LC3-II Protein Expression Research Articles

ASIC1/RIP1 accelerates atherosclerosis via disrupting lipophagy

IntroductionAtherosclerosis, a major contributor to cardiovascular disease, remains a significant health concern worldwide. While previous research has shown that acid-sensing ion channel 1 (ASIC1) impedes macrophage cholesterol efflux, its precise role in atherogenesis and the underlying mechanisms have remained elusive. ObjectivesThis study aimed to investigate the role of ASIC1 in atherosclerosis and its underlying mechanisms. MethodsFirst, data from a single-cell RNA sequencing (scRNA-seq) database were used to explore the relationships between ASIC1 differential expression and lipophagy in human atherosclerotic lesions. Finally, we validated the role of ASIC1/RIP1 signaling in lipophagy in vivo (human and mice) and in vitro (RAW264.7 and HTP-1 cells). ResultOur results demonstrated a significant increase in ASIC1 protein levels within CD68+ macrophages in both human aortic lesions and AopE-/- mouse lesion areas compared to nonlesion regions. Concurrently, there was a notable decrease in lipophagy, a crucial process for lipid metabolism. In vitro assays further elucidated that ASIC1 interaction with RIP1 (receptor-interacting protein 1) promoted the phosphorylation of RIP1 at serine 166 and transcription factor EB (TFEB) at serine 142, leading to disrupted lipophagy and increased lipid accumulation. Intriguingly, all these events were reversed upon ASIC1 deficiency and RIP1 inhibition. Furthermore, in ApoE-/- mouse models of atherosclerosis, silencing ASIC1 expression or inhibiting RIP1 activation not only significantly attenuated atherogenesis but also restored TFEB-mediated lipophagy in aortic tissues. This was evidenced by reduced TFEB Ser-142 phosphorylation, decreased LC3II and LAMP1 protein expression, increased numbers of lipophagosomes, and a decrease in lipid droplets. ConclusionOur findings unveil the critical role of macrophage ASIC1 in interacting with RIP1 to inhibit lipophagy, thereby promoting atherogenesis. Targeting ASIC1 represents a promising therapeutic avenue for the treatment of atherosclerosis.

Moschus ameliorates glutamate-induced cellular damage by regulating autophagy and apoptosis pathway

Alzheimer's disease (AD), a neurodegenerative disorder, causes short-term memory and cognition declines. It is estimated that one in three elderly people die from AD or other dementias. Chinese herbal medicine as a potential drug for treating AD has gained growing interest from many researchers. Moschus, a rare and valuable traditional Chinese animal medicine, was originally documented in Shennong Ben Cao Jing and recognized for its properties of reviving consciousness/resuscitation. Additionally, Moschus has the efficacy of “regulation of menstruation with blood activation, relief of swelling and pain” and is used for treating unconsciousness, stroke, coma, and cerebrovascular diseases. However, it is uncertain whether Moschus has any protective effect on AD patients. We explored whether Moschus could protect glutamate (Glu)-induced PC12 cells from cellular injury and preliminarily explored their related action mechanisms. The chemical compounds of Moschus were analyzed and identified by GC–MS. The Glu-induced differentiated PC12 cell model was thought to be the common AD cellular model. The study aims to preliminarily investigate the intervention effect of Moschus on Glu-induced PC12 cell damage as well as their related action mechanisms. Cell viability, lactate dehydrogenase (LDH), mitochondrial reactive oxygen species, mitochondrial membrane potential (MMP), cell apoptosis, autophagic vacuoles, autolysosomes or autophagosomes, proteins related to apoptosis, and the proteins related to autophagy were examined and analyzed. Seventeen active compounds of the Moschus sample were identified based on GC–MS analysis. In comparison to the control group, Glu stimulation increased cell viability loss, LDH release, mitochondrial damage, loss of MMP, apoptosis rate, and the number of cells containing autophagic vacuoles, and autolysosomes or autophagosomes, while these results were decreased after the pretreatment with Moschus and 3-methyladenine (3-MA). Furthermore, Glu stimulation significantly increased cleaved caspase-3, Beclin1, and LC3II protein expression, and reduced B-cell lymphoma 2/BAX ratio and p62 protein expression, but these results were reversed after pretreatment of Moschus and 3-MA. Moschus has protective activity in Glu-induced PC12 cell injury, and the potential mechanism might involve the regulation of autophagy and apoptosis. Our study may promote research on Moschus in the field of neurodegenerative diseases, and Moschus may be considered as a potential therapeutic agent for AD.

Association of Subclinical Thyroid Dysfunction with Cognitive Impairment in Rats: The Role of Autophagy.

