Expression Of Autophagy-associated Proteins Research Articles

Regulation of platelet activation and thrombus formation in acute non-ST segment elevation myocardial infarction: Role of Beclin1.

This study aims to investigate the mechanism of platelet activation-induced thrombosis in patients with acute non-ST segment elevation myocardial infarction (NSTEMI) by detecting the expression of autophagy-associated proteins in platelets of patients with NSTEMI. A prospective study was conducted on 121 patients with NSTEMI who underwent emergency coronary angiography and optical coherence tomography. The participants were divided into two groups: the ST segment un-offset group (n = 64) and the ST segment depression group (n = 57). We selected a control group of 60 patients without AMI during the same period. The levels of autophagy-associated proteins and the expression of autophagy-associated proteins in platelets were measured using immunofluorescence staining and Western blot. In NSTEMI, the prevalence of red thrombus was higher in the ST segment un-offset myocardial infarction (STUMI) group, whereas white thrombus was more common in the ST segment depression myocardial infarction (STDMI) group. Furthermore, the platelet aggregation rate was significantly higher in the white thrombus group compared with the red thrombus group. Compared with the control group, the autophagy-related protein expression decreased, and the expression of αIIbβ3 increased in NSTEMI. The overexpression of Beclin1 could activate platelet autophagy and inhibit the expression of αIIbβ3. The results suggested that the increase in platelet aggregation rate in patients with NSTEMI may be potentially related to the change in autophagy. And the overexpression of Beclin1 could reduce the platelet aggregation rate by activating platelet autophagy. Our findings demonstrated that Beclin1 could be a potential therapeutic target for inhibiting platelet aggregation in NSTEMI.

Polyvinylpyrrolidone/chitosan-loaded dihydromyricetin-based nanofiber membrane promotes diabetic wound healing by anti-inflammatory and regulating autophagy-associated protein expression

The healing of wounds in diabetics is commonly delayed by recurring infections and persistent inflammation at the wound site. For this reason, we conducted a study using the electrospinning technique to create nanofiber membranes consisting of polyvinylpyrrolidone/chitosan (PVP/CS) and incorporated dihydromyricetin (DHM) into them. Infrared Fourier transform spectroscopy and scanning electron microscopy were used to analyze the nanofiber membrane. Experimental results in vitro have shown that PVP/CS/DHM has exceptional properties such as hydrophilicity, porosity, water vapor transport rate, antioxidant capacity, and antibacterial activity. Moreover, our study has demonstrated that the application of PVP/CS/DHM can significantly improve wound healing in diabetic mice. After an 18-day treatment period, a remarkable wound closure rate of 88.63 ± 1.37 % was achieved. The in vivo experiments revealed that PVP/CS/DHM can promote diabetic wound healing by suppressing the activation of TLR4/MyD88/NF-κB signaling pathway and enhancing autophagy-related protein as well as CD31 and HIF-1α expression in skin tissues. This study showed that PVP/CS/DHM is a promising wound dressing.

TRAF6 triggers Mycobacterium-infected host autophagy through Rab7 ubiquitination.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an E3 ubiquitin ligase that is extensively involved in the autophagy process by interacting with diverse autophagy initiation and autophagosome maturation molecules. However, whether TRAF6 interacts with lysosomal proteins to regulate Mycobacterium-induced autophagy has not been completely characterized. Herein, the present study showed that TRAF6 interacted with lysosomal key proteins Rab7 through RING domain which caused Rab7 ubiquitination and subsequently ubiquitinated Rab7 binds to STX17 (syntaxin 17, a SNARE protein that is essential for mature autophagosome), and thus promoted the fusion of autophagosomes and lysosomes. Furthermore, TRAF6 enhanced the initiation and formation of autophagosomes in Mycobacterium-induced autophagy in both BMDMs and RAW264.7 cells, as evidenced by autophagic flux, colocalization of LC3 and BCG, autophagy rates, and autophagy-associated protein expression. Noteworthy to mention, TRAF6 deficiency exacerbated lung injury and promoted BCG survival. Taken together, these results identify novel molecular and cellular mechanisms by which TRAF6 positively regulates Mycobacterium-induced autophagy.

Metal-DNA Nanocomplexes Enhance Chemo-dynamic Therapy by Inhibiting Autophagy-Mediated Resistance.

