was improved. The expression of serum SP was increased and serum VIP was decreased. Colonic c-kit and p62 were up-regulated. The expressions of beclin1 and Atg5 were down-regulated. Emodin can improve colonic dysmotility and promote the recovery of colonic motility and intestinal defecation in diabetic rats. Its mechanism may involved with up-regulating the expression of C-kit and P62, down-regulating the expression of Beclin1 and Atg5 in colon, which are associated with colon over-autophagy of Cajal interstitial cell (ICC).

Ultrastructural and Molecular Evidence of Macroautophagy in Functioning PitNETs and Experimental Pituitary Tumors

Introduction: Macroautophagy is a lysosome-mediated degradation process that controls the quality of cytoplasmic components and organelles, with its regulation depending on autophagy-related proteins (Atg) and with Beclin1/Atg6 and microtubule-associated protein light chain 3 (LC3/Atg8) being key players in the mammalian autophagy. As reports on this mechanism in the field of pituitary neuropathology and neuroendocrinology are scarce, our study analyzed the ultrastructural signs of macroautophagy and the expression of Beclin1 and LC3 proteins in human functioning PitNETs and in experimental pituitary tumors. Methods: A group of humans functioning PitNETs and an experimental lactotroph model in rats of the F344 strain stimulated with estradiol benzoate (BE) were used. Ultrastructural and molecular evidence of the macroautophagic process was evaluated using different techniques. Results: In functioning PitNETs cohort, 60% exhibited evidence of macroautophagy, with a significant difference found for Beclin1 and LC3 between macro- and micro-PitNETs (p < 0.05). In the experimental model, the expression of both Beclin1 and LC3 proteins was immunopositive in normal and tumoral glands when analyzed by immunofluorescence, Western blot, and immunohistochemistry. In the experimental model, protein expression was associated with increased glandular size and weight. Conclusions: Our study revealed evidence of macroautophagy at the pituitary level and the important role of Beclin1 and LC3 in the progression of functioning PitNETs, implying that this mechanism participate in regulating pituitary cell growth.

Corilagin alleviates intestinal ischemia/reperfusion injury by relieving oxidative stress and apoptosis via AMPK/Sirt1-autophagy pathway.

Intestinal ischemia/reperfusion (II/R) injury is a common pathological process with high clinical morbidity and mortality. Autophagy plays an important role in the pathological development of II/R. Corilagin (CA) is a natural ellagitannin with various pharmacological effects such as autophagy regulation, antioxidant, and antiapoptosis. However, whether CA alleviates II/R injury is still unclear. In this study, we had found that CA significantly attenuated II/R induced intestinal tissue pathological damage, oxidative stress, and cell apoptosis in rats. Further studies showed that CA significantly promoted AMPK phosphorylation and sirt1 expression, and thus activated autophagy by upregulating protein expression of autophagy-related proteins Beclin1 and LC3II and promoting SQSTM1/P62 degradation both in vivo and in vitro. Inhibition of AMPK phosphorylation by its inhibitor compound C(CC) significantly abolished CA-mediated autophagy activation and the relievable effects on oxidative stress and apoptosis in vitro, suggesting the excellent protective activity of CA against II/R injury via AMPK/Sirt1-autophagy pathway. These findings confirmed the potent effects of CA against II/R injury, and provided novel insights into the mechanisms of the compound as a potential candidate for the treatment of II/R.

The Mechanism of Aerobic Exercise Regulating the PI3K/Akt-mTOR Signaling Pathway Intervenes in Hippocampal Neuronal Apoptosis in Vascular Dementia Rats.