We investigated the effect of subclinical hyperthyroidism and subclinical hypothyroidism on cognitive function in rats and the role of autophagy in this process. Forty Wistar rats were randomized into normal control (NC), hyperthyroidism (Hyper), hypothyroidism (Hypo), subclinical hyperthyroidism (sHyper), and subclinical hypothyroidism (sHypo) groups. Cognitive function (spatial learning and memory) was tested by the Morris water maze test. Hippocampal histopathology was analyzed by H&E staining, and expression levels of caspase-3 in hippocampal CA1 neurons were measured. In addition, immunoblot analysis was performed to detect hippocampal autophagy-related proteins. Escape latency from day 1 to day 4 was significantly longer in the Hypo, Hyper, and sHyper groups than in the NC group (P < 0.01). In addition, the number of rats crossing the virtual platform was significantly lower in the Hypo, Hyper, and sHyper groups than in the NC group (P < 0.01). Compared with the NC group, all four groups had significantly lower residence time in the target quadrant (P < 0.05). Beclin-1 and LC3-II protein expression in hippocampal tissues was significantly higher in the Hyper and sHyper groups than in the NC group (P < 0.01). Beclin-1 and LC3-II protein expression in hippocampal tissues did not significantly differ between the sHypo group and NC group (P > 0.05). Subclinical thyroid dysfunction in rats might lead to cognitive impairment. Subclinical hyperthyroidism might be associated with excessive activation of autophagy and hippocampal neuron damage and necrosis.

Regulatory Mechanism of Mangiferin Combined with Bortezomib on Malignant Biological Behavior of Burkitt Lymphoma and Its Effect on Expression of CXC Chemokine Receptors

To analyze the effects of mangiferin combined with bortezomib on the proliferation, invasion, apoptosis and autophagy of human Burkitt lymphoma Raji cells, as well as the expression of CXC chemokine receptors (CXCRs) family, and explore the molecular mechanism between them to provide scientific basis for basic research and clinical work of Burkitt lymphoma. Raji cells were intervened with different concentrations of mangiferin and bortezomib alone or in combination, then cell proliferation was detected by CCK-8 assay, cell invasion ability was detected by Transwell chamber method, cell apoptosis was detected by Annexin V/PI double-staining flow cytometry, apoptosis, autophagy and Akt/mTOR pathway protein expression were detected by Western blot, and the expression changes of CXCR family was detected by real-time quantitative PCR (RT-qPCR). Different concentrations of mangiferin intervened Raji cells for different time could inhibit cell viability in a concentration- and time-dependent manner (r =-0.682, r =-0.836). When Raji cells were intervened by combination of mangiferin and bortezomib, compared with single drug group, the proliferation and invasion abilities were significantly decreased, while the apoptosis level was significantly increased (P <0.01). Mangiferin combined with bortezomib could significantly up-regulate the expression of pro-apoptotic protein Bax and down-regulate the expression of anti-apoptotic protein Bcl-2 after intervention in Raji cells. Caspase-3 was also hydrolyzed and activated, and then induced the apoptosis of Raji cells. Mangiferin combined with bortezomib could up-regulate the expression of LC3Ⅱ protein in Raji cells, and the ratio of LC3Ⅱ/LC3Ⅰ in cells was significantly up-regulated compared with single drug or control group (P <0.01). Mangiferin combined with bortezomib could significantly inhibit the phosphorylation levels of Akt and mTOR, inhibit the proliferation and invasion of Raji cells by inhibiting Akt/mTOR pathway, and induce cell autophagy and apoptosis. Mangiferin and bortezomib could down-regulate the expressions of CXCR4 and CXCR7 mRNA after single-agent intervention in Raji cells, and the down-regulations of CXCR4 and CXCR7 mRNA expression were more significant when the two drugs were combined (P <0.01). Mangiferin alone or combined with bortezomib had no significant effect on CXCR5 mRNA expression in Raji cells (P >0.05), while the combination of the two drugs could down-regulate the expression of CXCR3 (P <0.05). Mangiferin combined with bortezomib can synergistically inhibit the proliferation and invasion of Raji cells, and induce autophagy and apoptosis. The mechanism may be related to the inhibition of Akt/mTOR signaling pathway, down-regulation of anti-apoptotic protein Bcl-2 and up-regulation of pro-apoptotic protein Bax, and the inhibition of the expression of CXCR family.

Bacterial lipoprotein plays an important role in the macrophage autophagy and apoptosis induced by Salmonella typhimurium and Staphylococcus aureus.

This study aimed to determine the role of bacterial lipoprotein (BLP) in autophagy and apoptosis. Western blot was used to examine autophagy biomarkers in mouse bone marrow-derived macrophages (BMDMs) after infection with Salmonella typhimurium (S. typhimurium) and Staphylococcus aureus (S. aureus) and BLP stimulation. In BMDMs, enhanced protein expression of LC3-II was observed after S. typhimurium or S. aureus infection (P < 0.05) and BLP stimulation (P < 0.05). Autophagy inhibition by chloroquine resulted in increased levels of LC3-Ⅱ and p62 protein (P < 0.05). Persistently upregulated expressions of Atg3 and Atg7 were observed following BLP stimulation (P < 0.05), and knockdown of Atg3 or Atg7 significantly attenuated BLP-enhanced protein expression of LC3-Ⅱ in BMDMs. Furthermore, we found that the autophagy inhibitor 3-methyladenine prevented BLP- and infection-induced macrophage apoptosis. BLP is not only required for autophagy and apoptosis activation in macrophages but also for regulating the balance between autophagy and apoptosis.

