Expression Levels Of Light Chain 3 Research Articles

PJ‑001, a small‑molecule proteolysis‑targeting chimera, ameliorates atopic dermatitis‑like inflammation in mice by inhibiting the JAK2/STAT3 pathway and repairing the skin barrier.

Atopic dermatitis (AD) is a common allergic skin disease, and its pathogenesis involves genetic and environmental factors, as well as the immune response and skin barrier. PJ-001 is a small-molecule proteolysis-targeting chimera, which can degrade proteins related to the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway. In the present study, 0.5% 2,4-dinitrofluorobenzene was used to induce a mouse model of AD. Following treatment with PJ-001, the number of scratches and the severity of skin damage in the AD mice were recorded. Pathological changes in skin lesions were observed with hematoxylin and eosin staining. The expression levels of JAK2/STAT3, Toll-like receptor 4/nuclear factor-κB (TLR4/NF-κB), Beclin 1 and microtubule-associated protein 1 light chain 3 (LC3) were detected using western blotting. Furthermore, reverse transcription-PCR was used to detect the mRNA expression levels of filaggrin (FLG) and keratin 17, and the change in interleukin-10 levels in the splenic tissue of the mice. Compared with in the control group, the model group exhibited severe skin lesions. Following treatment with PJ-001, the AD-like inflammation in mice decreased. The expression levels of LC3 II/LC3 I and Beclin 1 were significantly reduced (P<0.01), and the expression levels of JAK2, STAT3, TLR4 and NF-κB were significantly downregulated (P<0.001). Additionally, the mRNA expression levels of FLG were significantly upregulated (P<0.001). These results indicated that PJ-001 may alleviate the skin condition in a mouse model of AD. The underlying mechanism may involve inhibition of the JAK/STAT signaling pathway, thereby suppressing the release of inflammatory factors, reducing excessive autophagy at the site of skin lesions, and enhancing the skin barrier function. In conclusion, PJ-001 could be considered a potential therapeutic option for AD.

Effects of electroacupuncture on the expression of β-amyloid and autophagy-related proteins in hippocampal cells of mice with Alzheimer's disease

To investigate the effect of electroacupuncture (EA) on the expression of β-amyloid (Aβ) and autophagy-related proteins in hippocampal cells of Alzheimer's disease (AD) model mice, so as to explore its underlying mechanisms. Eighteen male APP/PS1 transgenic mice (6 months old) were randomly divided into model and EA groups, with 9 mice in each group. Nine male C57BL/6 wild-type mice of the same age were chosen as the normal group. Mice in the EA group were treated with acupuncture on "Baihui" (GV20) and EA (1 Hz/50 Hz, 1 mA) on bilateral "Yongquan" (KI1), once every other day, 20 min each time for a total of 21 times. After the interventions, the spatial learning and memory ability were observed by Morris water maze test. The autophagy-related pathological changes in hippocampus were observed by transmission electron microscopy. The expressions of microtublue associated protein 1 light chain 3 (LC3) and Aβ in hippocampus were observed by immunofluorescence and the expression levels of LC3 and p62 proteins were detected by Western blot. Compared with the normal group, the escape latency was prolonged (P<0.01), the residence time in the original quadrant platform was shor-tened (P<0.05), the positive expressions of LC3 and Aβ, the expression levels of LC3Ⅱ and p62 proteins, and the ratio of LC3Ⅱ/LC3Ⅰ proteins in hippocampus were increased (P<0.01, P<0.05) in the model group. Compared with the model group, the escape latency was shortened (P<0.05), the residence time in the original quadrant platform was prolonged (P<0.05), the positive expressions of LC3 and Aβ, the expression levels of LC3Ⅱ and p62 proteins, and the ratio of LC3Ⅱ/LC3Ⅰ proteins in hippocampus were decreased (P<0.05) in the EA group. The transmission electron microscopy showed that the structure of neurons was normal in the normal group, a large number of autolysosomes and autophagosomes existed in hip-pocampal nerve cells in the model group, and only a small number of autophagosomes were observed in the EA group. EA can reduce the expression levels of autophagy-related proteins LC3 and p62 in APP/PS1 transgenic mice, improve the hip-pocampal autophagy state, reduce intracellular Aβ aggregation, and thus improve the learning and memory ability.