The purpose of this paper is to explore the mechanism of aerobic exercise regulating autophagy through the PI3K/Akt-mTOR signaling pathway and its participation in apoptosis, to protect the hippocampal nerves from damage in vascular dementia rats. Thirty-six healthy male SD rats were randomly divided into a sham group, a model group, and a model exercise group. A neurobehavioral assessment was used to determine the memory and exploration abilities of the rats. A TUNEL assay was used to detect hippocampal neuron apoptosis. Immunohistochemical and Western blot analyses were used to analyze LC3Ⅱ and the beclin-1 protein. An RT-PCR detected the differential expression of mRNA. The results of the neurobehavioral tests showed that the platform latency time of the rats with vascular dementia was prolonged. Aerobic exercise significantly shortens the swimming time of rats in platform latency. The TUNEL results showed that the TUNEL-positive cells of the hippocampal neurons in the model group increased; the expression of pro-apoptotic genes caspase-3 and Bax mRNA was up-regulated, and the expression of Bcl-2 mRNA was down-regulated. Aerobic exercise reduced hippocampal neuronal apoptosis, up-regulated Bcl-2 mRNA, and down-regulated caspase-3 and Bax mRNA. The LC3Ⅱ and Beclin-1 proteins, detected by immunohistochemistry and a Western blot analysis, showed that the protein expression in the hippocampi of rats with vascular dementia increased. Aerobic exercise reduced LC3Ⅱ and Beclin-1 protein expression. The results of the RT-PCR showed similar changes. Aerobic exercise could improve the learning and memory abilities of vascular dementia rats, moderately regulate the process of autophagy, reduce the TUNEL-positive cells of hippocampal neurons, repair damaged hippocampal neurons by regulating the autophagy signaling pathway PI3K/Akt-mTOR, and improve hippocampal function.

Chaihu Shugan San promotes gastric motility in rats with functional dyspepsia by regulating Drp-1-mediated ICC mitophagy

Context Chaihu Shugan San (CHSGS) was effective in the treatment of functional dyspepsia (FD). Objective To investigate the mechanism of CHSGS in FD through dynamin-related protein 1 (Drp-1)-mediated interstitial cells of cajal (ICC) mitophagy. Materials and methods Forty Sprague-Dawley (SD) rats were randomly divided into control, model, mdivi-1, mdivi-1 + CHSGS and CHSGS groups. Tail-clamping stimulation was used to establish the FD model. Mdivi-1 + CHSGS and CHSGS groups were given CHSGS aqueous solution (4.8 g/kg) by gavage twice a day. Mdivi-1 (25 mg/kg) was injected intraperitoneally once every other week for 4 w. Mitochondrial damage was observed by corresponding kits and related protein expressions were assessed by Immunofluorescence and (or) Western Blot. Results Compared with the mean value of the control group, superoxide dismutase (SOD) and citrate synthase (CS) in the model group were decreased by 11% and 35%; malondialdehyde (MDA) and reactive oxygen species (ROS) were increased by 1.2- and 2.8-times; ckit fluorescence and protein expressions were decreased by 85% and 51%, co-localization expression of LC3 and voltage dependent anion channel 1 (VDAC1), Drp-1 and translocase of the outer mitochondrial membrane 20 (Tom20) were increased by 10.1- and 5.4-times; protein expressions of Drp-1, Beclin-1, and LC3 were increased by 0.5-, 1.4-, and 2.5-times whereas p62 was decreased by 43%. After mdivi-1 and (or) CHSGS intervention, the above situation has been improved. Discussion and conclusion CHSGS could improve mitochondrial damage and promote gastric motility in FD rats by regulating Drp-1-mediated ICC mitophagy.

Protective Effect of Buyang Huanwu Decoction on Cerebral Ischemia/Reperfusion Injury by Inhibiting Autophagy Based on AMPK/UlK1 Signaling Pathway