Chemo-dynamic therapy (CDT) based on the Fenton or Fenton-like reaction has emerged as a promising approach for cancer treatment. However, autophagy-mediated self-protection mechanisms of cancer cells pose a significant challenge to the efficacy of CDT. Herein, we developed metal-DNA nanocomplexes (DACs-Mn) to enhance CDT via DNAzyme inhibition of autophagy. Specifically, Mn-based catalyst in DACs-Mn was used to generate highly hydroxyl radicals (⋅OH) that kill cancer cells, while the ATG5 DNAzyme incorporated into DACs-Mn inhibited the expression of autophagy-associated proteins, thereby improving the efficacy of CDT. By disrupting the self-protective pathway of cells under severe oxidative stress, this novel approach of DACs-Mn was found to synergistically enhance CDT in both in vitro and in vivo models, effectively amplifying tumor-specific oxidative damage. Notably, the Metal-DNA nanocomplexes can also induce immunogenic cell death (ICD), thereby inhibiting tumor metastasis. Specifically, in a bilateral tumor model in mice, the combined approach of CDT and autophagy inhibition followed by immune checkpoint blockade therapy shown significant potential as a novel and effective treatment modality for primary and metastatic tumors.

Interleukin‑22 alleviates arginine‑induced pancreatic acinar cell injury via the regulation of intracellular vesicle transport system: Evidence from proteomic analysis.

Acute pancreatitis (AP) is a severe inflammatory condition characterized by the activation of pancreatic enzymes within acinar cells, leading to tissue damage and inflammation. Interleukin (IL)-22 is a potential therapeutic agent for AP owing to its anti-inflammatory properties and ability to promote tissue repair. The present study evaluated the differentially expressed proteins in arginine-induced pancreatic acinar cell injury following treatment with IL-22, and the possible mechanisms involved in IL-22-mediated alleviation of AP. AR42J cells were stimulated using L-arginine to establish an acinar cell injury model in vitro and the damaged cells were subsequently treated with IL-22. The characteristics of the model and the potential therapeutic effects of IL-22 were examined by CCK-8 assay, flow cytometry, TUNEL assay, transmission electron microscopy and ELISA. Differentially expressed proteins in cells induced by arginine and treated with IL-22 were assessed using liquid chromatography-mass spectrometry. The identified proteins were further subjected to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis to elucidate their functional roles. The present study demonstrated that arginine-stimulated cells showed significant pathological changes resembling those in AP, which were alleviated after IL-22 treatment. Proteomic analysis then demonstrated that in IL-22-treated cells, proteins related to the formation and fusion of autophagosomes with lysosomes were significantly downregulated, whereas endocytosis related proteins were enriched in the upregulated proteins. After IL-22 treatment, western blotting demonstrated reduced expression of autophagy-associated proteins. In conclusion, by inhibiting the formation and fusion of autophagosomes with lysosomes, IL-22 may have mitigated premature trypsinogen activation, subsequently minimizing acinar cell injury induced by L-arginine. This was accompanied by concurrent upregulation of endocytosis, which serves a pivotal role in sustaining regular cellular material transport and signal propagation. This research underscored the potential of IL-22 in mitigating arginine-induced AR42J injury, which could be valuable in refining treatment strategies for AP.

Wuzi Yanzong administration alleviates Sertoli cell injury by recovering AKT/mTOR-mediated autophagy and the mTORC1–mTROC2 balance in aging-induced testicular dysfunction

Ethnopharmacological relevanceWuzi Yanzong Prescription (WZ), a classic traditional Chinese medicine formula, has the properties of kidney nourishing and essence strengthening, and it is widely used to treat male infertility with a long history. Sertoli cells are injured with aging, resulting in testicular dysfunction, and WZ effectively rejuvenates the age-related decline of testicular function. However, whether the therapeutic effects of WZ on aging-related testicular dysfunction are dependent on the restoration of Sertoli cell function remains unclear. Aim of the studyIn a mouse model of natural aging, we explored the protective effects of WZ and its potential mechanisms. Materials and methodsFifteen-month-old C57BL/6 mice were randomized to receive either standard diet or WZ (2 and 8 g/kg) for 3 months. Meanwhile, 10 1-month-old mice were considered the adult control group and received standard diet for 3 months. The testis and epididymis were rapidly collected, and the sperm quality, testicular histology, Sertoli cell numbers, tight junction (TJ) ultrastructure, and blood–testis barrier-associated protein expression and localization were assessed. ResultsWZ significantly increased sperm concentration and sperm viability, improved the degenerative histomorphology and elevated the seminiferous epithelium height. Furthermore, WZ increased the number of Sertoli cells, restored the ultrastructure of the Sertoli cell TJ, and upregulated the expression of TJ-associated proteins (zonula occludens-1 and Claudin11), ectoplasm specialized-associated proteins (N-Cadherin, E-Cadherin and β-Catenin), and gap junction-associated protein (connexin 43), but did not affect the expression of Occludin and cytoskeletal protein (Vimentin). In addition, WZ did not change the localization of zonula occludens-1 and β-Catenin in aged testis. Moreover, WZ increased the expression of autophagy-associated proteins (light chain 3 beta and autophagy related 5) and decreased the expression of p62, phosphorylated mammalian target of rapamycin, and phosphorylated AKT in Sertoli cells. Finally, we found that WZ attenuated mTOR complex 1 (mTORC1) activity and upregulated mTORC2 activity, as evidenced by inhibition of the expression of the regulatory-associated protein of mTOR, phosphorylated p70 S6K, and phosphorylated ribosomal protein s6 and enhancement of the expression of Rictor in the Sertoli cells of aging mice. ConclusionsWZ improves the injury of Sertoli cells by restoring AKT/mTOR-mediated autophagy and the mTORC1–mTROC2 balance in Sertoli cells during aging. Our findings provide a new mechanism of WZ in the treatment of aging-induced testicular dysfunction.