Neuroprotective effect and mechanism of cPLA2 inhibitor increases autophagic flux on spinal cord injury

To investigate the mechanism of cytosolic phospholipase A2(cPLA2) inhibitor to improve neurological function after spinal cord injury (SCI). Thirty-six 3 months old female SD rats, with body mass (280±20) g, were divided into three groups (n=12):sham group, SCI group, and SCI+ arachidonyl trifluoromethyl ketone(AACOCF3) group. Balloon compression SCI model was established in all three groups. In the sham model group, the spinal cord compression model was created after the balloon was placed without pressure treatment, and the remaining two groups were pressurized with the balloon for 48 h. After successful modeling, rats in the SCI+AACOCF3 group were injected intraperitoneally with AACOCF3, a specific inhibitor of cPLA2. The remaining two groups of rats were injected intraperitoneally with saline. The animals were sacrificed in batches on 7 and 14 days after modeling, respectively. And the damaged spinal cord tissues were sampled for pathomorphological observation, to detect the expression of cPLA2 and various autophagic fluxPrelated molecules and test the recovery of motor function. Spinal cord histomorphometry examination showed that the spinal cord tissue in the sham group was structurally intact, with normal numbers and morphology of neurons and glial cells. In the SCI group, spinal cord tissue fractures with large and prominent spinal cord cavities were seen. In the SCI+AACOCF3 group, the spinal cord tissue was more intact than in the SCI group, with more fused spinal cord cavities, more surviving neurons, and less glial cell hyperplasia. Western blot showed that the sham group had the lowest protein expression of LC3-Ⅱ, Beclin 1, p62, and cPLA2 compared with the SCI and SCI+AACOCF3 groups (P<0.05) and the highest protein expression of LC3-Ⅰ (P<0.05). P62 and cPLA2 expression in the SCI group were higher than in the SCI+AACOCF3 group (P<0.05). Behavioral observations showed that the time corresponding to BBB exercise scores was significantly lower in both the SCI and SCI+AACOCF3 groups than in the sham group (P<0.05). Scores at 3, 7, and 14 days after pressurization were higher in the SCI+AACOCF3 group than in the SCI group (P<0.05). cPLA2 inhibitors can reduce neuronal damage secondary to SCI, promote neurological recovery and improve motor function by improving lysosomal membrane permeability and regulating autophagic flux.

Catalpol Ameliorates Myocardial I/R Injury Through PI3k/Akt/P53 Signaling-mediated Anti-autophagy and Anti-apoptosis

Background Myocardial infarction (MI) is a leading cardiovascular disease worldwide, with high mortality. Purpose The study aimed to observe the mechanism of catalpol inhibiting apoptosis and autophagy in H9c2 cells after ischemia/reperfusion (I/R) injury. Materials and Methods We grouped H9c2 cells into 4 groups: control, I/R injury, catalpol (I/R injury +1 ug/mL catalpol treatment), and wortmannin (I/R injury + 1 ug/mL catalpol +150 nM wortmannin treatment) groups. Both catalpol and wortmannin group cells were given drug treatment 30 min before I/R injury. At 2h post-I/R insult, we used flow cytometry for detecting cellular apoptosis and reactive oxygen species (ROS) levels. We identified p-PI3K, p-Akt, PI3K, Akt, p53, Bcl-2, Caspase-3, Bax, beclin1, LC3II, and LC3I protein expression levels. Results I/R significantly increased the apoptosis rate and ROS level of H9c2 cells, increased expression levels of p53, LC3II/LC3I, and Caspase-3, and decreased p-Akt/Akt, beclin1, Bcl-2/Bax, and p-PI3K/PI3K expression levels. Catalpol can reduce the expressions of p53, Caspase-3, and LC3II/LC3I ( p &lt; 0.5). Catalpol can increase the expression levels of Akt/ p-Akt, Bax/ Bcl-2, and beclin1. Also, it can inhibit apoptosis and autophagy levels of H9c2 cells ( p &lt; 0.5). Wortmannin, a PI3 K-specific inhibitor, was able partially to block the catalpol’s and anti-autophagic and anti-apoptotic effects. Conclusion Catalpol can inhibit apoptosis, reduces excessive autophagy, and alleviates the effects of myocardial ischemia-reperfusion through PI3K/Akt/p53 pathway.

Enhancing Efficacy of Albumin-Bound Paclitaxel for Human Lung and Colorectal Cancers through Autophagy Receptor Sequestosome 1 (SQSTM1)/p62-Mediated Nanodrug Delivery and Cancer therapy.