LRPPRC regulates metastasis and glycolysis by modulating autophagy and the ROS/HIF1-α pathway in retinoblastoma

Retinoblastoma (RB) is the most common intraocular tumor among children. Leucine-rich pentatricopeptide repeat (PPR)-motif-containing protein (LRPPRC), a suppressor gene of autophagy, has been proven to play a regulatory role in tumor progression. However, little is known about functional roles and mechanisms of LRPPRC in RB progression. First, we performed a detailed analysis for RB and normal control. The expression of LRPPRC in the RB tissues was significantly higher than that in normal tissues. Moreover, LRPPRC suppression could repress tumor cell migration, invasion, glycolysis, and reactive oxygen species (ROS)/hypoxia-inducible factor-1α (HIF1-α) pathway activation by mediating autophagy. Furthermore, overexpression of HIF1-α partially reversed the above changes induced by LRPPRC knockdown. The regulation of LRPPRC on tumor metastasis and glycolysis was also validated by a xenograft tumor assay. In summary, LRPPRC could regulate metastasis and glycolysis of RB by mediating autophagy suppression and further activating the ROS/HIF1-α pathway, and LRPPRC could be a promising prognostic biomarker for RB.

Autophagy dysfunction may be involved in the pathogenesis of ankylosing spondylitis.

The present study aimed to investigate the expression and significance of the mRNA of genes associated with autophagy and long non-coding RNA (lncRNA) GAS5 in peripheral blood mononuclear cells (PBMCs) of patients with ankylosing spondylitis (AS). The mRNA levels of microtubule-associated protein light chain 3 (LC3), Beclin1, autophagy-related gene (ATG)3, ATG5, ATG12, ATG 16 ligand 1 (ATG16L1) and lncRNA growth arrest-specific 5 (GAS5) in PBMCs from 60 patients with AS and 30 healthy controls (HC) were examined by reverse transcription-quantitative PCR. The correlations between the levels of LC3, Beclin1, ATG3, ATG5, ATG12 and ATG16L1 mRNA as well as lncRNA GAS5 levels with disease activity and laboratory parameters in patients with AS were determined by Spearman correlation analysis. In addition, the diagnostic value of lncRNA GAS5 for AS was explored through establishing a receiver operating characteristic (ROC) curve. The results indicated that, compared to the HCs, patients with AS had lower expression levels of LC3, ATG5, ATG12, ATG16L1 and lncRNA GAS5 in their PBMCs. Compared with those in patients with inactive AS, the levels of ATG5 and ATG12 were lower than those in patients with active AS. Of note, ATG5 and ATG12 mRNA levels were negatively correlated with disease activity indexes. lncRNA GAS5 was positively correlated with the expression of Beclin1, ATG3, ATG5, ATG12 and ATG16L1. The area under the ROC curve for the use of lncRNA GAS5 expression to diagnose AS was 0.808 with a 95% CI of 0.714-0.902. In conclusion, patients with AS had decreased expression of genes associated with autophagy and lncRNA GAS5. The extent of the reduction in ATG5 and ATG12 expression levels in patients with AS was correlated with the disease severity and activity. Furthermore, lncRNA GAS5 was a diagnostic indicator of AS.

The effect of selenium on the autophagy of macrophage infected by Staphylococcus aureus

Selenium can alleviate the inflammatory reaction infected by Staphylococcus aureus (S. aureus). However, the role of selenium on the autophagy in RAW264.7 macrophages infected by S. aureus has not been reported. The goal of this study was to clarify the effect of selenium on the autophagy and related inflammatory pathways (MAPK and NF-κB) in RAW264.7 macrophages infected by S. aureus. RAW264.7 macrophages were co-treated with Na2SeO3 and S. aureus. The expression of related inflammatory pathways (MAPK and NF-κB) and autophagy-related proteins were detected by Western blotting. The microtubule-binding protein light chain 3 (LC3) puncta were measured with immunofluorescence staining. The ultrastructure of RAW264.7 macrophages infected by S. aureus was detected by transmission electron microscope (TEM). And plate counting method was used to detect the proliferation of S. aureus in RAW264.7 macrophages. The results showed that the expression levels of LC3 II increased and the expression levels of p62 decreased after adding selenium, compared with S. aureus infection group. Compared with S. aureus infection group, the intracellular LC3 puncta and autophagic vesicles, autophagosomes, and autolysosomes increased with selenium supplementation. The number of S. aureus proliferation decreased with addition of selenium, compared with S. aureus infection group. Selenium could significantly inhibit the phosphorylation of MAPK and NF-κB signaling pathway key proteins, compared with S. aureus infection group. In summary, selenium could promote the autophagy in macrophages infected by S. aureus, alleviate the blockade of autophagic flow, depress the transcription of MAPK and NF-κB signaling pathways, and inhibit the proliferation of S. aureus in RAW264.7 macrophages.