The aim of the paper is to explore whether AMPK/UlK1 signaling pathway is involved in regulating autophagy and alleviating cerebral ischemia/reperfusion (I/R) injury. On this basis, study the regulatory effect of Buyang Huanwu Decoction (BHD) on AMPK/UlK1 signaling pathway, aiming to clarify the molecular mechanism of BHD in alleviating cerebral I/R injury and the protective effect of BHD containing serum (BHDS) on OGD/R neurons. The research group adopted the following methods: Neurons cultured for 7 days in vitro were randomly divided into normal control group, model group (OGD/R), AMPK activator (AICAR) group (3 mmol/L), Buyang Huanwu Decoction medicated serum (BHDS) group (3%), Buyang Huanwu Decoction medicated serum + AMPK activator (BHDS + AICAR) group. The protein expressions of p-AMPK, AMPK, p-UlK1, UlK1, Beclin-1 and LC3B in neurons were detected by Western blot, the fluorescence intensity and apoptosis rate of neuron autophagosome CYTO-ID staining were detected by flow cytometry, the fluorescence intensity and morphological changes of neuron LC3B were detected by immunofluorescence double labeling method, the morphological changes of neurons were observed by inverted microscope, and the survival rate of neurons was detected by CCK-8 method. Neuron OGD/R model and the optimal concentration of BHDS. The neurons in the model group were subjected to hypoxia and glucose deprivation for 1 h, 2 h, 3 h, 4 h and reoxygenation and glucose deprivation for 24 h. The survival rate of neurons and the release rate of l DH to determine whether the model was successfully established. And the results show that BHDS group, the survival rate of OGD/R neurons was significantly increased, and the apoptosis rate was significantly decreased. Among them, 3% BHDS intervention at the same time of hypoxia glucose deprivation for 3 h and reoxygenation glucose for 24 h was the best condition. In BHDS group, the levels of p-AMPK/AMPK, p-UlK1/UlK1, beclin-1, CYTO-ID and LC3B fluorescence intensity, and the transformation from LC3B-Ⅰ to LC3B-Ⅱ of OGD/R neurons were significantly decreased. So we can draw a conclusion that BHD can inhibit autophagy and alleviate cerebral I/R injury through AMPK/UlK1 signaling pathway.

Leucine mediates cognitive dysfunction in early life stress-induced mental disorders by activating autophagy.

To explore the relationship between leucine in cerebrospinal fluid (CSF) and cognitive dysfunction in rats with early life stress (ELS) induced mental illness, and pathophysiological mechanism involved. The maternal separation (MS), an animal paradigm used widely as a preclinical model of ELS which is one of the important risk factors for mental disorders. Behavioral experiments including open-field test, sucrose preference, object recognition and Morris water maze tests, Nissl staining, transmission electron microscopy and WES were employed in the present study. The behavioral results showed that MS rats were more prone to cognitive impairment and depression-and-anxiety-like behaviors than controls, including spatial self-exploration ability, memory ability, and spatial learning and memory function. Nissl staining analysis indicated that the number of neurons in the CA1 and CA3 regions of the hippocampus significantly decreased and the arrangement of nerve cells was abnormal. The leucine levels were decreased in the CSF of MS rats and highly correlated with the number of hippocampal neurons, and yet leucine supplementation improved the degree of MS-induced cognitive impairment. Furthermore, there were autophagosomes in the hippocampus of the low-leucine diet rats of the control and MS group but not in the high-leucine diet MS group by transmission electron microscopy. The protein expression of Beclin-1 in the hippocampus was significantly increased in the MS normal diet group and MS low-leucine diet group, yet decreased in the MS high-leucine diet group compared with the MS low-leucine diet group. Meanwhile, the Bcl-2/Bax ratio was significantly decreased in the control low-leucine diet group, MS normal diet group and MS low-leucine diet group. Ultimately, in vitro experiments suggested that leucine deficiency could activate neuronal autophagy including enhanced LC3II/LC3I and mRFP-GFP-LC3, which was consistent with the in vivo results, and the cell apoptosis rate and lactate dehydrogenase (LDH) cytotoxicity were also increased with leucine deficiency, while the above effects could be partly reversed by autophagy inhibitor treatment. MS model caused adult male rats to be susceptible to cognitive dysfunction, which may regulate autophagy in hippocampal neurons through leucine metabolism in CSF.

Methyltransferase 3 beta (DNMT3B) inhibition by decitabine promotes endometrial carcinoma cell apoptosis by autophagy induction