Mitochondria-targeted combination treatment strategy counteracts myocardial reperfusion injury of aged rats by modulating autophagy and inflammatory response.

Aging, as a recognized risk factor for ischemic heart disease, interferes with protective mechanisms and abolishes the optimal effectiveness of cardioprotective interventions, leading to the loss of cardioprotection following myocardial ischemia/reperfusion (I/R) injury. This study was designed to explore the possible interaction of aging with cardioprotection induced by combination therapy with coenzyme Q10 (CoQ10) and mitochondrial transplantation in myocardial I/R injury of aged rats. Male Wistar rats (n = 72, 400-450g, 22-24 months old) were randomized into groups with/without I/R and/or CoQ10 and mitochondrial transplantation, alone or in a combinational mode. An in vivo model of myocardial I/R injury was established by left anterior descending coronary artery occlusion and re-opening. Mitochondria were isolated from donor rats and injected intramyocardially (150µl of the mitochondrial suspension containing 2 × 105±0.3 × 105 mitochondria) at the onset of reperfusion in recipient groups. CoQ10 (20mg/kg/day) was injected intramuscularly for 7 days before I/R operation. Lastly, myocardial function, cTn-I level, expression of autophagy-associated proteins (Beclin1, p62, and LC3-II/LC3-I), and the levels of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) were assessed. Co-application of CoQ10 and mitotherapy concomitantly improved myocardial function and decreased cTn-I level in aged reperfused hearts (P < .001). This combination therapy also modulated autophagic activity and decreased pro-inflammatory cytokines (P < .01 to P < .001). This combinational approach induced noticeable cardioprotection in comparison with monotherapies-received groups. We found that combination of CoQ10 and mitochondrial transplantation attenuated myocardial I/R injury in aged rats, in part by modulating autophagy and inflammatory response, hence, appears to restore aging-related loss of cardioprotection in aged patients.

LncRNA SNHG7 promotes non-small cell lung cancer progression and cisplatin resistance by inducing autophagic activity.

Cisplatin (DDP) is among the most widely used chemotherapeutic drugs for non-small cell lung cancer (NSCLC), yet the frequent emergence of chemoresistance serves as a major barrier to the treatment of this tumor type. Long non-coding RNAs (lncRNAs) have recently been shown to influence the ability of cells to resist particular chemotherapy drugs. The present study was developed to explore the role of the lncRNA SNHG7 as a regulator of NSCLC cell chemosensitivity. Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to measure SNHG7 expression in NSCLC tissues from patients that were sensitive/resistant to DDP, correlations between SNHG7 expression levels and the patients' clinicopathological characteristics were assessed, and the prognostic relevance of SNHG7 expression was examined via the Kaplan-Meier approach. In addition, SNHG7 expression was assessed in NSCLC cell lines that were DDP-sensitive or -resistant, while western blotting and immunofluorescence staining were employed to detect autophagy-associated protein expression in A549, A549/DDP, HCC827, and HCC827/DDP cells. NSCLC cell chemoresistance was quantified via the Cell Counting Kit-8 (CCK-8) assay approach, and flow cytometry was used to detect the apoptotic death of these tumor cells. The chemosensitivity of xenograft tumors in vivo was further assessed to validate the functional importance of SNHG7 as a regulator of NSCLC DDP resistance. Relative to paracancerous tissues, NSCLC tumors exhibited SNHG7 upregulation, and this lncRNA was further upregulated in DDP-resistant patients compared to chemosensitive patients. Consistently, higher SNHG7 expression levels were correlated with worse patient survival outcomes. DDP-resistant NSCLC cells were also found to exhibit higher levels of SNHG7 expression than chemosensitive cells, and knocking down this lncRNA enhanced the sensitivity of these cells to DDP treatment, resulting in impaired proliferation and higher rates of apoptotic death. Knocking down SNHG7 was also sufficient to suppress microtubule associated protein 1 light chain 3 beta (LC3B) and Beclin1 protein levels and promote p62 upregulation in vitro. The silencing of this lncRNA additionally inhibited the resistance of NSCLC xenograft tumors to DDP treatment in vivo. SNHG7 can promote malignant behaviors and DDP resistance in NSCLC cells at least partly via the induction of autophagic activity.