Selective autophagy is a defense mechanism by which foreign pathogens and abnormal substances are processed to maintain cellular homeostasis. Sequestosome 1 (SQSTM1)/p62, a vital selective autophagy receptor, recruits ubiquitinated cargo to form autophagosomes for lysosomal degradation. Nab-PTX is an albumin-bound paclitaxel nanoparticle used in clinical cancer therapy. However, the role of SQSTM1 in regulating the delivery and efficacy of nanodrugs remains unclear. Here we showed that SQSTM1 plays a crucial role in Nab-PTX drug delivery and efficacy in human lung and colorectal cancers. Nab-PTX induces SQSTM1 phosphorylation at Ser403, which facilitates its incorporation into the selective autophagy of nanoparticles, known as nanoparticulophagy. Nab-PTX increased LC3-II protein expression, which triggered autophagosome formation. SQSTM1 enhanced Nab-PTX recognition to form autophagosomes, which were delivered to lysosomes for albumin degradation, thereby releasing PTX to induce mitotic catastrophe and apoptosis. Knockout of SQSTM1 downregulated Nab-PTX-induced mitotic catastrophe, apoptosis, and tumor inhibition in vitro and in vivo and inhibited Nab-PTX-induced caspase 3 activation via a p53-independent pathway. Ectopic expression of SQSTM1 by transfection of an SQSTM1-GFP vector restored the drug efficacy of Nab-PTX. Importantly, SQSTM1 is highly expressed in advanced lung and colorectal tumors and is associated with poor overall survival in clinical patients. Targeting SQSTM1 may provide an important strategy to improve nanodrug efficacy in clinical cancer therapy. This study demonstrates the enhanced efficacy of Nab-PTX for human lung and colorectal cancers via SQSTM1-mediated nanodrug delivery.

Enhanced efficacy with reduced toxicity of tripterygium glycoside tablet by compatibility with total glucosides of paeony for rheumatoid arthritis therapy

Background and aimsDrug-induced liver injury remains a critical issue to hinder clinical application of Tripterygium Glycosides Tablets (TGTs) for rheumatoid arthritis (RA) therapy. Combination of TGTs with Total Glucosides of Peony (TGP) may be the most common therapeutic strategy for enhancing TGTs’ efficacy and reducing its toxicity. Herein, we aimed to investigate the efficacy-enhancing and toxicity-reducing properties and mechanisms of TGT-TGP combination. MethodsBoth TGT-induced acute and chronic liver injury animal models were established. ELISA, transmission electron microscopy, immunohistochemistry, western blot and quantitative PCR were performed to determine the efficacy, toxicity and regulatory mechanisms of TGT-TGP combination. ResultsThe compatibility of TGP significantly reduced the abnormal serum ALT and AST levels, and improved liver histopathological changes in both acute and chronic DILI animal models induced by TGTs, with the most effective dosage of TGP-M (medium-dose TGP, 450 mg/kg). Additionally, TGP and TGT synergistically alleviated joint swelling and improved the elevation of serum inflammatory factors, in line with the positive changes in joint histopathological features of collagen induced arthritis mice, with the same effective dosage of TGP-M following 5 weeks’ drug combination treatment. Mechanically, TGT significantly increased the number of autophagosomes and the expression of LC3II protein while reducing p62 protein expression in the liver tissues, which were significantly reversed by the compatibility with TGP, similar to the findings based on the inflamed joint tissues. ConclusionsThese findings suggest an enhanced efficacy with reduced toxicity of TGT by the compatibility with TGP for RA therapy, possibly through regulating various autophagy-related proteins.

Role of PI3K/Akt/mTOR pathway-mediated macrophage autophagy in affecting the phenotype transformation of lung fibroblasts induced by silica dust exposure.

The phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway is one of the main signaling pathways related to autophagy. Autophagy plays a key role in the formation of silicosis fibrosis. The phenotypic transformation of lung fibroblasts into myofibroblasts is a hallmark of the transition from the inflammatory phase to the fibrotic phase in silicosis. This study aims to investigate whether the PI3K/Akt/mTOR pathway affects the phenotypic transformation of silicosis-induced lung fibroblasts into myofibroblasts via mediating macrophage autophagy. The human monocytic leukemia cell line THP-1 cells were differentiated into macrophages by treating with 100 ng/mL of phorbol ester for 24 h. Macrophages were exposed to different concentrations (0, 25, 50, 100, 200, 400 μg/mL) and different times (0, 6, 12, 24, 48 h) of SiO2 dust suspension. The survival rate of macrophages was measured by cell counting kit-8 (CCK-8) method. Enzyme linked immunosorbent assay (ELISA) was used to measure the contents of transforming growth factor-β1 (TGF-β1) and tumor necrosis factor-α (TNF-α) in the cell supernatant. The co-culture system of macrophages and HFL-1 cells was established by transwell. A blank control group, a SiO2 group, a LY294002 group, a SC79 group, a LY294002+SiO2 group, and a SC79+SiO2 group were set up in this experiment. Macrophages in the LY294002+SiO2 group were pretreated with LY294002 (PI3K inhibitor) for 18 hours, and macrophages in the SC79+SiO2 group were pretreated with SC79 (Akt activator) for 24 hours, and then exposed to SiO2 (100 μg/mL) dust suspension for 12 hours. The expression of microtubule-associated protein 1 light chain 3 (LC3) protein in macrophages was detected by the immunofluorescence method. The protein expressions of PI3K, Akt, mTOR, Beclin-1, LC3 in macrophages, and collagen III (Col III), α-smooth muscle actin (α-SMA), fibronectin (FN), matrix metalloproteinase-1 (MMP-1), tissue metalloproteinase inhibitor-1 (TIMP-1) in HFL-1 cells were measured by Western blotting. After the macrophages were exposed to SiO2 dust suspension of different concentrations for 12 h, the survival rates of macrophages were gradually decreased with the increase of SiO2 concentration. Compared with the 0 μg/mL group, the survival rates of macrophages in the 100, 200, and 400 μg/mL groups were significantly decreased, and the concentrations of TGF-β1 and TNF-α in the cell supernatant were obviously increased (all P<0.05). When 100 μg/mL SiO2 dust suspension was applied to macrophages, the survival rates of macrophages were decreased with the prolonged exposure time. Compared with the 0 h group, the survival rates of macrophages were significantly decreased (all P<0.05), the concentrations of TGF-β1 and TNF-α in the cell supernatant were significantly increased, and the protein expression levels of Beclin-1 and LC3II were increased markedly in the 6, 12, 24, and 48 h groups (all P<0.05). Immunofluorescence results demonstrated that after exposure to SiO2 (100 μg/mL) dust for 12 h, LC3 exhibited punctate aggregation and significantly higher fluorescence intensity compared to the blank control group (P<0.05). Compared with the blank control group, the protein expressions of Col III, FN, α-SMA, MMP-1, and TIMP-1 in HFL-1 cells were up-regulated in the SiO2 group (all P<0.05). Compared with the SiO2 group, the protein expressions of PI3K, Akt, and mTOR were down-regulated and the protein expressions of LC3II and Beclin-1 were up-regulated in macrophages (all P<0.05), the contents of TNF-α and TGF-β1 in the cell supernatant were decreased (both P<0.01), and the protein expressions of Col III, FN, α-SMA, MMP-1, and TIMP-1 in HFL-1 cells were down-regulated (all P<0.05) in the LY294002+SiO2 group. Compared with the SiO2 group, the protein expressions of PI3K, Akt, and mTOR were up-regulated and the protein expressions of LC3II and Beclin-1 were down-regulated in macrophages (all P<0.05), the contents of TNF-α and TGF-β1 in the cell supernatant were increased (both P<0.01), and the protein expressions of Col III, FN, α-SMA, MMP-1, and TIMP-1 in HFL-1 cells were up-regulated (all P<0.05) in the SC79+SiO2 group. Silica dust exposure inhibits the PI3K/Akt/mTOR pathway, increases autophagy and concentration of inflammatory factors in macrophages, and promotes the phenotype transformation of HFL-1 cells into myofibroblasts. The regulation of the PI3K/Akt/mTOR pathway can affect the autophagy induction and the concentration of inflammatory factors of macrophages by silica dust exposure, and then affect the phenotype transformation of HFL-1 cells into myofibroblasts induced by silica dust exposure.

Yiqi Qingre Xiaozheng formula protects against diabetic nephropathy by restoring autophagy in mice

To evaluate the protective effect of Yiqi Qingre Xiaozheng formula (YQQRXZF) via the regulation of autophagy in diabetic nephropathy (DN) mice induced by injection of streptozotocin (STZ) after high fat diet (HFD). The composition of YQQRXZF was analyzed by high performance liquid chromatography-electrospray ionization/mass spectrometry (HPLC-ESI/MSn). The DN model was induced by intraperitoneally injection of 50 mg/kg STZ within 5 days, followed by HFD feeding for 8 weeks in C57BL/6J mice. Mice were randomly separated into DN group, YQQRXZF group, irbesartan group, and control group. Blood glucose was calculated, and body weight was measured every 2 weeks. An enzyme-linked immunosorbent assay was used to measure the urinary albumin-to-creatinine ratio (uACR) before and after treatment, and the serum concentrations of total cholesterol (TC), low-density lipoprotein (LDL), triglycerides (TG), high-density lipoprotein (HDL), blood urea nitrogen (BUN), and serum creatinine (Scr) were measured. In addition, hematoxylin-eosin (H&E) staining, periodic acid-Schiff (PAS) staining, Masson’s trichrome staining, and transmission electron microscopy (TEM) were used to observe pathological changes in renal tissue. Autophagy levels were determined by immunofluorescence staining. In this study, 21 dominant chemical constituents were identified in YQQRXZF. The treatment group reduced uACR in a more significant way than the DN group (P = .018). The treatment group reduced TC, TG, and LDL concentrations after YQQRXFF treatment (P = .021, P = .014 and P = .026, respectively). H&E, PAS, and Masson staining showed that pathological damage to mice kidneys improved, the volume of renal glomeruli was reduced, and glomerular sclerosis and fibrosis were reduced. Immunofluorescence analysis revealed that expressions of LC3-Ⅱprotein increased in the treatment group (P = .013). In contrast, the protein expressions of P62 were reduced after treatment (P = .025). YQQRXZF effectively attenuates kidney injury, probably by regulating autophagy.

