Valproate modulates the activity of multidrug resistance efflux pumps, as a chemoresistance factor in gastric cancer cells.

BACKGROUND: Drug resistance is one of the most critical problems in gastric cancer therapy. This study was performed to investigate the valproic acid effects on the proliferation of sensitive and resistant cell lines of human gastric cancer, and to explore the mechanism of the agent on multi drug resistance and apoptosis genes. METHODS: The cytotoxicity effect of valproic acid on the EPG85.257 and EPG85.257RDB cells was assessed by the MTT assay, and the IC50 concentration was evaluated. Apoptosis, genotoxicity, and drug resistance pump activity were evaluated using comet assay, Real-time PCR, and flow cytometry, respectively. Cell proliferation was assayed using a scratch test. RESULTS: Dose-dependent toxicity was recorded after treatment of cells with valproic acid. Valproic acid represented a significant growth inhibition on EPG85.257 cells with IC50 values of 5.84 µM and 4.78 µM after 48 h and 72 h treatment, respectively. In contrast, the drug-resistant counterpart represented 8.7 µM and 7.02 µM IC50 values after the same treatment time. Valproic acid induced PTEN, Bcl2, P53, Bax, P21, and caspase3 expression in EPG85.257 cells, whereas p21, p53, PTEN, and ABCB1 were overexpressed in EPG5.257RDB. Valproic acid hindered cell migration in both cell lines (P < 0.01). Valproate genotoxicity was significantly higher in the parent cells than in their resistant EPG85.257RDB counterparts. Valproate led to a 62% reduction in the daunorubicin efflux of the MDR1 pump activity. CONCLUSIONS: Valproate can affect drug resistance in gastric cancer via a unique mechanism independent of MDR1 expression.

Homogeneous Polyporus polysaccharide exerts anti-bladder cancer effects via autophagy induction.

CONTEXT: Polyporus polysaccharide (PPS), the leading bioactive ingredient extracted from Polyporus umbellatus (Pers.) Fr. (Polyporaceae), has been demonstrated to exert anti-bladder cancer and immunomodulatory functions in macrophages. OBJECTIVE: To explore the effects of homogeneous Polyporus polysaccharide (HPP) on the proliferation and autophagy of bladder cancer cells co-cultured with macrophages. MATERIALS AND METHODS: MB49 bladder cancer cells and RAW264.7 macrophages were co-cultured with or without HPP intervention (50, 100, or 200 µg/mL) for 24 h. The cell counting kit-8 (CCK-8) assay and 5-ethynyl-2″-deoxyuridine (EdU) staining evaluated MB49 cell proliferation. Monodansylcadaverine (MDC) staining and transmission electron microscopy (TEM) observed autophagosomes. Western blotting detected the expression levels of autophagy-related proteins and PI3K/Akt/mTOR pathway proteins. RESULTS: HPP inhibited the proliferation of MB49 cells co-cultured with RAW264.7 cells but not MB49 cells alone. HPP altered the expression of autophagy-related proteins and promoted the formation of autophagosomes in MB49 cells in the co-culture system. Autophagy inhibitors 3-methyladenine (3-MA) and chloroquine (CQ) not only antagonized HPP-induced autophagy but also attenuated the inhibitory effects of HPP on MB49 cell proliferation in the co-culture system. HPP or RAW264.7 alone was not sufficient to induce autophagy in MB49 cells. In addition, HPP suppressed the protein expression of the PI3K/Akt/mTOR pathway in MB49 cells in the co-culture system. DISCUSSION AND CONCLUSIONS: HPP induced bladder cancer cell autophagy by regulating macrophages in the co-culture system, resulting in the inhibition of cancer cell proliferation. The PI3K/Akt/mTOR pathway was involved in HPP-induced autophagy in the co-culture system.

Liuwei Dihuang pills attenuate ovariectomy-induced bone loss by alleviating bone marrow mesenchymal stem cell (BMSC) senescence via the Yes-associated protein (YAP)-autophagy axis.