Phosphorylated mTORC1 represses autophagic-related mRNA translation in neurons exposed to ischemia-reperfusion injury.

The sequential reactivation of mechanistic target of rapamycin (mTOR) inhibited autophagic flux in neurons exposed to oxygen-glucose deprivation/reperfusion (OGD/R), which was characterized by reduction of autophagosome formation and restriction of autolysosome degradation. However, its detailed molecular mechanism was still unknown. In this study, we further explore the existing form of mTOR and its suppression on the transcriptional levels of related mRNA from neurons exposed to ischemia-reperfusion injury. The OGD/R or middle cerebral artery occlusion/reperfusion (MCAO/R)-treated neurons was used to simulate ischemia/reperfusion injury . Autophagy flux was monitored by means of microtubule-associated protein 1 light chain 3 (LC3) and p62. The reactivation of mTOR was determined by phosphorylation of ribosomal protein S6 kinase 1 (S6K1). Then the inhibitors of mTOR were used to confirm its existence form. Finally, the mRNA transcription levels were analyzed to observe the negative regulation of mTOR. The sequential phosphorylation of mTOR contributed to the neuronal autophagy flux blocking. mTOR was re-phosphorylated and existed as mTOR complex 1 (mTORC1), which was supported by phosphorylation of S6K1 at Thr 389 in neurons. In addition, the phosphorylation of S6K1 was decreased roughly by applying mTORC1 inhibitors, rapamycin and torin 1. However, the administration of mTORC1/2 inhibitor PP242 could recover the phosphorylation of S6K1, which suggested that mTORC2 was involved in the regulation of mTORC1 activity. In paralleling with reactivation of mTORC1, related mRNA transcription was repressed in neurons under ischemia-reperfusion exposure in vivo and in vitro. The mRNA expression levels of LC3, Stx17, Vamp8, Snap29, Lamp2a, and Lamp2b were decreased in neurons after reperfusion, comparing with ischemia-treated neurons. The reactivated mTORC1 could suppress the transcription levels of related mRNA, such as LC3, Stx17, Vamp8, Snap29, Lamp2a, and Lamp2b. The research will expand the horizons that mTOR would negatively regulate autophagy at transcription and post-translation levels in neurons suffering ischemia-reperfusion injury.

Lung fibrosis and changes in autophagy-related proteins in rats exposed to silica dust

Objective: To explore the changes in the autophagy marker microtubule-associated protein 1 light chain 3 (LC3) and yeast autophagy-related gene 6 (Beclin1) in rat lungs exposed to free silica (SiO(2)) dust for different periods. Methods: A total of 72 male specific pathogen-free Wistar rats were randomly divided into solvent control group and SiO(2) model group. The SiO(2) model group received one-time non-exposed intratracheal instillation of suspension of SiO(2) particles to establish a model of silicosis. The solvent control group received an equal amount of saline. Six rats each were sacrificed at 1, 7, 14, 21, 28, and 60 days after model establishment. The pathological changes and fibrosis of rat lungs at different time points were evaluated by H&E staining and Masson staining, respectively. Enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of transforming growth factor-β (TGF-β) , interleukin-1 (IL-1) , and tumor necrosis factor-α (TNF-α) in lung tissue homogenate. Western blot was used to determine the relative expression levels of LC3 and Beclin1 in the lung tissue. Results: The results of H&E staining showed that the model group had continuous inflammation in the lung tissue from day 1 to day 60, and the inflammatory scores were significantly higher in the model group than in the control group (P<0.05) . The results of Masson staining showed that rats in the model group had a small amount of collagen fibers in the lung tissue on day 14 and a large amount of collagen fibers on day 60. The fibrosis score was significantly higher in the model group than in the control group (P<0.05) . No collagen fibrosis was observed in the lung tissue in the control group. The results of ELISA showed that the model group had significantly higher levels of IL-1, TNF-α, and TGF-β in lung tissue homogenate than the control group at each time point after exposure (P<0.05) . The results of Western blot showed that the model group had decreased expression of Beclin1 protein in the lung tissue on days 7, 14, 21, 28, and 60, which was significantly higher than that of the control group (P<0.05) . The model group also had a decreased ratio of LC3II/LC3I on days 1, 7, 14, 21, 28, and 60, which were significantly higher than that of the control group (P<0.05) . Conclusion: In the rat model of silicosis induced by free SiO(2) dust, the expression levels of autophagy-related proteins, LC3 and Beclin1, are correlated with different stages of silicosis. In the early stage of silicosis, the lung tissue has inflammation, substantially increased ratio of LC3II/LC3I and expression of Beclin1, and active autophagy. With the progression of silicosis, the ratio of LC3II/LC3I and expression level of Beclin1 gradually decrease and autophagy becomes weak.