Imatinib Mesylate Induces Necroptotic Cell Death and Impairs Autophagic Flux in Human Cardiac Progenitor Cells.

The receptor tyrosine kinase inhibitor imatinib improves patient cancer survival but is linked to cardiotoxicity. This study investigated imatinib’s effects on cell viability, apoptosis, autophagy, and necroptosis in human cardiac progenitor cells in vitro. Imatinib reduced cell viability (75.9 ± 2.7% vs. 100.0 ± 0.0%) at concentrations comparable to peak plasma levels (10 µM). Imatinib reduced cells’ TMRM fluorescence (74.6 ± 6.5% vs. 100.0 ± 0.0%), consistent with mitochondrial depolarisation. Imatinib increased lysosome and autophagosome content as indicated by LAMP2 expression (2.4 ± 0.3-fold) and acridine orange fluorescence (46.0 ± 5.4% vs. 9.0 ± 3.0), respectively. Although imatinib increased expression of autophagy-associated proteins and also impaired autophagic flux, shown by proximity ligation assay staining for LAMP2 and LC3II (autophagosome marker): 48 h of imatinib treatment reduced visible puncta to 2.7 ± 0.7/cell from 11.3 ± 2.1 puncta/cell in the control. Cell viability was partially recovered by autophagosome inhibition by wortmannin, with the viability increasing 91.8 ± 8.2% after imatinib-wortmannin co-treatment (84 ± 1.5% after imatinib). Imatinib-induced necroptosis was associated with an 8.5 ± 2.5-fold increase in mixed lineage kinase domain-like pseudokinase activation. Imatinib-induced toxicity was rescued by RIP1 inhibition: 88.6 ± 3.0% vs. 100.0 ± 0.0% in the control. Imatinib applied to human cardiac progenitor cells depolarises mitochondria and induces cell death through necroptosis, recoverable by RIP1 inhibition, with a partial role for autophagy.

Zuo-Gui-Wan Aqueous Extract Ameliorates Glucocorticoid-Induced Spinal Osteoporosis of Rats by Regulating let-7f and Autophagy

ObjectiveThis study proposes to explore the protective effect of Zuo-Gui-Wan (ZGW) aqueous extract on spinal glucocorticoid-induced osteoporosis (GIOP) in vivo and in vitro, and the underlying mechanisms of ZGW in GIOP and osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) were conducted.MethodsIn vivo, SD rats were randomly divided into three groups: control group (CON), dexamethasone (DEXM) group, and ZGW group, which were given vehicle, DEXM injection, and ZGW intragastric administration at the same time. Vertebral bone microarchitecture, biomechanics, histomorphology, serum AKP activity, and the autophagosome of osteoblasts were examined. The mRNA expressions of let-7f, autophagy-associated genes (mTORC1, Beclin-1, ATG12, ATG5, and LC3), Runx2, and CTSK were examined. In vitro, the let-7f overexpression/silencing vector was constructed and transfected to evaluate the osteogenic differentiation of BMSCs. Western blot was employed to detect the expression of autophagy-associated proteins (ULK2, ATG5, ATG12, Beclin-1, LC3).ResultsIn vivo, ZGW promoted the bone quantity, quality, and strength; alleviated histological damage; increased the serum AKP activity; and reduced the autophagosome number in osteoblasts. Moreover, ZGW increased the let-7f, mTORC1, and Runx2 mRNA expressions and reduced the Beclin-1, ATG12, ATG5, LC3, and CTSK mRNA expressions. In vitro, bioinformatics prediction and dual luciferase reporter gene assay verified that let-7f targeted the binding to ULK2 and negatively regulated the ULK2 expression. Furthermore, by let-7f overexpression/silencing, ZGW may promote osteoblast differentiation of BMSCs by regulating let-7f and autophagy as evidenced by Western blot (ULK2, ATG5, ATG12, Beclin-1, LC3).ConclusionsZGW may ameliorate GC-induced spinal osteoporosis by promoting osteoblast differentiation of BMSCs by activation of let-7f and suppression of autophagy.

Diagnostic Value and Prognostic Evaluation of Autophagy-Related Protein Expression Level in Sepsis Complicated with Acute Respiratory Distress Syndrome.