CONTEXT: Liuwei Dihuang pill (LWDH) has been used to treat postmenopausal osteoporosis (PMOP). OBJECTIVE: To explore the effects and mechanisms of action of LWDH in PMOP. MATERIALS AND METHODS: Forty-eight female Sprague-Dawley rats were divided into four groups: sham-operated (SHAM), ovariectomized (OVX), LWDH high dose (LWDH-H, 1.6 g/kg/d) and LWDH low dose (LWDH-L, 0.8 g/kg/d); the doses were administered after ovariectomy via gavage for eight weeks. After eight weeks, the bone microarchitecture was evaluated. The effect of LWDH on the differentiation of bone marrow mesenchymal stem cells (BMSCs) was assessed via osteogenesis- and lipogenesis-induced BMSC differentiation. The senescence-related biological indices were also detected using senescence staining, cell cycle analysis, quantitative real-time polymerase chain reaction and western blotting. Finally, the expression levels of autophagy-related proteins and Yes-associated protein (YAP) were evaluated. RESULTS: LWDH-L and LWDH-H significantly modified OVX-induced bone loss. LWDH promoted osteogenesis and inhibited adipogenesis in OVX-BMSCs. Additionally, LWDH decreased the positive ratio of senescence OVX-BMSCs and improved cell viability, cell cycle, and the mRNA and protein levels of p53 and p21. LWDH upregulated the expression of autophagy-related proteins, LC3, Beclin1 and YAP, in OVX-BMSCs and downregulated the expression of p62. DISCUSSION AND CONCLUSIONS: LWDH improves osteoporosis by delaying the BMSC senescence through the YAP-autophagy axis.

Biological functions and structural biology of Plasmodium falciparum autophagy-related proteins: The under-explored options for novel antimalarial drug design.

Malaria remains a threat to global public health and the available antimalarial drugs are undermined by side effects and parasite resistance, suggesting an emphasis on new potential targets. Among the novel targets, Plasmodium falciparum autophagy-related proteins (PfAtg) remain a priority. In this paper, we reviewed the existing knowledge on the functions and structural biology of PfAtg including the compounds with inhibitory activity toward P. falciparum Atg8-Atg3 protein-protein interaction (PfAtg8-PfAtg3 PPI). A total of five PfAtg (PfAtg5, PfAtg8, PfAtg12, PfAtg18, and Rab7) were observed to have autophagic and/or non-autophagic roles. Available data showed that PfAtg8 has conserved hydrophobic pockets, which allows it to interact with PfAtg3 to form PfAtg8-PfAtg3 PPI. Additionally, 2-bromo-N-(4-pyridin-2-yl-1,3-thiazol-2-yl) benzamide was identified as the most powerful inhibitor of PfAtg8-PfAtg3 PPI. Due to the dearth of knowledge in this field, we hope that the article would open an avenue to further research on the remaining PfAtg as possible drug candidates.

Treponema pallidum membrane protein Tp47 promotes angiogenesis through ROS-induced autophagy.

BACKGROUND: Pathological angiogenesis is an important manifestation of syphilis, but the underlying mechanism of Treponema pallidum subspecies pallidum (T. pallidum)-induced angiogenesis is poorly understood. OBJECTIVES: The objective of this study is to investigate the role and related mechanism of the T. pallidum membrane protein Tp47 in angiogenesis. METHODS: The proangiogenic activity of recombinant T. pallidum membrane protein Tp47 in human umbilical vein endothelial cells (HUVECs) was assessed by tube formation assay, three-dimensional angiogenesis analysis and experiments with a zebrafish embryo model. The effects of mitochondrial ROS and NADPH oxidase on intracellular ROS induced by Tp47 were further investigated. Furthermore, the levels of autophagy-related proteins and autophagic flux were measured. Finally, the role of ROS-induced autophagy in angiogenesis was studied. RESULTS: Tp47 promoted tubule formation and the formation of angiogenic sprouts in vitro. In addition, a significant increase in the number of subintestinal vessel branch points in zebrafish injected with Tp47 was observed using a zebrafish embryo model. Tp47 also significantly increased intracellular ROS levels in a dose-dependent manner. Tp47-induced tube formation and angiogenic sprout formation were effectively prevented by the ROS inhibitor NAC. In addition, Tp47 enhanced the production of mitochondrial ROS and expression of the NADPH oxidase-related proteins Nox2 and Nox4. The production of mitochondrial ROS and intracellular ROS was reduced by the NADPH oxidase inhibitors DPI and apocynin. Furthermore, Tp47 significantly increased expression of the autophagy-related proteins P62 and Beclin 1 and the LC3-II/LC3-I ratio and promoted an increase in autophagic flux, which could be effectively rescued by coincubation with the ROS inhibitor NAC. Further intervention with the autophagy inhibitor BafA1 significantly inhibited tube formation and angiogenic sprout formation. CONCLUSIONS: Tp47-induced NADPH oxidase enhanced intracellular ROS production via mitochondrial ROS and promoted angiogenesis through autophagy mediated by ROS. These findings may contribute to our understanding of pathological angiogenesis in syphilis.