Autophagy regulates endometrial cancer cell apoptosis and inhibits endometrial cancer development; however, the specific underlying mechanisms are unclear. The effects of DNA methylation on endometrial cancer cell autophagy were explored in HEC-1B cells using decitabine, a DNA methylase inhibitor. Real-time fluorescence quantitative PCR (RT-qPCR) and western blot (WB) analyses were used to detect the mRNA and protein expression of DNA methylase (DNMT3B), autophagy-related genes LC3B and Beclin-1, and apoptosis-related genes Bax and Bcl-2. High-purity and high-quality RNA was obtained by nano-magnetic bead extraction, to ensure highly accurate RT-qPCR and WB results. MTT cell viability assay, scratch assay, and Transwell migration assay were utilized to detect the proliferation and migration of HEC-1B cells. The results showed that decitabine significantly inhibited the expression of DNMT3B protein and mRNA, with 25 μM decitabine being the most effective. In subsequent experiments, this concentration of decitabine was used. The low DNMT3B expression significantly inhibited the expression of Beclin-1 and LC3B proteins (***P &lt;0.001 and *P &lt;0.05) and mRNA (*P &lt;0.05 and *P &lt;0.05). Decitabine promoted the mRNA and protein expression of apoptotic factor Bax (*P &lt; 0.05 and *P &lt; 0.05), while inhibiting the mRNA protein expression of anti-apoptotic factor Bcl-2 (***P &lt;0.001 and *P &lt;0.05). MTT assay results showed decreased proliferation of HEC-1B cells following inhibition of DNMT3B expression by decitabine, which was significantly different from that of the NC group (*P &lt;0.05). The results of the cell scratch and Transwell migration assays showed reduced migration ability of HEC-1B cells compared to that observed in the NC group (**P &lt;0.01 and *P &lt;0.05). These results show that decitabine inhibits DNMT3B expression, and low DNMT3B levels regulate the expression of autophagy- and apoptosis-related factors, thereby inhibiting the proliferation and migration of endometrial cancer cells.

The roles of AMPK/mTOR autophagy pathway in the acute kidney injury-induced acute lung injury.

Acute kidney injury (AKI) is one of the most challenging clinical problems in kidney disease due to serious complications and high mortality rate, which can lead to acute lung injury (ALI) through inflammatory reactions and oxidative stress. Adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway has been reported to be involved in the development of renal ischemia-reperfusion through autophagy and it remains unclear whether AMPK/mTOR pathway has an effect on the AKI-induced ALI. In this study, we aimed to investigate the effects of autophagy-related AMPK/mTOR signaling pathway on inflammatory factors and oxidative stress in an AKI-induced ALI model. The 48 male Sprague-Dawley rats were divided into four groups randomly: (i) sham, (ii) ischemia/reperfusion injury (IRI), (iii) IRI + rapamycin (RA), and (iv) IRI + 3-methyladenine (3-MA). Unilateral flank incisions were made and right kidneys were excised. The left kidney was subjected to 60 min of ischemia followed by 12, 24, 48, and 72 h of reperfusion. The levels of Scr, blood urea nitrogen (BUN), Wet/Dry ratio, indexes of inflammation, and oxidative stress were assayed. Histological examinations were performed. The protein expression of AMPK, mTOR, LC3-II/LC3-I ratio, and Beclin-1, ULK1 was evaluated by western blotting and immunohistochemistry. Compared to the rats from the sham group, IRI rats showed significantly pulmonary damage after AKI with increased Scr, BUN, Wet/Dry ratio, indexes of inflammation, and oxidative stress. The expression of AMPK, LC3-II/LC3-I ratio, Beclin-1, and ULK1 and were increased, while p62 and mTOR were decreased. In addition, RA treatment significantly attenuated lung injury by promoting autophagy through the activation of the AMPK/mTOR pathway, and 3-MA treatment exhibited adverse effects inversely. Therefore, the activation of the AMPK/mTOR pathway after renal IRI induction could significantly attenuate kidney injury and following AKI-induced ALI by inducing autophagy, which alienates inflammation, oxidative stress, and apoptosis.

Autophagy-related gene and protein expressions during blastocyst development.

This study aims to examine the expression of autophagic genes and proteins during blastocyst development and differentiation. This is a prospective cohort study. Between March 2018 and November 2019, 30 females aged 30.13 ± 4.83years underwent an intracytoplasmic sperm injection (ICSI) cycle atMadina Fertility Center.ICSI was used to develop and incubate 82 leftover embryos to day 5. Then, the embryos were divided into two groups based on their developmental structure: group D (n = 49) included embryos that developed into blastocysts, whereas group A (n = 33) included arrested embryos. These embryos were used to investigate the autophagic gene and protein expressions. The current study was approved by the Clinical Trial Ethical Committee of the Faculty of Medicine, Alexandria University, following the ethical standards of scientific research (Registration no. 0303721). Embryos that developed into blastocysts on day 5 (group D) had significantly higher relative expression of the LC3 gene (1.11 ± 0.52) and beclin-1 gene (1.43 ± 0.34) and beclin-1 protein expression (3.8 ± 0.028) than those that did not develop into blastocysts on day 5 (group A) [0.72 ± 0.18 (P = 0.03), 0.35 ± 0.12 (P = 0.0001), and 3.14 ± 0.05, (P = 0.0001), respectively]. In contrast, mTOR and PIK3C3 protein expression wassignificantly higher in group A (arrested embryos) than those in group D (developed embryos) (P = 0.007 and P = 0.0001, respectively). Furthermore, the expression of the eIF4E gene was significantly lower in group D embryos (0.32 ± 0.07) than that in group A embryos (4.38 ± 1.16) (P = 0.0001). This work identifiesautophagy as awell regulated process required tomaintain cell allocation anddifferentiation during late preimplantationembryo developmental stages.

