Marker Protein LC3 Research Articles

A potential strategy for bladder cancer treatment: inhibiting autophagy to enhance antitumor effects of Nectin-4-MMAE

Research and development on Nectin-4 antibody-drug conjugates (ADC) have been greatly accelerated since the approval of enfortumab vedotin to treat uroepithelial cancer. During the course of this study, we identified that autophagy serves as a cytoprotective mechanism during Nectin-4-MMAE treatment and proposed a strategy to enhance the antitumor effects of Nectin-4-MMAE in bladder cancer. Nectin-4-MMAE rapidly internalized into bladder cancer cells in 30 minutes and released MMAE, inducing the onset of caspase-mediated apoptosis and leading to the inhibition of tumor cell growth. Transcriptomics showed significant alterations in autophagy-associated genes in bladder cancer cells treated with Nectin-4-MMAE, which suggested autophagy was activated by Nectin-4-MMAE. Furthermore, autophagy activation was characterized by ultrastructural analysis of autophagosome accumulation, immunofluorescence of autophagic flux, and immunoblotting autophagy marker proteins SQSTM1 and LC3 I/II. Importantly, inhibiting autophagy by LY294002 and chloroquine significantly enhances the cytotoxicity effects of Nectin-4-MMAE in bladder cancer cells. Additionally, we detected the participation of the AKT/mTOR signaling cascade in the induction of autophagy by Nectin-4-MMAE. The combination of Nectin-4-MMAE and an autophagy inhibitor demonstrated enhanced antitumor effects in the HT1376 xenograft tumor model. After receiving a single dose of Nectin-4-MMAE, the group that received the combination treatment showed a significant decrease in tumor size compared to the group that received only one type of treatment. Notably, one mouse in the combination treatment group achieved complete remission of the tumor. The combination group exhibited a notable rise in apoptosis and necrosis, as indicated by H&E staining and immunohistochemistry (cleaved caspase-3, ki67). These findings demonstrated the cytoprotective role of autophagy during Nectin-4-MMAE treatment and highlighted the potential of combining Nectin-4-MMAE with autophagy inhibitors for bladder cancer treatment.

Modulation of autophagy affected tumorigenesis induced by the envelope glycoprotein of JSRV

Ovine pulmonary adenocarcinoma (OPA), caused by the jaagsiekte sheep retrovirus (JSRV), is a chronic, progressive, and contagious lung tumor that seriously affects sheep production. It also represents a valuable animal model for several human lung adenocarcinomas. However, little is known about the role of autophagy in OPA tumorigenesis. Here, Western blotting combined with transmission electron microscopy examination and Cyto-ID dye staining was employed for evaluation of changes of autophagic levels. The results of the present study showed that expression of the autophagy marker proteins Beclin-1 and LC3 was decreased in OPA lung tissues, as well as in cells overexpressing the envelope glycoprotein of JSRV (JSRV Env). Reduced numbers of autophagosomes were also observed in cells overexpressing JSRV Env, although assessment of autophagic flux showed that JSRV Env overexpression did not block the formation of autophagosomes, suggesting increased degradation of autolysosomes. Last, mouse xenograft experiments indicated that inhibition of autophagy by 3-methyladenine suppressed both tumor growth and the epithelial-to-mesenchymal transition. In conclusion, JSRV, through JSRV Env, takes advantage of the autophagy process, leading to the development of OPA.

Hydrogen inhalation enhances autophagy via the AMPK/mTOR pathway, thereby attenuating doxorubicin-induced cardiac injury.