Activation of the Akt/FoxO3 signaling pathway enhances oxidative stress-induced autophagy and alleviates brain damage in a rat model of ischemic stroke.

Autophagy has been implicated in stroke. Our previous study showed that the FoxO3 transcription factor promotes autophagy after transient cerebral ischemia/reperfusion (I/R). However, whether the Akt/FoxO3 signaling pathway plays a regulatory role in autophagy in cerebral I/R-induced oxidative stress injury is still unclear. The present study aims to investigate the effects of the Akt/FoxO3 signaling pathway on autophagy activation and neuronal injury in vitro and in vivo. By employing LY294002 or insulin to regulate the Akt/FoxO3 signaling pathway, we found that insulin pretreatment increased cell viability, decreased reactive oxygen species production, and enhanced the expression of antiapoptotic and autophagy-related proteins following H2O2 injury in HT22 cells. In addition, insulin significantly decreased neurological deficit scores and infarct volume and increased the expression of antiapoptotic and autophagy-related proteins following I/R injury in rats. However, LY294002 showed the opposite effects under these conditions. Altogether, these results indicate that Akt/FoxO3 signaling pathway activation inhibited oxidative stress-mediated cell death through activation of autophagy. Our study supports a critical role for the Akt/FoxO3 signaling pathway in autophagy activation in stroke.

Melatonin Protects against Primary Ovarian Insufficiency by Activating the PI3K/Akt/mTOR Pathway and Inhibiting Autophagy.

OBJECTIVE: Primary ovarian insufficiency (POI), which refers to the occurrence of ovarian insufficiency before the age of 40, is indicated by menstrual cycle changes as a precursor and is accompanied by menstrual disorders, elevated gonadotropin levels, and decreased estrogen levels. The incidence of POI is reportedly increasing worldwide and this disease markedly reduces the quality of life and affects the physical and mental health of patients. Treatment options for POI include hormone replacement therapy; however, its efficacy remains unsatisfactory. Therefore, exploring hormonal drugs with superior curative effects and clarifying the molecular mechanism underlying POI pathogenesis could afford new directions for POI therapy. METHODS: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays were used to detect the effects of melatonin (MT) on cell survival and mortality. Flow cytometry was performed to examine the effect of MT on apoptosis. The impact of MT on autophagosome formation was examined using electron microscopy, whereas the expression of autophagy-related proteins and phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway-related proteins following MT intervention was detected by western blotting. RESULTS: (1) MT exerted a protective effect on ovarian granulosa cells subjected to serum starvation. (2) MT inhibited serum starvation-induced apoptosis of ovarian granulosa cells. (3) MT inhibited serum starvation-induced autophagosome formation in ovarian granulosa cells. (4) MT inhibited the expression of autophagy-related proteins LC3II/I and Agt5. (5) MT suppressed autophagy in ovarian granulosa cells by activating the PI3K/Akt/mTOR signaling pathway. CONCLUSION: Collectively, our results demonstrate that MT can inhibit excessive autophagy in ovarian granulosa cells by activating the PI3K/Akt/mTOR pathway, thereby exerting its protective effect against POI.

LncRNA MEG3 promotes the sensitivity of bortezomib by inhibiting autophagy in multiple myeloma.

BACKGROUND: Bortezomib resistance hampers the long-term survival of multiple myeloma (MM) patients. Our previous study has proved that downregulated lncRNA MEG3 is associated with the poor clinical outcome in MM. However, the effect of MEG3 on the sensitivity of bortezomib in MM and its possible molecular mechanism remains muddled. METHODS: In this study, CCK8 and flow cytometry techniques were used to assess cell viability in MEG3 overexpressed MM cells after bortezomib treatment. The expression of autophagy-related protein LC3 and p62 were distinguished by Western blot, and the mCherry-GFP-LC3 puncta reflecting autophagy level was observed under fluorescence microscope. RNA immunoprecipitation (RIP) technology was used to detect the binding relationship of MEG3 and ATG2B to PTBP1. RESULTS: Increased toxicity of bortezomib and diminished autophagy level were found in MEG3 overexpressed MM cells. Mechanistically, we discovered that RNA-binding protein PTBP1 could bind to MEG3 and ATG2B by RIP assay. Upregulation of MEG3 promoted PTBP1 expression and inhibited the expression level of ATG2B, suggesting that MEG3 recruited PTBP1 and then decayed ATG2B expression. CONCLUSION: In summary, our study illustrated that MEG3 increased bortezomib sensitivity by hindering autophagy through the PTBP1/ATG2B axis, providing a new therapeutic target for bortezomib-resistant MM patients.