Effect of calcitonin gene-related peptide on MC3T3-E1 osteoblast apoptosis and autophagy induced by serum starvation

To study the effect of calcitonin gene-related peptide (CGRP) on apoptosis and autophagy of mouse MC3T3-E1 osteoblast and their interaction and to further clarify protective mechanism of CGRP on osteoblasts. MC3T3-E1 osteoblasts of mouse were cultured in vitro. Western blot and flow cytometry were used to detect expressions of microtubule-associated protein 1 light chain 3 (LC3) and P62 protein of MC3T3-E1 osteoblasts cultured with serum culture and serum-free (serum starvation) culture. Western blot was also used to detect expressions of LC3 and P62 protein of MC3T3-E1 osteoblast cultured at different concentrations (10⁻¹⁰, 10⁻⁹, 10⁻⁸, and 10⁻⁷ mol·L⁻¹) or without added CGRP. MC3T3-E1 osteoblasts were treated with 10⁻⁸ mol·L⁻¹ CGRP at different times (2, 6, 12, 24, 48, and 72 h), protein expression levels of LC3 were assessed by Western blot and flow cytometry, and changes in autophagosome in cells were detected by monodansylcadaverin staining. Autophagy inhibitor 3-methyladenine (3-MA) was used to pretreat MC3T3-E1 osteoblasts. Cells were then treated with or without CGRP for 24 h. Flow cytometry was used to detect apoptosis level. Under serum starvation conditions, LC3Ⅱ expression and apoptosis of osteoblasts increased compared with that of serum culture. Under 3-MA pretreatment and serum starvation conditions, LC3Ⅱ expression of osteoblasts increased compared with that of serum culture (P<0.01). Compared with serum culture, serum starvation culture with or without CGRP significantly increased expression level of LC3 and reduced expression level of P62. LC3Ⅱ/Ⅰ of osteoblasts was the highest under serum starvation and 10⁻⁸ mol·L⁻¹ CGRP conditions. Serum starvation and 10⁻⁸ mol·L⁻¹ CGRP culture inhibited apoptosis of osteoblasts and promoted synthesis of autophagosome. Apoptosis of osteoblasts increased after 3-MA pretreatment, and CGRP reversed inhibitory effects of 3-MA CGRP on apoptosis. CGRP can increase autophagy of MC3T3-E1 osteoblasts under serum starvation conditions. CGRP may also inhibit apoptosis of MC3T3-E1 osteoblasts by promoting autophagy. .

Vitamin D induces autophagy of pancreatic β-cells and enhances insulin secretion.

Epidemiological evidence indicates that vitamin D is involved in defense against diabetes; however, the precise underlying mechanism remains to be elucidated. In the present study, the effect of vitamin D on the pathogenesis of diabetes was investigated, with an emphasis on its direct effect on pancreatic β‑cells. A streptozotocin (STZ)‑induced type 1 diabetes mellitus (T1DM) mouse model and MIN6 mouse insulinoma β‑cells were subjected to vitamin D treatment. Histopathological analysis of pancreatic islets was performed to investigate insulitis, and reverse transcription-quantitative polymerase chain reaction and western blotting were used to determine the mRNA and protein expression levels of markers of autophagy [microtubule-associated protein 1A/1B‑light chain 3 (LC3) and Beclin 1] and regulation of apoptosis [B-cell lymphoma 2 (Bcl-2)]. Apoptosis of MIN6 cells was examined by flow cytometry following annexin V/propidium iodide labeling. The secretion of insulin was measured by ELISA. The results revealed that vitamin D reduced the incidence of T1DM, enhanced insulin secretion and relieved pancreatic inflammation in STZ‑treated mice. Furthermore, vitamin D increased the mRNA expression levels of LC3 and Beclin 1, and increased Bcl‑2 protein expression levels in STZ‑treated MIN6 cells, while decreasing the apoptosis rate. The results of the present study demonstrated, for the first time to the best of our knowledge, that vitamin D induces autophagy and suppresses apoptosis of pancreatic β‑cells, as well as preventing insulitis. These findings regarding vitamin D provide insights into its involvement in diabetes, and suggest a potential novel strategy for the treatment of diabetes via agents enhancing autophagy in pancreatic β-cells.