Identification of Fangjihuangqi Decoction as a late-stage autophagy inhibitor with an adjuvant anti-tumor effect against non-small cell lung cancer

BackgroundClinically, although chemotherapy is one of the most commonly used methods of treating tumors, chemotherapeutic drugs can induce autophagic flux and increase tumor cell resistance, leading to drug tolerance. Therefore, theoretically, inhibiting autophagy may improve the efficacy of chemotherapy. The discovery of autophagy regulators and their potential application as adjuvant anti-cancer drugs is of substantial importance. In this study, we clarified that Fangjihuangqi Decoction (FJHQ, traditional Chinese medicine) is an autophagy inhibitor, which can synergistically enhance the effect of cisplatin and paclitaxel on non-small cell lung cancer (NSCLC) cells.MethodsWe observed the changes of autophagy level in NSCLC cells under the effect of FJHQ, and verified the level of the autophagy marker protein and cathepsin. Apoptosis was detected after the combination of FJHQ with cisplatin or paclitaxel, and NAC (ROS scavenger) was further used to verify the activation of ROS-MAPK pathway by FJHQ.ResultsWe observed that FJHQ induced autophagosomes in NSCLC cells and increased the levels of P62 and LC3-II protein expression in a concentration- and time-gradient-dependent manner, indicating that autophagic flux was inhibited. Co-localization experiments further showed that while FJHQ did not inhibit autophagosome and lysosome fusion, it affected the maturation of cathepsin and thus inhibited the autophagic pathway. Finally, we found that the combination of FJHQ with cisplatin or paclitaxel increased the apoptosis rate of NSCLC cells, due to increased ROS accumulation and further activation of the ROS-MAPK pathway. This synergistic effect could be reversed by NAC.ConclusionCollectively, these results demonstrate that FJHQ is a novel late-stage autophagy inhibitor that can amplify the anti-tumor effect of cisplatin and paclitaxel against NSCLC cells.

Effect of peroxiredoxin 1 on the regulation of trophoblast function by affecting autophagy and oxidative stress in preeclampsia

PurposePE is a pregnancy-specific syndrome and one of the main causes of maternal, fetal, and neonatal mortality. PRDX1 is an antioxidant that regulates cell proliferation, differentiation, and apoptosis. The aim of this study is to investigate the effect of PRDX1 on the regulation of trophoblast function by affecting autophagy and oxidative stress in preeclampsia.MethodsWestern blotting, RT-qPCR, and immunofluorescence were used to examine the expression of PRDX1 in placentas. PRDX1-siRNA was transfected to knockdown PRDX1 in HTR-8/SVneo cells. The biological function of HTR-8/SVneo cells was detected by wound healing, invasion, tube formation, CCK-8, EdU, flow cytometry, and TUNEL assays. Western blotting was used to detect the protein expression of cleaved-Caspase3, Bax, LC3II, Beclin1, PTEN, and p-AKT. DCFH-DA staining was used to detect ROS levels by flow cytometry.ResultsPRDX1 was significantly decreased in placental trophoblasts in PE patients. Following the exposure of HTR-8/SVneo cells to H2O2, PRDX1 expression was significantly decreased, LC3II and Beclin1 expression was notably increased, and ROS level was also markedly increased. PRDX1 knockdown impaired migration, invasion, and tube-formation abilities and promoted apoptosis, which was accompanied by an increased expression of cleaved-Caspase3 and Bax. PRDX1 knockdown induced a significant decrease in LC3II and Beclin1 expression, along with an elevated p-AKT expression and a decreased PTEN expression. PRDX1 knockdown increased intracellular ROS levels, and NAC attenuated PRDX1 knockdown-induced apoptosis.ConclusionPRDX1 regulated trophoblast function through the PTEN/AKT signaling pathway to affect cell autophagy and ROS level, which provided a potential target for the treatment of PE.

Cadmium accelerates autophagy of osteocytes by inhibiting the PI3K/AKT/mTOR signaling pathway.