Cornin protects against cerebral ischemia/reperfusion injury by preventing autophagy via the PI3K/Akt/mTOR pathway.

BACKGROUND: Ischemia stroke is the leading cause of disability, which is a consequence of vascular occlusion. The purpose of this study is to investigate the effect of cornin which is isolated from the fruit of Verbena officinalis L, against astrocytes autophagy induced by cerebral ischemia/reperfusion (CI/R) injury in vitro and in vivo and its potential mechanism. METHODS: Cornin at dose of 2.5, 5 and 10 mg/kg were intravenously injected to MCAO rats at 15 min after reperfusion. The infarction volume, blood-brain barrier (BBB), neurological severity score (mNSS), and autophagy related protein were used to evaluated the protective effects and potential mechanism of cornin in autophagy with or without phosphoinositide-3 kinase (PI3K)inhibitor LY294002 and mammalian target of rapamycin (mTOR) small interfering RNA (siRNA) at 24 h after CI/R injury. The potential protective effects and mechanism of cornin at concention of 10 ~ 1000 nM were also evaluated in oxygen glucose deprivation/reperfusion (OGD/R) in U87 cells. RESULTS: The results suggest that cornin at dose of 5 or 10 mg/kg significantly reduce the cerebral infarction volume and blood-brain barrier (BBB) leakage, and improve neurological recovery in MCAO rats. Cleaved caspase-3 and Bax levels were significantly decreased, while B-cell lymphoma-2 (Bcl-2) and the apoptosis regulator ratio (Bcl-2/Bax) were markedly increased when treated with 2.5-10 mg/kg cornin. The obvious decreased expressions of glial fibrillary acidic protein (GFAP), myosin-like BCL2 interacting protein (Beclin-1) and microtubule-associated protein light chain 3 II (LC3-II) and increased of neuronal nuclei (NeuN), sequestosome-1 (p62), phosphorylated mTOR (p-mTOR), and phosphorylated Akt (p-Akt) were observed in MCAO rats treated with 10 mg/kg cornin, which was counteracted by LY294002. The expression of autophagy-related proteins with or without LY294002 and mTOR siRNA presented the similar results as in vitro in OGD/R in U87 cells. CONCLUSIONS: These results indicate that cornin improved neurological recovery after cerebral ischemia injury by preventing astrocytes autophagy induced by CI/R via the PI3K/Akt/mTOR signaling pathway.

Neuregulin-4 attenuates diabetic cardiomyopathy by regulating autophagy via the AMPK/mTOR signalling pathway.

BACKGROUND: Diabetic cardiomyopathy is characterized by left ventricle dysfunction, cardiomyocyte apoptosis, and interstitial fibrosis and is a serious complication of diabetes mellitus (DM). Autophagy is a mechanism that is essential for maintaining normal heart morphology and function, and its dysregulation can produce pathological effects on diabetic hearts. Neuregulin-4 (Nrg4) is an adipokine that exerts protective effects against metabolic disorders and insulin resistance. The aim of this study was to explore whether Nrg4 could ameliorate DM-induced myocardial injury by regulating autophagy. METHODS: Four weeks after the establishment of a model of type 1 diabetes in mice, the mice received Nrg4 treatment (with or without an autophagy inhibitor) for another 4 weeks. The cardiac functions, histological structures and cardiomyocyte apoptosis were investigated. Autophagy-related protein levels along with related signalling pathways that regulate autophagy were evaluated. In addition, the effects of Nrg4 on autophagy were also determined in cultured primary cardiomyocytes. RESULTS: Nrg4 alleviated myocardial injury both in vivo and in vitro. The autophagy level was decreased in type 1 diabetic mice, and Nrg4 intervention reactivated autophagy. Furthermore, Nrg4 intervention was found to activate autophagy via the AMPK/mTOR signalling pathway. Moreover, when autophagy was suppressed or the AMPK/mTOR pathway was inhibited, the beneficial effects of Nrg4 were diminished. CONCLUSION: Nrg4 intervention attenuated diabetic cardiomyopathy by promoting autophagy in type 1 diabetic mice. Additionally, Nrg4 induced autophagy via the AMPK/mTOR signalling pathway.