Tanshinone IIA Improves Acute Gouty Arthritis in Rats through Regulating Neutrophil Activation and the NLRP3 Inflammasome.

To investigate the prevention and treatment effect of tanshinone IIA (TIIA) on acute gouty arthritis (AGA) and its mechanism. The anti-AGA effect of TIIA was observed in vivo and in vitro. Neutrophils were isolated from the abdominal cavity of mice, and the anti-AGA effect of TIIA was investigated in a rat model of MSU-induced AGA. The pathological changes of the ankle joint tissues were assessed by H&E. Cytokine and chemokine expression were determined by ELISA and RT-qPCR. The NLRP3 inflammasome pathway protein levels in the ankle joint tissues were evaluated via western blotting. Neutrophil migration was evaluated in air pouch and transwell assays. Immunohistochemistry and immunofluorescence analysis evaluate the release of myeloperoxidase (MPO), neutrophil elastase (NE), and citrullination of histone H3 (CitH3). Beclin-1 and LC3B expressions were determined using western blotting and immunofluorescence. Key Findings. Treatment with TIIA alleviated synovial hyperplasia and neutrophil infiltration, regulated cytokine and chemokine expressions, and inhibited NLRP3 activation in AGA rats, neutrophil migration, MPO, NE, and CitH3 expression, and LC3B and Beclin-1 protein expression. These results demonstrate that TIIA can effectively enhance AGA by focusing on the neutrophils and NLRP3 inflammasome, demonstrating that TIIA may act as a potential helpful agent for AGA.

Nano-selenium Alleviates Cadmium-Induced Mouse Leydig Cell Injury, via the Inhibition of Reactive Oxygen Species and the Restoration of Autophagic Flux.

Cadmium (Cd) is a well-known environmental pollutant that can contribute to male reproductive toxicity through oxidative stress. Nano-selenium (Nano-se) is an active single body of selenium with strong antioxidant properties and low toxicity. Some studies have addressed the potential ameliorative effect of Nano-se against Cd-induced testicular toxicity; however, the underlying mechanisms remain to be investigated. This study aimed to explore the protective effect of Nano-se on Cd-induced mouse testicular TM3 cell toxicity by regulating autophagy process. We showed that cadmium exposure to TM3 cells inhibited cell viability and elevated the level of reactive oxygen species (ROS) generation. Morphology observation by transmission electron microscope and the presence of mRFP-GFP-LC3 fluorescence puncta demonstrated that cadmium increased autophagosome formation and accumulation in TM3 cells, resulting in blocking the autophagic flux of TM3 cells. Meanwhile, cadmium remarkably increased the ratio of LC3-II to LC3-I protein expression (2.07 ± 0.31) and the Beclin-1 protein expression (1.97 ± 0.40) in TM3 cells (P < 0.01). Pretreatment with Nano-se significantly reduced Cd-induced TM3 cell toxicity (P < 0.01). Furthermore, Nano-se treatment reversed Cd-induced ROS production and autophagosome accumulation, and autophagy as evidenced by the ratio of LC3-II to LC3-I and Beclin-1 expression. In addition, ROS scavenger, N-acetyl-L-cysteine (NAC) or autophagy inhibitor, 3-methyladenine (3-MA) reversed cadmium-induced ROS generation, autophagosome accumulation, and autophagy-related protein expression levels, which confirmed that cadmium induced TM3 cell injury via ROS signal pathway and blockage of autophagic flux. Collectively, our results reveal that Nano-se attenuates Cd-induced TM3 cell toxicity through the inhibition of ROS production and the amelioration of autophagy disruption.