Doxorubicin is a drug widely used in clinical cancer treatment, but severe cardiotoxicity limits its clinical application. Autophagy disorder is an important factor in the mechanism of doxorubicin-induced cardiac injury. As the smallest molecule in nature, hydrogen has various biological effects such as anti-oxidation, anti-apoptosis and regulation of autophagy. Hydrogen therapy is currently considered to be an emerging therapeutic method, but the effect and mechanism of hydrogen on doxorubicin-induced myocardial injury have not been determined. The purpose of this study was to investigate the protective effect of hydrogen inhalation on doxorubicin-induced chronic myocardial injury and its effect and mechanism on autophagy. In this study, we established a chronic heart injury model by intraperitoneal injection of doxorubicin in rats for 30days, accumulating 20mg/kg. The effect of hydrogen inhalation on the cardiac function in rats was explored by echocardiography, Elisa, and H&E staining. To clarify the influence of autophagy, we detected the expression of LC3 and related autophagy proteins in vivo and in vitro by immunofluorescence and western blot.In order to further explore the mechanism of autophagy, we added pathway inhibitors and used western blot to preliminarily investigate the protective effect of hydrogen inhalation on myocardial injury caused by doxorubicin. Hydrogen inhalation can improve doxorubicin-induced cardiac function decline and pathological structural abnormalities in rats. It was confirmed by immunofluorescence that hydrogen treatment could restore the expression of autophagy marker protein LC3 (microtubule-associated protein 1 light chain 3) in cardiomyocytes reduced by doxorubicin, while reducing cardiomyocyte apoptosis. Mechanistically, Western blot results consistently showed that hydrogen treatment up-regulated the ratio of p-AMPK (phosphorylated AMP-dependent protein kinase) to AMPK and down-regulated p-mTOR (phosphorylated mammalian target of rapamycin) and mTOR ratio. These results suggest that hydrogen inhalation can activate autophagy through the AMPK/mTOR pathway and protect against myocardial injury induced by doxorubicin. Hydrogen inhalation therapy may be a potential treatment for doxorubicin-induced myocardial injury.

Discovering Potential Anti-Oral Squamous Cell Carcinoma Mechanisms from Kochiae Fructus Using Network-Based Pharmacology Analysis and Experimental Validation.

The natural product Kochiae Fructus (KF) is the ripe fruit of Kochia scoparia (L.) Schrad and is renowned for its anti-inflammatory, anticancer, anti-fungal, and anti-pruritic effects. This study examined the anticancer effect of components of KF to assess its potential as an adjuvant for cancer treatment. Network-based pharmacological and docking analyses of KF found associations with oral squamous cell carcinoma. The molecular docking of oleanolic acid (OA) with LC3 and SQSTM1 had high binding scores, and hydrogen binding with amino acids of the receptors suggests that OA is involved in autophagy, rather than the apoptosis pathway. For experimental validation, we exposed SCC-15 squamous carcinoma cells derived from a human tongue lesion to KF extract (KFE), OA, and cisplatin. The KFE caused SCC-15 cell death, and induced an accumulation of the autophagy marker proteins LC3 and p62/SQSTM1. The novelty of this study lies in the discovery that the change in autophagy protein levels can be related to the regulatory death of SCC-15 cells. These findings suggest that KF is a promising candidate for future studies to provide insight into the role of autophagy in cancer cells and advance our understanding of cancer prevention and treatment.

Rab2 stimulates LC3 lipidation on secretory membranes by noncanonical autophagy

The Golgi complex is a highly dynamic organelle that regulates various cellular activities and yet maintains a distinct structure. Multiple proteins participate in Golgi structure/organization including the small GTPase Rab2. Rab2 is found on the cis/medial Golgi compartments and the endoplasmic reticulum-Golgi intermediate compartment. Interestingly, Rab2 gene amplification occurs in a wide range of human cancers and Golgi morphological alterations are associated with cellular transformation. To learn how Rab2 ‘gain of function’ influences the structure/activity of membrane compartments in the early secretory pathway that may contribute to oncogenesis, NRK cells were transfected with Rab2B cDNA. We found that Rab2B overexpression had a dramatic effect on the morphology of pre- and early Golgi compartments that resulted in a decreased transport rate of VSV-G in the early secretory pathway. We monitored the cells for the autophagic marker protein LC3 based on the findings that depressed membrane trafficking affects homeostasis. Morphological and biochemical studies confirmed that Rab2 ectopic expression stimulated LC3-lipidation on Rab2-containing membranes that was dependent on GAPDH and utilized a non-canonical LC3-conjugation mechanism that is nondegradative. Golgi structural alterations are associated with changes in Golgi-associated signalling pathways. Indeed, Rab2 overexpressing cells had elevated Src activity. We propose that increased Rab2 expression facilitates cis Golgi structural changes that are maintained and tolerated by the cell due to LC3 tagging, and subsequent membrane remodeling triggers Golgi associated signaling pathways that may contribute to oncogenesis.