Metformin alleviates crystalline silica-induced pulmonary fibrosis by remodeling endothelial cells to mesenchymal transition via autophagy signaling.

Silicosis is a severe progressive lung disease without effective treatment methods. Previous evidence has demonstrated that endothelial cell to mesenchymal transition (EndoMT) plays an essential role in pulmonary fibrosis, and pulmonary fibrosis is associated with dysregulation of autophagy, while the relationship between autophagy and EndoMT has not yet been adequately studied. Herein, we established a mouse model of silicosis, and we found that the pharmacological induction of the AMPK/mTOR-dependent pathway using 100 mg/kg Metformin (Met) enhanced autophagy in vivo, and results of the Western blot showed that autophagy-related proteins, LC3 II/I ratio, and Beclin-1 increased while p62 decreased. In addition, Met treatment attenuated silica-induced pulmonary inflammation and decreased collagen deposition by suppressing EndoMT, and the proliferation of human umbilical vein endothelial cells (HUVECs) was also inhibited. Notably, the tube forming assay showed that Met also protected the vascular endothelial cells from silica-induced morphological damage. In conclusion, Met can alleviate inflammatory response and collagen deposition in the process of pulmonary fibrosis induced by silica via suppressing EndoMT through the AMPK/mTOR signaling pathway.

Formosanin C induces autophagy-mediated apoptosis in multiple myeloma cells through the PI3K/AKT/mTOR signaling pathway.

OBJECTIVES: Multiple myeloma (MM) is an incurable plasma cell malignancy associated with poor survival. Novel therapeutic drugs are urgently needed to improve MM therapy and patient outcomes. This study aimed to investigate the effect of formosanin C (FC), a Chinese medicine monomer, on MM in vitro and disclose the underlying molecular mechanism. METHODS: The effect of FC on the viability, proliferation, apoptosis, and autophagy of MM cell lines (NCI-H929 and ARP1) was studied through CCK-8, colony formation, flow cytometry, GFP-LC3, and western blotting assays, respectively. A pharmacological approach and network pharmacology technology were implemented to explore the potential mechanisms of the action of FC on MM cells. RESULTS: FC efficiently suppressed the viability and colony-forming capacity, but promoted the number of autophagic vacuoles with GFP-LC3 localization and the percentage of apoptotic cells in MM cells. Additionally, FC significantly increased the levels of the autophagy-related proteins LC3-Ⅱ and Beclin 1, as well as the apoptosis-related proteins Bax and cleaved caspase-3, but blocked the expression of the proapoptotic protein Bcl-2 in the cells; these effects were reversed by an inhibitor of autophagy, 3-methyladenine. What's more, we found that the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway was involved in the FC-mediated inhibition of MM. Pharmacological inhibition of this pathway dramatically relieved FC-triggered excessive expression of autophagy-related proteins and rescued MM cells from FC-induced apoptosis. CONCLUSION: Our findings indicate that FC exhibits an anti-MM effect by activating cell autophagy through the PI3K/AKT/mTOR signaling pathway.

Lipopolysaccharide Inhibits Autophagy and Promotes Inflammatory Responses via p38 MAPK-Induced Proteasomal Degradation of Atg13 in Hepatic Stellate Cells.