Objective To explore the diagnostic value and prognostic evaluation of the autophagy-related protein expression level among patients with sepsis comorbid with acute respiratory distress syndrome. Methods A total of 182 sepsis patients were admitted to Naval Medical Center from March 2016 to April 2020 and divided into the acute respiratory distress syndrome and non-ARDS groups. Immunoblotting was employed to identify the expression of autophagy-associated protein from participants' peripheral blood mononuclear cells. Multivariate linear regression analysis was used to examine the association between mortality and the protein expression in sepsis complicated with acute respiratory distress syndrome. Results Among the 182 patients with sepsis included in this study, 82 patients had acute respiratory distress syndrome and 100 patients did not have acute respiratory distress syndrome. We observed that microtubule-related protein 1A/1B LC3II, Beclin-1, RAB7, and LAMP2 protein expression was significantly decreased in septic patients with ARDS, and p62 was significantly increased. Further receiver operating characteristic curve analysis showed that autophagy-related proteins had a high recognition ability in sepsis complicated with acute respiratory distress syndrome. LAMP2 protein was the best among them, and its specificity was up to 91.46%. In this study, 38 of the 82 patients with sepsis complicated with acute respiratory distress syndrome died, with a mortality rate of 46.34%. We found that the autophagy level was further inhibited in the patients with death, LC3II, Beclin-1, and RAB7. However, the lysosomal-associated membrane protein 2 levels in the survival patients were remarkably higher than that in the dead patients. In addition, the p62 level was lower in survival patients as well. Our results indicated age and SOFA score were the independent risk factors for mortality in septic patients with acute respiratory distress syndrome. Conclusion The autophagy level is significantly inhibited in septic patients with acute respiratory distress syndrome, and autophagy-associated proteins LC3II, Beclin-1, RAB7, LAMP2, and p62 have good value for the diagnosis and prognosis evaluation of sepsis comorbid with acute respiratory distress syndrome.

Metformin ameliorates polycystic ovary syndrome in a rat model by decreasing excessive autophagy in ovarian granulosa cells via the PI3K/AKT/mTOR pathway.

Polycystic ovary syndrome (PCOS) is a common gynecological disease accompanied by a variety of clinical features, including anovulation, hyperandrogenism, and ovarian abnormalities, resulting in infertility. PCOS affects approximately 6%-15% of all reproductive-age women worldwide. Metformin, a popular drug used to treat PCOS in patients, has beneficial effects in reducing hyperandrogenism and inducing ovulation; however, the mechanisms by which metformin ameliorates PCOS are not clear. Hence, we aimed to explore the mechanisms of metformin in treating PCOS. In the present study, we first treated a letrozole-induced PCOS rat model with metformin, detected the pathological recovery of PCOS, and then assessed the effects of metformin on H2O2-induced autophagy in ovarian granulosa cells (GCs) by detecting the level of oxidative stress and the expression of autophagy-associated proteins and key proteins in the PI3K/AKT/mTOR pathway. We demonstrated that metformin ameliorated PCOS in a rat model by downregulating autophagy in GCs, and metformin decreased the levels of oxidative stress and autophagy in H2O2-induced GCs and affected the PI3K/AKT/mTOR signaling pathway. Taken together, our results indicate that metformin ameliorates PCOS in a rat model by decreasing excessive autophagy in GCs via the PI3K/AKT/mTOR pathway, and this study provides evidence for targeted reduction of excessive autophagy of ovarian granulosa cells and improvement of PCOS.

Long-term ketamine administration induces bladder damage and upregulates autophagy-associated proteins in bladder smooth muscle tissue.

Long-term ketamine abuse can cause significant lower urinary tract symptoms in humans, termed ketamine-associated cystitis (KC). Here, we established a model of long-term (6 months) ketamine administration in wild-type (C57BL/6) mice. We elucidated the pathological effects of ketamine in the bladder and investigated changes in autophagy-associated protein expression (i.e., LC3, Beclin-1, and P62) and inflammatory cytokines (i.e., IL-6 and IL-1β) in the bladder smooth muscle tissue. Long-term ketamine administration reduced the number of layers in the bladder mucosal epithelial cells (4-5 layers in the saline group vs. 2-3 layers in the ketamine groups), but increased the number of mast cells and collagen fibers. LC3-II/LC3-I, Beclin-1, IL-6, and IL-1β protein expression in the bladder smooth muscle tissues of ketamine-treated mice was significantly increased. The mRNA and protein levels of P62 in the Ket-60 mg/kg group were also significantly increased, but not the Ket-30 mg/kg group. Our results reveal that long-term ketamine administration can cause cystitis-like pathological changes in mice, and the disordered autophagy in the bladder tissue may be involved in the persistent bladder damage following long-term administration of ketamine at 60 mg/kg.

FUNDC1 Regulates Autophagy by Inhibiting ROS-NLRP3 Signaling to Avoid Apoptosis in the Lung in a Lipopolysaccharide-Induced Mouse Model.