Autophagy: A newly discovered protective mechanism in the marine rotifer Brachionus plicatilis in response to BDE-47 exposure

2,2′,4,4′-Tetrabromodiphenyl ether (BDE-47) is a persistent organic pollutant that spreads widely in the marine environment. Our previous studies found that it had adverse effects on the marine rotifer Brachionus plicatilis and caused a series of stress responses. The present study was performed to verify the occurrence of autophagy and explore its role in B. plicatilis’ coping with BDE-47 exposure. Rotifers were exposed to 0.05, 0.2, 0.8, and 3.2 mg/L BDE-47 for 24 h, respectively. Detections of the autophagy marker protein LC3 by western blot and autophagosomes by MDC staining demonstrated the occurrence of autophagy. The levels of autophagy were significantly increased in BDE-47-treated groups with a peak in 0.8 mg/L group. A series of indicators responded to BDE-47 exposure, including reactive oxygen species (ROS), GSH/GSSG ratio, superoxide dismutase (SOD) activity, and malonaldehyde (MDA), collectively indicating the occurrence of oxidative stress. The potential interplay between autophagy and oxidative stress in B. plicatilis was explored in the 0.8 mg/L group through a series of additions. The ROS level was significantly decreased by the addition of the ROS generation inhibitor diphenyleneiodonium chloride, to a level even lower than that in the blank control, and concomitantly, autophagosome was almost undetectable, indicating that a certain level of ROS was essential for the occurrence of autophagy. Autophagy was weakened by the addition of the autophagy inhibitor 3-methyladenine coincident with the great elevation of ROS, indicating that activated autophagy contributed to reducing the ROS level. Additional proof of this relation was obtained from the direct opposite effects of the autophagy inhibitor bafilomycin A1 and the autophagy activator rapamycin: the former increased the MDA content significantly, whereas the latter decreased it significantly. The combined results suggested that autophagy alleviated oxidative stress and might be a newly discovered protective mechanism in B. plicatilis coping with BDE-47 exposure.

Dexmedetomidine improves acute lung injury by activating autophagy in a rat hemorrhagic shock and resuscitation model

Dexmedetomidine (DEX) can reduce lung injury in a hemorrhagic shock (HS) resuscitation (HSR) model in rats by inhibiting inflammation. Here, we aimed to investigate if these effects of DEX are due to autophagy activation. Therefore, we established HSR rat models and divided them into four groups. HS was induced using a blood draw. The rats were then resuscitated by reinjecting the drawn blood and saline. The rats were sacrificed 24 h after resuscitation. Lung tissues were harvested for histopathological examination, determination of wet/dry lung weight ratio, and detection of the levels of autophagy-related marker proteins LC3, P62, Beclin-1, and the ATG12-ATG5 conjugate. The morphological findings of hematoxylin and eosin staining in lung tissues and the pulmonary wet/dry weight ratio showed that lung injury improved in HSR + DEX rats. However, chloroquine (CQ), an autophagy inhibitor, abolished this effect. Detecting the concentration of autophagy-related proteins showed that DEX administration increased LC3, ATG12-ATG5, and Beclin-1 expression and decreased P62 expression. The expression levels of these proteins were similar to those in the HSR group after CQ + DEX administration. In summary, DEX induced autophagic activation in an HSR model. These findings suggest that DEX administration partially ameliorates HSR-induced lung injury via autophagic activation.

Physiologic Functions and Therapeutic Applications of α7 Nicotinic Acetylcholine Receptor in Brain Disorders.