Cadmium (Cd) can damage bone cells and cause osteoporosis. Osteocytes are the most numerous bone cells and also important target cells for Cd-induced osteotoxic damage. Autophagy plays important role in the progression of osteoporosis. However, osteocyte autophagy in Cd-induced bone injury is not well characterized. Thus, we established a Cd-induced bone injury model in BALB/c mice and a cellular damage model in MLO-Y4 cells. Aqueous Cd exposure for 16 months showed an increase in plasma alkaline phosphatase (ALP) activity and increase in urine calcium (Ca) and phosphorus (P) concentrations in vivo. Moreover, expression level of autophagy-related microtubule-associated protein 1A/1B-light chain 3 II (LC3II) and autophagy-related 5 (ATG5) proteins were induced, and the expression of sequestosome-1 (p62) was reduced, along with Cd-induced trabecular bone damage. In addition, Cd inhibited the phosphorylation of mammalian target of rapamycin (mTOR), protein kinase B (AKT), and phosphatidylinositol 3-kinase (PI3K). In vitro, 80 μM Cd concentrations exposure upregulated LC3II protein expression, and downregulated of p62 protein expression. Similarly, we found that treatment with 80 μM Cd resulted in a reduction in the phosphorylation levels of mTOR, AKT, and PI3K. Further experiments revealed that addition of rapamycin, an autophagy inducer, enhanced autophagy and alleviated the Cd-induced damage to MLO-Y4 cells. The findings of our study reveal for the first time that Cd causes damage to both bone and osteocytes, as well as induces autophagy in osteocytes and inhibits PI3K/AKT/mTOR signaling, which could be a protective mechanism against Cd-induced bone injury.

Different effects of fenoldopam and angiotensin II on autophagy in vascular smooth muscle cells

Both dopamine and angiotensin II are essential neurotransmitters/hormones with pleiotropic functions in vascular smooth muscle cells (VSMCs). Autophagy, an intracellular self-degradative process that delivers cytoplasmic constituents to lysosomes, plays an important role in physiological homeostasis in VSMCs. Fenoldopam (Fen), a dopamine D 1 /D 5 receptor agonist, increases autophagic flux in renal proximal tubule cells, however, in VSMCs the roles of these two neurotransmitters on autophagy are unknown. In rat aortic VSMCs, Fen increased autophagy, determined by the protein expression of microtubule-associated protein 1 light chain (LC)3-II and autophagy related 5 (ATG5), in a concentration- and time-dependent manner. By contrast, angiotensin II (Ang II), the endogenous AT 1 R agonist, decreased the protein expression of LC3-II and ATG5 in a concentration-and time-dependent manner. Further studies showed that Fen (1 μM, 15 min) increased while Ang II (100 nM, 15 min) decreased cyclic AMP (cAMP) production in VSMCs (Vehicle: 100±35.4%, Fen: 513.7±95.8%, Ang II: 24.6±8.0%, n=6). Pre-treatment with Rp-cAMPS (50 μM, 6 hr), a PKA inhibitor, attenuated the Fen-mediated increase in LC3-II protein expression (Fen: 1 μM, 6 hr) (Vehicle: 100±15.0%, Fen: 251.8±37.2%, Fen+Rp-cAMP: 96.9±16.7%, n=6) and reversed the Ang II-mediated decrease in autophagy (Ang II: 100 nM, 6 hr) (Vehicle: 100±8.9%, Ang II: 44.9±6.3%, Ang II+Rp-cAMP: 95.4±11.0%, n=4). These results demonstrate distinct roles of Fen and Ang II on autophagy through regulation of cAMP/PKA signaling by their corresponding receptors in VSMCs. This study was supported by National Institutes of Health of US and National Natural Science Foundation of China. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

"Trichosanthis Fructus-Allii Macrostemonis Bulbus" combination inhibits NLRP3 inflammasome activation and reduces inflammatory response in RAW264.7 macrophage by inducing autophagy

This study aims to explore the effect of &quot;Trichosanthis Fructus-Allii Macrostemonis&quot; combination(GX) on the activation of NOD-, LRR-, and pyrin domain-containing protein 3(NLRP3) inflammasome, the release of inflammatory cytokines, and the level of autophagy in RAW264.7 macrophage damaged by lipopolysaccharide(LPS), and the mechanism of GX against inflammatory response in macrophages. To be specific, LPS was used to induce the injury of RAW264.7 cells. Cell Counting Kit-8(CCK-8) assay was employed to measure the survival rate of cells, and Western blot to detect the protein expression of NLRP3, apoptosis-associated speck-like protein(ASC), cysteine-aspartic acid protease(caspase)-1, interleukin(IL)-18, IL-1β, microtubule-associated protein light chain 3(LC3)-Ⅱ, and selective autophagy junction protein p62/sequestosome 1 in RAW264.7 macrophages. ELISA was used to measure the levels of IL-18 and IL-1β in RAW264.7 cells. Transmission electron microscopy was applied to observe the number of autophagosomes in RAW264.7 cells. Immunofulourescence staining was used to detect the expression of LC3-Ⅱ and p62 in RAW264.7 cells. The result showed that GX significantly reduced the protein expression of NLRP3, ASC, and caspase-1 in RAW264.7 cells, significantly increased the protein expression of LC3Ⅱ, decreased the expression of p62, significantly inhibited the secretion of IL-18 and IL-1β, significantly increased the number of autophagosomes, significantly enhanced the immunofluorescence of LC3Ⅱ, and reduced the immunofluorescence of p62. Furthermore, 3-methyladenine(3-MA) could reverse the inhibitory effect of GX on NLRP3, ASC, and caspase-1 and reduce the release of IL-18 and IL-1β. In summary, GX can increase of the autophagy activity of RAW264.7 and inhibit the activation of NLRP3 inflammasome, thereby reducing the release of inflammatory cytokines and suppressing inflammatory response in macrophages.