The incidence and mortality of acute respiratory distress syndrome (ARDS) are high, but the relevant mechanism for this disorder remains unclear. Autophagy plays an important role in the development of ARDS. The mitochondrial outer membrane protein FUNDC1 is involved in hypoxia-mediated mitochondrial autophagy, which may contribute to ARDS development. This study explored whether FUNDC1 regulates autophagy by inhibiting ROS-NLRP3 signaling to avoid apoptosis in the lung in a lipopolysaccharide-induced mouse model. In this study, FUNDC1 knockout mice were constructed, and a lipopolysaccharide-induced mouse model was generated. HE staining of pathological sections from the lung, wet/dry lung measurements, myeloperoxidase concentration/neutrophil counts in BALF and survival time of mice were examined to determine the effect of modeling. The release of cytokines (TNF-α, IL-1β, IL-6, and IL-10) in response to LPS in the BALF and plasma was assessed using ELISA. The effects of oxidative stress (malondialdehyde, superoxide dismutase, catalase, glutathione peroxidase) in lung tissue in response to LPS were detected by biochemical analysis. Oxidative stress damage was validated by iNOS staining, and apoptosis was assessed by TUNEL staining after LPS. Finally, the expression of autophagy-associated proteins and inflammasome-associated proteins in lung tissue after LPS intervention was analyzed by western blot. We found that wild-type control, FUNDC1 knockout control, lipopolysaccharide-induced wild-type, and FUNDC1 knockout mouse models were used to investigate whether FUNDC1-mediated autophagy is involved in lung injury and its possible molecular mechanisms. Compared with the normal control group, lung tissue FUNDC1 and LC3 II increased and p62/SQSTM1 decreased after LPS intervention, and increased ROS levels led to a decrease in corresponding antioxidant enzymes along with an increased inflammatory response and apoptosis. Levels of autophagy in lipopolysaccharide-induced mice deficient in FUNDC1 were significantly decreased, but the expression of ROS and inflammatory factors in lung tissue was more severe than in lipopolysaccharide-induced wild-type mice, and the survival rate was significantly decreased. Western blot analysis showed that autophagy was significantly inhibited in the FUNDC1 KO+LPS group, and there was a significant increase in NLRP3, caspase-1, IL-1β, and ASC compared with the lipopolysaccharide-induced wild-type group. In summary, lipopolysaccharide-induced wild-type mice exhibit ROS-dependent activation of autophagy, and knocking out FUNDC1 promotes inflammasome activation and exacerbates lung injury.

Metformin ameliorates HMGB1-mediated oxidative stress through mTOR pathway in experimental periodontitis

Periodontitis is an oral chronic inflammatory disease. Inhibiting tissue destruction and promoting tissue regeneration are important means for the treatment of periodontitis. Metformin not only has hypoglycemic effect but also has anti-inflammatory effect. Metformin has been shown to inhibit oxidative stress and activate autophagy through AMPK/mTOR pathway. High mobility group box 1 (HMGB1) has been implicated in the pathogenesis of many inflammatory diseases including periodontitis, it can participate in the induction of oxidative stress. HMGB1 is an autophagy regulator under oxidative stress, which can activate mTOR pathway. However, it is not clear whether metformin is related to HMGB1 and its mechanism in the process of periodontitis. Cell viability and expression of inflammatory cytokines were clarified by Cell Counting Kit-8, real-time PCR and enzyme-linked immunosorbent assay. Western blot and immunofluorescence were conducted to determine HMGB1 intracellular localization and expression of autophagy-associated proteins in vitro. Experimental periodontitis mice model was induced by administering a ligature. Immunohistochemistry was performed to detect the expression and localization of HMGB1 in vivo. The results of CCK-8, real-time PCR, enzyme-linked immunosorbent assay, Western blot and immunofluorescence showed lipopolysaccharide (LPS) treatment inhibited cell viability, and increased HMGB1 expression at a dose-independent manner. Metformin can reduce the effect of LPS. It also improves autophagy pathway inhibited by LPS and down-regulates mTOR expression. In addition, metformin attenuated alveolar bone resorption induced by ligation. This study provides new evidence for that metformin is a potential drug for the treatment of periodontitis and HMGB1 may be a potential target for periodontal intervention.

Dexmedetomidine attenuates neuronal injury induced bycerebral ischemia‑reperfusion by regulating miR‑199a.