Accumulating data suggest that α7 nicotinic acetylcholine receptors (α7nAChRs) are an important therapeutic target for the treatment of Alzheimer's disease (AD) and schizophrenia. The homopentameric ligand-gated ion channel α7nAChR consists of five identical α7 subunits that are encoded by the CHRNA7 (cholinergic receptor nicotinic alpha7 subunit) gene. Moreover, α7nAChRs are densely distributed throughout the hippocampus, cortex, and thalamus brain regions, but sparsely in the striatum, forebrain, and medulla. Compared with other nAChRs, α7nAChR binds with low affinity to the naturally occurring neurotransmitter acetylcholine and the non-specific exogenous agonist nicotine, and with high affinity to the specific antagonists α-bungarotoxin and methyllycaconitine. Reports indicate that α7nAChR plays important roles in neurotransmitter release, cognitive functioning, and the cholinergic anti-inflammatory response. Genetic variations that alter CHRNA7 mRNA and protein expression or cause α7nAChR dysfunction are associated with many brain disorders. Our previous studies revealed that α7nAChR exerts neuroprotection in AD by acting as a cargo receptor for binding the autophagosomal marker protein LC3 and engulfing extracellular neurotoxic Aβ1-42 during autophagic degradation of the α7nAChR-Aβ1-42 complex. However, the role of α7nAChRs in other diseases remains unknown. Here, we review and summarize the essential characteristics and current findings concerning α7nAChRs in four common brain diseases (AD, Parkinson's disease, schizophrenia, and depression), which may elucidate the role of α7nAChRs and inform innovative research and novel treatments that target α7nAChRs in brain disease.

Effect of starvation and refeeding on reactive oxygen species, autophagy and oxidative stress in Chinese perch (Siniperca chuatsi) muscle growth.

In skeletal muscle, autophagy regulates the development and growth of muscle fibres and maintains the normal muscle metabolism. Under starvation and refeeding conditions, the effect of reactive oxygen species (ROS) levels on skeletal muscle autophagy is still unclear, although the excessive accumulation of ROS has been shown to increase autophagy in cells. The purpose of this study was to explore the effects of starvation and diet after starvation on the autophagy of adult Chinese perch muscle, and to determine the level of ROS in the muscle. We performed zero (Normal control), three and seven starvation treatments on adult Chinese perch, and returned to normal feeding for 3 days after starvation for 7 days. In the muscles of the adult Chinese perch muscle after 3 days of starvation, the autophagy marker protein LC3 and the number of autophagosomes remained basically the same as in the normal feeding situation. However, on starvation for 7 days, the mitochondrial autophagy was sensitive and the number of autophagosomes increased, but the antioxidant-related molecules (malondialdehyde, catalase, glutathione S-transferase, glutathione and anti-superoxide anion) decreased and the accumulation of ROS was obvious. In addition, the extended starvation time also increased the level of LC3 protein. However, by refeeding after starvation this nutritional stress resulted in a decrease in ROS levels and a partial restoration of antioxidant enzyme activity. Our data show that in the adult Chinese perch muscle, starvation could reduce the antioxidant activity through the accumulation of ROS, and that the number of autophagosomes continues to increase. Refeeding after starvation could effectively compensate for the level of ROS, and restore the mRNA abundance of antioxidant genes and the activity of antioxidant enzymes to reduce autophagy and improve feed efficiency. Further research should optimize starvation conditions to reduce autophagy in muscles and maintain normal muscle metabolism.

A LysR Transcriptional Regulator Manipulates Macrophage Autophagy Flux During Brucella Infection.

Brucella, the intracellular bacteria, have evolved subtle strategies to efficiently survive and replicate in macrophages. However, the virulence effector proteins involved are still unclear. LysR-type transcriptional regulators (lttrs) are the largest regulator family with diverse function in prokaryotes. However, very little is known about the role of LysR regulators in the Brucella spp. Here, a BSS2_II0858 gene, encoded as one of the LysR-type regulators, was studied. We successfully constructed a BSS2_II0858 deletion mutant, Δ0858, and complementation strain CΔ0858 in Brucella suis S2. The cell apoptosis induced by B. suis S2 and its derivatives were detected by flow cytometry. The autophagy was then assessed by immunoblot analysis using the IL3I/II and p62 makers. In addition, the autophagy flux was evaluated by double fluorescent labeling method for autophagy marker protein LC3. Our studies demonstrated that B. suis S2 and its derivatives inhibited the programmed cell death in early stage and promoted apoptosis in the later stage during infection in RAW264.7 cells. The BSS2_II0858 gene was found to play no role during apoptosis according to these results. Compared with the wild-type strain, Δ0858 mutant can stimulate the conversion of LC3-I to LC3-II and markedly inhibited the autophagy flux at early stage leading to obvious autophagosome accumulation. This study explored the function of BSS2_II0858 gene and may provide new insights for understanding the mechanisms involved in the survival of Brucella in macrophages.