Background: Inflammation plays a critical role in the progression of acute-on-chronic liver failure (ACLF). Atg13 is a vital regulatory component of the ULK1 complex, which plays an essential role in the initiation of autophagy. Previously, hepatic stellate cells (HSCs) were considered to be noninflammatory cells that contribute only to hepatic fibrosis. Recently, it has been found that HSCs can secrete inflammatory cytokines and participate in hepatic inflammation. Autophagy and proteasome-mediated degradation constitute two major means of protein turnover in cells. Autophagy has been shown to regulate inflammation, but it is unclear whether ubiquitin (Ub)-proteasome system (UPS) is involved in inflammatory responses in HSCs during ACLF. Methods: Clinical data were collected from ACLF patients, and surgically resected paraffin-embedded human ACLF liver tissue specimens were collected. The expression of Atg13 was assessed by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. Secretion of IL-1ß was assessed by ELISA. Atg13 was knocked down by siRNA in LX2 cells. Coimmunoprecipitation assay was used to detect protein binding and polyubiquitination of Atg13. In vitro tests with LX2 cells were performed to explore the effects and regulation of p38 MAPK, Atg13, UPS, autophagy, and inflammation. Results: Serum lipopolysaccharide (LPS) was positively associated with disease severity in ACLF patients, and p38 MAPK was overexpressed in ACLF liver tissue. We evaluated the role of Atg13 in HSC inflammation and explored the possible underlying mechanisms. Inflammatory factors were upregulated via activation of p38 MAPK and inhibition of autophagy in LX-2 cells. Expression of Atg13 was decreased in LPS-incubated LX2 cells. Atg13 knockdown markedly inhibited autophagy and promoted LPS-induced inflammation in LX2 cells. Our in vitro experiments also showed that LPS induced depletion of Atg13 via UPS, and this process was dependent on p38 MAPK. Conclusions: LPS induces proteasomal degradation of Atg13 via p38 MAPK, thereby participating in the aggravation of LPS-induced autophagy inhibition and inflammatory responses in LX2 cells. Atg13 serves as a mediator between autophagy and proteasome. Modulation of Atg13 or proteasome activity might be a novel strategy for treating HSC inflammation.

Berbamine hydrochloride inhibits bovine viral diarrhea virus replication via interfering in late-stage autophagy.

Bovine viral diarrhea virus (BVDV) is a harmful pathogen that easily causes large-scale infections and huge economic losses to the cattle industry. Berbamine hydrochloride (BBH) is a natural product extracted from berberis and has a wide range of pharmacological effects. However, the antiviral effect of BBH against BVDV needs to be further elucidated. This study aimed to evaluate the antiviral activities of BBH against BVDV infection. We mainly used RT-qPCR, Western blotting, immunofluorescence, and TEM assays to assess the inhibitory activity of BBH against BVDV. The results showed that BBH had an inhibitory effect on BVDV and higher inhibitory activity in the viral attachment and release in MDBK cells. This study found that BVDV could induce and use autophagy to replicate itself. Further results showed that BBH inhibited BVDV infection by inhibiting autophagy integrity in BVDV-infected cells, which was proven by the detection of autophagy-related proteins. Our data show that in BBH-treated BVDV-infected cells, the expression of p62 and LC3 increased over time. After the addition of an autophagy inhibitor, chloroquine (CQ), and an autophagy promoter, rapamycin (Rapa), we found that promoting autophagy was beneficial to the replication of BVDV, while inhibiting autophagy could reduce the number of infections by BVDV, which was evidenced by the expression of the BVDV E2 protein. Furthermore, BBH blocked the normal binding of LC3 and LAMP1 in BVDV-infected cells. In conclusion, BBH inhibited BVDV infection by inhibiting BVDV-induced autophagy in cells, and its inhibitory effect was obvious in the viral attachment and release stages. Therefore, our study provides a new idea for exploring novel anti-BVDV drugs.

Effect of icaritin on autophagy-related protein expression in TDP-43-transfected SH-SY5Y cells.

Objective: To study the protective effect and mechanism of icaritin (ICT) in a SH-SY5Y cells with virus-loaded TAR DNA-binding domain protein 43(TDP-43) by examining the effect of ICT on the expression of autophagy-related proteins in TDP-43-infected SH-SY5Y cells. Methods: A TDP-43-induced neuronal cell injury model was established by transfecting well-growing SH-SY5Y cells with virus loaded with the TDP-43 gene. The changes in cell viability were detected by the CCK-8 method. After successful transfection, the establishment of the model was verified by real-time quantitative PCR (qPCR) and Western blot methods. After the cells were subjected to drug intervention with ICT, the changes in the expression levels of TDP-43, cleaved Caspase-3, LC3 II/I, Beclin-1 and p62 were detected by Western blotting. Results: After ICT intervention, it was found that compared with that of the TDP-43 group, the cell viability of the TDP-43+ICT group increased, the expression level of TDP-43 decreased, and the expression levels of the apoptotic protein cleaved Caspase-3, autophagy protein Beclin-1, and LC3-II/I decreased, while the expression level of the autophagy protein p62 increased. Conclusion: ICT has a protective effect on the SH-SY5Y cell injury model transfected with TDP-43. This protective effect may be related to reducing the protein expression of TDP-43 and inhibiting autophagy.

Exogenous H2S Protects against Septic Cardiomyopathy by Inhibiting Autophagy through the AMPK/mTOR Pathway.