Effect of autophagy in cadmium chloride induced apoptosis of mouse spermatogenic cells

To study the effect of autophagy in cadmium chloride(CdCl_2)-induced apoptosis of mouse spermatocytes(GC-2 spd) cells and explore the underlying molecular mechanisms. The cells were treated with different concentrations of CdCl_2(0, 5 and 10 μmol/L) for 24 h. Hoechst33342 staining and monodansylcadaverine(MDC) were performed to explore the formation of autophagosomes and apoptotic bodies. The apoptosis of cadmium-treated cells was examined by TUNEL staining. Autophagy inhibitor 3-methyladenine(3-MA)(60 μmol/L), apoptotic inhibitorCaspase inhibitor Z-VAD-FMK( zVAD-FMK)(50 nmol/L), autophagy inducer rapamycin(RAPA)(50 nmol/L) and lysosomal inhibitor chloroquine(CQ)(10 μmol/L) were added to cell culture in the presence/absence of CdCl_2(10 μmol/L) to treat GC-2 spd cells for 24 h. The expression levels of autophagy-related proteins LC3, P62, and pro-apoptotic proteins cleaved Caspase-3 and cleaved Caspase-9 were examined by Western blot. Autophagosomes aggregated and the number of apoptotic cells increased after exposure to CdCl_2 for 24 h. Western blot result showed that in the 5 and 10 μmol/L CdCl_2 exposure groups, the protein expression levels of LC3II/LC3I increased to 9.23±0.81 and 12.15±0.80 compared with the control group(5.50±0.56)(P&lt;0.05), LC3II protein expression level increased to 3.35±0.14 and 3.47±0.32 compared with the control group(2.35±0.34)(P&lt;0.05), P62 protein expression level increased to 1.48±0.12 and 1.80±0.22 compared with the control group(0.83±0.09)(P&lt;0.05). Compared with the CdCl_2-treated group, the protein expression levels of LC3II/LC3I, LC3II, P62, cleaved Caspase-9 and cleaved Caspase-3 after 3-MA treatment decreased to 0.90±0.07(CdCl_2 group: 1.47±0.06), 1.57±0.14(CdCl_2 group: 2.45±0.29), 0.82±0.05(CdCl_2 group: 1.44±0.18), 0.18±0.01(CdCl_2 group: 0.28±0.01) and 0.61±0.84(CdCl_2 group: 1.15±0.04)(P&lt;0.05). Compared with the CdCl_2-treated group, the protein expression levels of cleaved Caspase-9 and cleaved Caspase-3 after zVAD-FMK treatment decreased to 0.12±0.01(CdCl_2 group: 0.28±0.01) and 0.34±0.01(CdCl_2 group: 1.15±0.04)(P&lt;0.05), while those of LC3II/LC3I, LC3II and P62 had no significant change(P&gt;0.05). Compared with the CdCl_2-treated group, RAPA enhanced cadmium-induced LC3II/LC3I, LC3II and P62 protein expressions to 2.22±0.21(CdCl_2 group: 1.56±0.06), 3.72±0.21(CdCl_2 group: 2.97±0.15) and 2.41±0.19(CdCl_2 group: 1.52±0.35)(P&lt;0.05). Western blot result showed that compared with the CdCl_2 group, the protein expressions of LC3II/LC3I, LC3II, P62 and cleaved Caspase-3 in the CdCl_2 and CQ treatment groups increased to 3.21±0.31(CdCl_2 group: 2.09±0.25), 4.49±0.43(CdCl_2 group: 2.72±0.26), 2.59±0.19(CdCl_2 group: 1.84±0.19) and 2.43±0.23(CdCl_2 group: 1.50±0.27)(P&lt;0.05). Cadmium chloride induces apoptosis of mouse spermatocyte cells by inhibiting autophagosome-lysosomal fusion and prompting abnormal aggregation of autophagosomes.

Effects of true-to-life PET nanoplastics using primary human nasal epithelial cells

Since inhalation is a relevant exposure route, studies using appropriate micro/nanoplastic (MNPLs) models, representative targeted cells, and relevant biomarkers of effect are required. We have used lab-made polyethylene terephthalate (PET)NPLs obtained from PET plastic water bottles. Human primary nasal epithelial cells (HNEpCs) were used as a model of the first barrier of the respiratory system. Cell internalization and intracellular reactive oxygen species (iROS) induction, as well as the effects on mitochondria functionality and in the modulation of the autophagy pathway, were evaluated. The data indicated significant cellular uptake and increased levels of iROS. Furthermore, a loss of mitochondrial membrane potential was observed in the exposed cells. Regarding the effects on the autophagy pathway, PETNPLs exposure significantly increases LC3-II protein expression levels. PETNPLs exposure also induced significant increases in the expression of p62. This is the first study showing that true-to-life PETNPLs can alter the autophagy pathway in HNEpCs.

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.

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.