Autophagy-Related Activation of Hepatic Stellate Cells Reduces Cellular miR-29a by Promoting Its Vesicular Secretion.

Background & AimsLiver fibrosis arises from long-term chronic liver injury, accompanied by an accelerated wound healing response with interstitial accumulation of extracellular matrix (ECM). Activated hepatic stellate cells (HSC) are the main source for ECM production. MicroRNA29a (miR-29a) is a crucial antifibrotic miRNA that is repressed during fibrosis, resulting in up-regulation of collagen synthesis.MethodsIntracellular and extracellular miRNA levels of primary and immortalized myofibroblastic HSC in response to profibrogenic stimulation by transforming growth factor β (TGFβ) or platelet-derived growth factor-BB (PDGF-BB) or upon inhibition of vesicular transport and autophagy processes were determined by quantitative polymerase chain reaction. Autophagy flux was studied by electron microscopy, flow cytometry, immunoblotting, and immunocytochemistry. Hepatic and serum miR-29a levels were quantified by using both liver tissue and serum samples from a cohort of chronic hepatitis C virus patients and a murine CCl4 induced liver fibrosis model.ResultsIn our study, we show that TGFβ and PDGF-BB resulted in decrease of intracellular miR-29a and a pronounced increase of vesicular miR-29a release into the supernatant. Strikingly, miR-29a vesicular release was accompanied by enhanced autophagic activity and up-regulation of the autophagy marker protein LC3. Moreover, autophagy inhibition strongly prevented miR-29a secretion and repressed its targets’ expression such as Col1A1. Consistently, hepatic miR-29a loss and increased LC3 expression in myofibroblastic HSC were associated with increased serum miR-29a levels in CCl4-treated murine liver fibrosis and specimens of hepatitis C virus patients with chronic liver disease.ConclusionsWe provide evidence that activation-associated autophagy in HSC induces release of miR-29a, whereas inhibition of autophagy represses fibrogenic gene expression in part through attenuated miR-29a secretion.

Modified LC3 Dot Quantification Method.

Since the discovery of autophagy genes and proteins in the early1990s, numerous previously unknown physiological and pathological functions have been discovered for autophagy. At the same time, precise monitoring of autophagy has become important, and western blotting and fluorescence microscopy of the marker protein LC3 iswidely used for this purpose. Here, we describe a modification of the widely used method, number of LC3 dots per cell. This protocol provides the proportion of vesicular LC3 staining over the total LC3 staining in the same cell. The approach is well suitable for quantification of endogenous LC3.

A 1,10-phenanthroline derivative selectively targeting telomeric G-quadruplex induces cytoprotective autophagy, causing apoptosis of gastric cancer cells

AimsThis study aimed to evaluate the ability of compound 13d to induce autophagy and to promote apoptosis of tumor cells and its interaction mechanism. Materials and methodsUsing CCK-8 assay, transwell assay, fluorescence resonance energy transfer melting analysis (FRET), transmission electron microscopy, flow cytometry assay, immunofluorescence assay, Western blot analysis, and wound healing assay. Key findingsThe results indicated that compound 13d could induce autophagy and apoptosis of gastric cancer cells. Moreover, the findings of CCK-8 assay, colony formation, migration and invasion assay, and wound healing assay revealed that compound 13d would effectively inhibit cell proliferation, migration, and invasion. Its IC50 value is about 2.4 μM against gastric cancer cells, which is similar to positive drug‑platinum. 13d specific induction of telomere G-quadruplex formation was proved in extracellular FRET melting assay, and indirectly affected telomerase activity. G-quadruplex formation promoted cell apoptosis and autophagy. Upon incorporating the autophagy inhibitors 3-MA and HCQ, the expression of the autophagy marker protein LC3 was then checked, suggesting that the compound 13d influences the autophagy flux. Furthermore, knocking down the autophagy-related gene Atg5 to reduce the level of autophagy enhances the anti-tumor activity and increases apoptotic cells' proportion. Mechanistic experiments have shown that blocking the Akt/m-TOR signal pathway plays a crucial role in autophagy and G-quadruplex induced telomere dysfunction. DNA damage is the leading cause of autophagy. Compound 13d combined with autophagy inhibitor can inhibit tumor cells more effectively. SignificanceOur findings demonstrate that compound 13d as a telomeric G-quadruplex ligand induces Telomere dysfunction, DNA damage response, autophagy, and apoptosis in gastric cancer cells by blocking the Akt/m-TOR signaling pathway.