Background: Given the cardioprotective role of autophagy, this study aimed to investigate the protective effect of exogenous H2S (NaHS) on infectious cardiomyopathy through the inhibition of the AMPK/mTOR pathway. Methods: In this study, sepsis models were established by cecal ligation and puncture (CLP) induction in vivo and intraperitoneal injection of NaHS was performed. Autophagy- and apoptosis-related proteins were observed by western blot, isolated myocardial tissue morphology was observed by hematoxylin-eosin (H&E) staining, and myocardial apoptosis was evaluated by the tunnel method. The ultrastructure of autophagy was observed by using an electron transmission electron microscope. Results: In an SD rat model of cecum ligation puncture-induced sepsis, the level of autophagy-related proteins was significantly increased, and hematoxylin and eosin staining showed irregular myocardial bands and swollen cardiomyocytes. Following NaHS treatment, the level of autophagy-related proteins decreased, and electron transmission microscopy revealed decreased autophagosomes. Echocardiography suggested an increase in ejection fraction and significant relief of myocardial inhibition. Conclusions: Our results suggest that NaHS treatment can attenuate the cellular damage caused by excessive autophagy through the AMPK/mTOR pathway.

BMI1 promotes the proliferation and inhibits autophagy of breast cancer cells by activating COPZ1.

PURPOSE: This study was designed to explore the role of COPZ1 in breast cancer as well as discuss its specific reaction mechanism. METHODS: With the help of RT-qPCR and western blot, the expression of BMI1 and COPZ1 were measured. Then, the proliferation, colony formation and apoptosis were evaluated by CCK-8, colony formation and TUNEL assays, separately. Luciferase reporter assay and ChIP were applied to assess the relative activity of COPZ1 promoter as well as its binding with BMI1. Moreover, western blot was utilized to measure the expression of proliferation-, apoptosis- and autophagy-related proteins. RESULTS: According to GEPIA2 database, COPZ1 was upregulated in breast cancer tissues and was associated with the poor prognosis (P = 0.03). Results obtained from RT-qPCR and western blot verified that COPZ1 expression was greatly increased at both mRNA and protein levels in breast cancer cells as compared to control cells (P < 0.05 or P < 0.001). COPZ1 knockdown inhibited the proliferation, induced the autophagy and promoted the apoptosis of breast cancer cells. HumanTFDB predicted the binding sites of BMI1 and COPZ1. The increased relative luciferase activity of COPZ1 promoter following BMI1 overexpression (P < 0.001) and the binding of BMI1 with COPZ1 promoter indicated that BMI1 could activate COPZ1. Further experiments suggested that the effects of COPZ1 knockdown on the proliferation, apoptosis and autophagy of breast cancer cells were reversed by BMI1 overexpression, implying that BMI1 promoted the proliferation and repressed the autophagy of breast cancer cells via activating COPZ1. CONCLUSIONS: To sum up, BMI1 exhibited promotive effects on the malignant progression of breast cancer through the activation of COPZ1. These findings might offer a preliminary theoretical basis for COPZ1 participation in autophagy in breast cancer cells.

Berberine Induces Autophagic Cell Death by Inactivating the Akt/mTOR Signaling Pathway.

The incidence of skin cancer has been increasing over the past decades, and melanoma is considered highly malignant because of its high rate of metastasis. Plant-derived berberine, an isoquinoline quaternary alkaloid, has been reported to possess multiple pharmacological effects against various types of cancer cells. Therefore, we treated melanoma B16F10 cells with berberine to induce cell death and understand the cell death mechanisms. The berberine-treated cells showed decreased cell viability, according to berberine concentration. However, western blot analysis of apoptosis-related marker proteins showed that the expression of Bcl-2, an apoptosis inhibitory protein, and the Bcl-2/Bax ratio were increased. Therefore, by adding 3-methyladenine to the berberine-treated cells, we investigated whether the reduced cell viability was due to autophagic cell death. The results showed that 3-methyladenine restored the cell viability decreased by berberine, suggesting autophagy. To clarify autophagic cell death, we performed transmission electron microscopy analysis, which revealed the presence of autophagosomes and autolysosomes in the cells after treatment with berberine. Next, by analyzing the expression of autophagy-related proteins, we found an increase in the levels of light chain 3A-II and Atg12-Atg5 complex in the berberine-treated cells. We then assessed the involvement of the Akt/mTOR signaling pathway and found that berberine inhibited the expression of phosphorylated Akt and mTOR. Our data demonstrated that berberine induces autophagic cell death by inactivating the Akt/mTOR signaling pathway in melanoma cells and that berberine can be used as a possible target for the development of anti-melanoma drugs.