As is well known, dexmedetomidine (DEX) serves a neuroprotective role in cerebral ischemia-reperfusion (CIR) injury, and microRNA (miR)-199a has been re-ported to be associated with IR injury. However, the association between DEX and miR-199a in CIR injury remains unknown. Thus, the aim of the present study was to verify whether the neuroprotective effect of DEX on cerebral ischemia-reperfusion rats is associated with miR-199a. A rat model of CIR was established, and the modified neurological severity score (mNSS) was evaluated. The effect of DEX on the patholog-ical structure of the cerebral cortex in CIR rats was observed by hematoxylin and eosin and Nissl staining. Reverse transcription-quantitative PCR was used to analyze the expression levels of miR-199a in brain tissue following intracerebroventricular injection of miR-199a antagomir. The co-expression of NeuN and microtubule-associated proteins 1A/1B light chain 3B in the cerebral cortex was analyzed by immunofluorescence staining. Western blotting and immunohistochemistry were performed to analyze the expression of autophagy-associated proteins in the brain tissue. DEX inhibited the expression of miR-199a, decreased the mNSS and improved pathological damage to the cerebral cortex. DEX also inhibited autophagy and expression levels of associated proteins and decreased nerve cell injury. In conclusion, DEX inhibited expression of miR-199a and improved neurocyte injury induced by CIR.

Fluorochloridone induces autophagy in TM4 Sertoli cells: involvement of ROS-mediated AKT-mTOR signaling pathway

BackgroundFluorochloridone (FLC), a selective pyrrolidone herbicide, has been recognized as a potential endocrine disruptor and reported to induce male reproductive toxicity, but the underlying mechanism is unclear. The aim of this study was to investigate the mechanism of FLC-induced reproductive toxicity on male mice with particular emphasis on the role of autophagy in mice’ TM4 Sertoli cells.MethodsAdult C57BL/6 mice were divided into one control group (0.5% sodium carboxymethyl cellulose), and four FLC-treated groups (3,15,75,375 mg/kg). The animals (ten mice per group) received gavage for 28 days. After treatment, histological analysis, sperm parameters, the microstructure of autophagy and the expression of autophagy-associated proteins in testis were evaluated. Furthermore, to explore the autophagy mechanism, TM4 Sertoli cells were treated with FLC (0,40,80,160 μM) in vitro for 24 h. Cell activity and cytoskeletal changes were measured by MTT assay and F-actin immunofluorescence staining. The formation of autophagosome, accumulation of reactive oxygen species (ROS), expression of autophagy marker proteins (LC3, Beclin-1 and P62) and AKT-related pathway proteins (AKT, mTOR) were observed. The ROS scavenger N-acetylcysteine (NAC) and AKT agonist (SC79) were used to treat TM4 cells to observe the changes of AKT-mTOR pathway and autophagy.ResultsIn vivo, it showed that FLC exposure caused testicular injuries, abnormality in epididymal sperm. Moreover, FLC increased the formation of autophagosomes, the accumulation of LC3II/LC3I, Beclin-1 and P62 protein, which is related to the degradation of autophagy. In vitro, FLC triggered TM4 cell autophagy by increasing the formation of autophagosomes and upregulating of LC3II/LC3I, Beclin-1 and P62 levels. In addition, FLC induced ROS production and inhibited the activities of AKT and mTOR kinases. The Inhibition of AKT/mTOR signaling pathways and the activation of autophagy induced by FLC could be efficiently reversed by pretreatment of NAC. Additionally, decreased autophagy and increased cell viability were observed in TM4 cells treated with SC79 and FLC, compared with FLC alone, indicating that FLC-induced autophagy may be pro-death.ConclusionTaken together, our study provided the evidence that FLC promoted autophagy in TM4 Sertoli cells and that this process may involve ROS-mediated AKT/mTOR signaling pathways.

FSTL1 aggravates cigarette smoke-induced airway inflammation and airway remodeling by regulating autophagy

BackgroundCigarette smoke (CS) is a major risk factor for Chronic Obstructive Pulmonary Disease (COPD). Follistatin-like protein 1 (FSTL1), a critical factor during embryogenesis particularly in respiratory lung development, is a novel mediator related to inflammation and tissue remodeling. We tried to investigate the role of FSTL1 in CS-induced autophagy dysregulation, airway inflammation and remodeling.MethodsSerum and lung specimens were obtained from COPD patients and controls. Adult female wild-type (WT) mice, FSTL1± mice and FSTL1flox/+ mice were exposed to room air or chronic CS. Additionally, 3-methyladenine (3-MA), an inhibitor of autophagy, was applied in CS-exposed WT mice. The lung tissues and serum from patients and murine models were tested for FSTL1 and autophagy-associated protein expression by ELISA, western blotting and immunohistochemical. Autophagosome were observed using electron microscope technology. LTB4, IL-8 and TNF-α in bronchoalveolar lavage fluid of mice were examined using ELISA. Airway remodeling and lung function were also assessed.ResultsBoth FSTL1 and autophagy biomarkers increased in COPD patients and CS-exposed WT mice. Autophagy activation was upregulated in CS-exposed mice accompanied by airway remodeling and airway inflammation. FSTL1± mice showed a lower level of CS-induced autophagy compared with the control mice. FSTL1± mice can also resist CS-induced inflammatory response, airway remodeling and impaired lung function. CS-exposed WT mice with 3-MA pretreatment have a similar manifestation with CS-exposed FSTL1± mice.ConclusionsFSTL1 promotes CS-induced COPD by modulating autophagy, therefore targeting FSTL1 and autophagy may shed light on treating cigarette smoke-induced COPD.