Effect and mechanism of Angelic Shaoyaosan mediated AMPK/SIRT1 positive feedback loop to promote autophagy and regulate the systemic inflammatory response in acute pancreatitis.

This research was carried out to investigate the effect and mechanism of Angelic Shaoyaosan mediated AMPK/SIRT1 positive feedback loop to promote autophagy and regulate systemic inflammatory response in acute pancreatitis. In this study, the rat pancreatic acini AR42J cells were chosen as the research object, the application of hyla induced pancreatic acinar cells made model for acute pancreatitis, application of different concentrations of angelica peony spread effect on building cells, thus divided into control group, built in the module, the low concentration group, concentration and high concentration groups, determined by MTT method was applied to explore the above categories in cell proliferation, cell apoptosis was measured by flow cytometry, the expression of inflammatory factors in cell supernatant was determined by enzyme-linked immunoassay, and the expression of autophagy marker proteins LC3- ? and P62 was determined by Western-Bolt method. In order to explore the relationship between AMPK and SIRT1, immunoco-precipitation method was used to determine the interaction between AMPK and SIRT1, and dual luciferase experiment was used to explore the effect of AMPK on SIRT1. The AICAR group, BLM-275 group and negative control group were established. To explore the effect of SIRT1 on AMPK, we established SRT 1720 group, EX-527 group and control group. Direct binding between AMPK and SIRT1 should be determined by chromatin co-precipitation assay. In order to further explore the effect of AMPK/SIRT1 positive feedback loop on the systemic inflammatory response of acute pancreatitis, this study selected the medium-concentration Danggui Shaoyajiao SAN group as the control group (group C), and applied AMPK inhibitor BLM-275 and SIRT1 inhibitor EX 527 to the effect of medium-concentration Danggui Shaoyajiao SAN cells, respectively. The expression of autophagy marker proteins LC3- ? and P62 in groups A and B were determined by the Western-Bolt method. Results showed that compared with the control group, the cell survival rate, the expression of AMPK, SIRT1 and LC3-II in the model group were decreased, and the apoptosis rate of iNOS, IL-2, TNF-?, P62 and apoptosis were increased in the model group (P<0.05). the levels of iNOS, IL-2, TNF-?, P62 and cell survival rate in low, medium and high concentration groups decreased gradually, while the expressions of AMPK, SIRT1, LC3-II and cell apoptosis rate increased (P<0.05). The levels of iNOS, IL-2 and TNF-? in the three groups were gradually decreased with the increase of the concentration (P<0.05). Immunoprecipitation showed that AMPK and SIRT1 could bind to each other in cells. The double luciferase experiment indicated that the reporter gene containing the SIRT1 binding site was constructed. The luciferase activity was increased in THE AICAR group and decreased in the BLM-275 group (P<0.05). The reporter gene containing the AMPK promoter binding site was constructed. The luciferase activity in SRT1720 group was increased, while that in EX-527 group was decreased. SIRT1 could directly bind to the AMPK promoter. SIRT1 and LC3- ? protein expressions in group A were down-regulated, and P62 protein was increased (P<0.05). The protein expressions of AMPK and LC3- ? in group B were down-regulated, and the protein expression of P62 was increased (P<0.05). It concluded that AMPK can directly bind to activate SIRT1 expression, and SIRT1 expression can also activate AMPK, forming a positive feedback loop between the two. Therefore, Angelic Shaoyaodong decoction can mediate AMPK/SIRT1 positive feedback pathway to promote autophagy and regulate systemic inflammatory response in acute pancreatitis.