Effects of bone marrow-derived mesenchymal stem cells on the autophagic activity of alveolar macrophages in a rat model of silicosis.

The aim of the present study was to evaluate the effects of bone marrow-derived mesenchymal stem cells (BMSCs) on the expression of the autophagy-associated proteins, microtubule-associated protein light chain 3 (LC-3) and autophagy-related gene Beclin-1 (Beclin-1), in alveolar macrophages (AMs) in a rat model of silicosis. Furthermore, the study investigated the molecular mechanisms underlying the effects of BMSC treatment. A population of 60 adult female Sprague-Dawley (SD) rats were allocated at random into three groups, namely the control, model and BMSC treatment groups (n=20 per group). BMSCs were isolated from five male SD rats (age, 6-8 weeks) and cultured in vitro. The silicosis model was established using a single 1.0-ml infusion of silicon dioxide suspension administered via non-exposed tracheal intubation. Rats in the BMSC treatment group received a 1.0-ml transplantation of BMSCs (1×106/ml). The rats were sacrificed on days 1, 7, 14 and 28 after modeling, and AMs were extracted from the rats using bronchoalveolar lavage. Third-generation BMSCs were identified using flow cytometry with fluorescein isothiocyanate staining, and the morphological characteristics of the AMs were observed using hematoxylin and eosin staining. The expression levels of LC-3 and Beclin-1 were determined using immunocytochemistry sand western blot analysis. The expression levels of LC-3 and Beclin-1 were found to be increased at all the time points in the model group. LC-3 and Beclin-1 levels began to increase at day 1, peaked at day 14 and decreased after day 28; however, the levels remained elevated compared with the basal expression levels. The AMs of the BMSC treatment group exhibited significantly alleviated pathological symptoms compared with the model group AMs, as indicated by significantly decreased expression levels of LC-3 and Beclin-1 at each time point. Therefore, the results indicated that autophagy was promoted in the AMs of the silicosis model rats. Furthermore, treatment with BMSCs was demonstrated to reduce the expression levels of LC-3 and Beclin-1, subsequently inhibiting autophagic activity and mitigating the damage associated with silicosis.

Involvement of autophagy inhibition in Brucea javanica oil emulsion-induced colon cancer cell death

Brucea javanica oil emulsion (BJOE), the petroleum ether extract of B. javanica emulsified by phospholipid, is widely used in China as an anticancer agent. The extracts from B. javanica induce cancer cell death by various mechanisms; however, it is not known whether these mechanisms involve autophagy, which is an important process in cancer development and treatment. Thus, the current study aimed to investigate whether BJOE modulates autophagy in HCT116 human colon cancer cells and whether modulation of autophagy is an anticancer mechanism of BJOE. Immunoblotting was employed to analyze the protein expression levels of microtubule-associated protein light-chain 3 (LC3), a specific protein marker of autophagy, in HCT116 cancer cells following exposure to BJOE. The apoptosis rate of the HCT116 cancer cells was detected by performing an Annexin V-fluorescein isothiocyanate/propidium iodide assay. According to the effect of BJOE administration on autophagy in the HCT116 cancer cells (induction or suppression), a functionally opposite agent (autophagy suppressor or inducer) was applied to counteract this effect, and the apoptosis rate of the cancer cells was detected again. The role of autophagy (pro-survival or pro-death) was demonstrated by comparing the rates of apoptotic cancer cells prior to and following the counteraction. The results revealed that BJOE suppressed the protein expression levels of LC3, including the LC3-I and LC3-II forms, and induced apoptosis in the HCT116 cancer cells with a high level of basal LC3. The apoptosis-inducing activity of BJOE was significantly attenuated when autophagy was induced by the administration of trehalose, an autophagy inducer. The data indicates that autophagy inhibition is involved in BJOE-induced cancer cell death, and that this inhibition may be a potential anticancer mechanism of BJOE.