Gelatine nanostructured lipid carrier encapsulated FGF15 inhibits autophagy and improves recovery in spinal cord injury

Gelatine nanostructured lipid carriers (GNLs) have attracted increasing attention due to their biodegradable status and capacity to capture various biologically active compounds. Many studies demonstrated that fibroblast growth factor therapies after spinal cord injury (SCI) can be used in the future for the recovery of neurons. In this study, the therapeutic effects of GNL-encapsulated fibroblast growth factor 15 (FGF15) and FGF15 were compared in SCI. The FGF15-GNLs had 88.17 ± 1.22% encapsulation efficiency and 4.82 ± 0.12% loading capacity. The effects of FGF15-GNLs and FGF15 were assessed based on the Basso–Beattie–Bresnahan (BBB) locomotion scale, inclined plane test and footprint analysis. Immunofluorescent staining was used to identify the expression of autophagy-associated proteins, GFAP (glial fibrillary acidic protein) and neurofilament 200 (NF200). FGF15-GNLs use enhanced the repair after SCI compared to the effect of FGF15. The suppression of autophagy-associated proteins LC3-II and beclin-1, and p62 enhancement by FGF15-GNLs treatment were more pronounced. Thus, the effects of FGF15-GNLs on the recovery after SCI are related to the inhibition of autophagy and glial scar, and promotion of nerve regeneration in SCI.

Flavokawain B and Doxorubicin Work Synergistically to Impede the Propagation of Gastric Cancer Cells via ROS-Mediated Apoptosis and Autophagy Pathways.

Simple SummaryAmong various kinds of treatment strategies for cancers, combination therapy has attracted significant attention due to its beneficial effects than the individual effects of the same compounds. Based on this idea, this study has investigated the synergistic effects of combination treatment of a natural anti-cancer agent flavokawain B (FKB) and a chemotherapeutic agent Doxorubicin on human gastric cancer cells and the underlying molecular mechanisms were deciphered through in vitro and in vivo approaches. Experimental data obtained in this study provided promising application prospects of FKB + Doxrubicin combination treatment in human gastric cancer cells.Chalcone flavokawain B (FKB) possesses a chemopreventive and anti-cancer activity. Doxorubicin is a chemotherapeutic DNA intercalating agent widely used in malignancy treatment. The present study investigated whether synergistic effects exist between the combination of FKB (1.25–5 µg/mL) and doxorubicin (0.5 µg/mL) on the apoptosis and autophagy in human gastric cancer (AGS) cells, and the possible in vitro and in vivo mechanisms. The MTT assay measured cell viability. Various apoptotic-, autophagy-associated protein expression was determined by the Western blot technique. FKB+doxorubicin synergy was estimated by the Chou-Talalay combination index (CI) method. In vivo studies were performed on BALB/c mice. Results showed that compared to FKB/doxorubicin treatments, low doses of FKB+doxorubicin suppressed AGS cell growth. FKB potentiated doxorubicin-induced DNA fragmentation, apoptotic cell death, and enhanced doxorubicin-mediated mitochondrial, death receptor pathways. FKB+doxorubicin activated increased LC3-II accumulation, p62/SQSTM1 expression, and AVO formation as compared to the FKB/doxorubicin alone treatments indicating autophagy in these cells. The death mechanism in FKB+doxorubicin-treated AGS cells is due to the activation of autophagy. FKB+doxorubicin-mediated dysregulated Bax/Bcl-2, Beclin-1/Bcl-2 ratios suggested apoptosis, autophagy induction in AGS cells. FKB+doxorubicin-induced LC3-II/AVOs downregulation was suppressed due to an apoptotic inhibitor Z-VAD-FMK. Whereas, 3-methyladenine/chloroquine weakened FKB+doxorubicin-induced apoptosis (decreased DNA fragmentation/caspase-3). Activation of ERK/JNK may be involved in FKB+doxorubicin-induced apoptosis and autophagy. FKB+doxorubicin-triggered ROS generation, but NAC attenuated FKB+doxorubicin-induced autophagic (LC3 accumulation) and apoptotic (caspase-3 activation and PARP cleavage) cell death. FKB+doxorubicin blocked gastric cancer cell xenografts in nude mice in vivo as compared to FKB/doxorubicin alone treatments. FKB and doxorubicin wielded synergistic anti-tumor effects in gastric cancer cells and is a promising therapeutic approach.