Oridonin relieves depressive-like behaviors by inhibiting neuroinflammation and autophagy impairment in rats subjected to chronic unpredictable mild stress.

Major depressive disorder (MDD) is a severe life-threatening disorder with increasing prevalence. However, the mechanistic interplay between depression, neuroinflammation, and autophagy is yet to be demonstrated. This study investigated the effect of Oridonin on CUMS-induced depression, neuroinflammation, and autophagy impairment. Male 4-week-old Sprague-Dawley rats were subjected to chronic unpredictable mild stress (CUMS), some of which were injected with Oridonin, fluoxetine (FLX), or their combination at different durations of CUMS. CUMS significantly increased the levels of cytokines (IL-1ß, IL-18, and caspase-1), reduced autophagy-related protein levels (Beclin-1, p62, Atg5, and LC3B), and caused microglia cells activation. Oridonin prevented and reversed the depressive-like behavior. Furthermore, it has a stronger and longer-lasting antidepressant effect than FLX. And the antidepressant effect of Oridonin in combination with fluoxetine was greater than that of high-dose fluoxetine alone. In addition, Oridonin significantly normalized autophagy-related protein levels, and reduced levels of cytokines by blocking the interaction between NLRP3 and NEK7. Similarly, Oridonin abolished levels of cytokines and reversed autophagy impairment in LPS-activated BV2 cells. All these results supported our hypothesis that Oridonin possesses potent anti-depressive action, which might be mediated via inhibition of neuroinflammation and autophagy impairment by blocking the interaction between NLRP3 and NEK7.

[Effects of Zhongfeng capsule on autophagy related proteins expressions of brain tissue in rats with cerebral ischemia/reperfusion injury].

Objective: To investigate the effects of Zhongfeng capsule on the autophagy-related proteins expression in rats with cerebral ischemia/reperfusion injury (CI/ RI), and to explore its neural protection mechanisms of the decoction. Methods: Rat middle cerebral artery ischemia/reperfusion injury model (ischemia for 2 h, reperfusion for 24 h) was prepared by the improved line plug method. Sixty male SD rats were randomly divided into sham operation group, model group, butylphthalide group(0.054 g/kg), Zhongfeng capsule high-dose groups (1.08 g/kg), Zhongfeng capsule middle-dose groups (0.54 g/kg), Zhongfeng capsule low-dose groups (0.27 g/kg), with 10 rats in each group. Rats were treated with Zhongfeng capsule by gavage once a day for 10 days. The rats were sacrificed and the brain tissue was obtained after the experiment in each group. Score neurological deficit was evaluated after 24 h of the last intervention in rat of each group. The pathological changes of brain tissue were observed by HE staining. The serum levels of estradiol (E2) and follicle stimulating hormone (FSH) were determined by ELISA. The expressions of key genes and proteins of PI3K/Akt/Beclin1 signaling pathway in brain tissue were detected by qRT-PCR and Western blot respectively. Results: Compared with the sham operation group, the body weight and protein expressions of p-PI3k and p-Akt in brain tissue of rats were decreased significantly in the model group, while the brain index, neurological deficit score, gene and protein expressions of Beclin1 and LC3 were increased markedly in the model group(P＜0.05 or P＜0.01). In the model group, nerve cells of brain tissue were loosely packed, interstitial edema, triangular in shape, nuclear pyknosis and dark-blue staining were observed. Compared with the model group, the body weight of rats was increased obviously, the neurological deficit score was decreased significantly and the pathological injury of brain tissue was alleviated evidently in high-dose of Zhongfeng capsule group (P＜0.05). The brain index, the gene and protein expressions of Beclin1 and LC3 were decreased apparently in Zhongfeng capsule treatment groups(P＜0.05 or P＜0.01), while the expressions of p-PI3k and p-Akt in brain tissue were increased evidently in Zhongfeng capsule treatment groups(P＜0.05 or P＜0.01). Conclusion: Zhongfeng capsule can inhibit autophagy and improve brain neurons lesion of CIRI rats, the mechanism may be related to regulate the expression of Beclin1 and LC3 in PI3K/Akt/Beclin1 signaling pathway.