Therapeutic effect of co-culture of rat bone marrow mesenchymal stem cells and degenerated nucleus pulposus cells on intervertebral disc degeneration

BackgroundAfter non-contact co-culture of bone marrow mesenchymal stem cells (BMSCs) with nucleus pulposus cells (NPCs), exosomes secreted by BMSCs were able to ameliorate the degree of disc degeneration. The reason for this is, at least in part, that exosomes from BMSCs achieve by affecting the level of autophagy in NPCs, while the components in exosomes are diverse and their specific mechanism of action is still unclear. PurposeHere, we aimed to explore the therapeutic effect of co-culture of BMSCs and NPCs on NPCs and explore its specific mechanism of action. Study design/SettingIn vitro study. MethodsRat NPCs and BMSCs were isolated and cultured in vitro. The serum deprivation experiment (using oxygen, glucose, and serum deprivation [OGD]) simulates the pathological state of low blood supply of the intervertebral disc in vivo. We used apoptotic cell staining and flow cytometry to study the effect of BMSCs on the apoptosis rate of rat NPCs, and the apoptotic proteins active-caspase-3, active-caspase-9, autophagy marker proteins LC3 and Beclin 1 were further detected using Western blot analysis. The expression levels of the pro-apoptotic protein Bax and the apoptosis-inhibiting protein Bcl2 were measured. The differentially expressed miRNAs were screened in a gene expression profiling chip. Then qRT-PCR was used to detect the effect of different treatment methods on miR-155 expression. The effect of anti-miR-155 antibodies on autophagy was studied by flow cytometry and transmission electron microscopy. A luciferase reporter assay was used to study the direct interaction between miR-155 and BACH1 mRNA, which was analyzed by TargetScan software, and the results were verified by Western blotting. ResultsCompared with the OGD group, the expression level of miR-155 and the NPC autophagy level significantly increased; the HO-1 protein expression increased; and the Bach1 protein expression, degeneration index, and apoptosis index all significantly decreased in the co-culture group. After BMSCs transfected with anti-miR-155 were co-cultured with NPCs, the miR-155 expression in the cells was significantly reduced, the HO-1 protein expression and the level of cell autophagy was reduced. However, Bach1 protein expression, NPC degeneration index, and apoptosis index increased. After being inhibited by the autophagy inhibitor wortmannin, the cell degeneration index and apoptosis rate significantly improved. ConclusionIn the OGD model, BMSCs can significantly increase the viability, the level of autophagy, and reduce the level of apoptosis in rat NPCs. BMSC exosomes increase miR-155 expression in NPCs, which targets Bach1 and in turn upregulates HO-1 expression, activates autophagy in NPCs, inhibits the apoptosis level, and improves intervertebral disc degeneration. Clinical SignificanceOur experiment shows that it is maybe feasible to treat disc degeneration with drugs. At the same time, compared with BMSC injection method of treatment, side effects of drug therapy are smaller, and can be controlled, it also provides a new way for intervertebral disc degeneration drug treatment.

Effects of &amp;lt;i&amp;gt;Buyang Huanwu&amp;lt;/i&amp;gt; Decoction on Endoplasmic Reticulum Stress-Autophagy Response and GADD34 Expression in Mice with CIRI

<i>Objective</i>: To investigate the effects of <i>Buyang Huanwu Decoction</i> on endoplasmic reticulum stress-autophagic response and the expression of GADD34 in ischemic penumbra of mice with CIRI. <i>Methods</i>: The mice were randomly divided into sham operation group, model group and <i>Buyang Huanwu Decoction</i> low-, medium-, and high-dosage groups. The mice were treated by gavage for 7 days before modeling and 24 hours after modeling. CIRI model was established by the thread embolization method. The neurological function was evaluated on the 48 hours after modeling; TTC staining was used to observe the cerebral infarct volume; HE staining was used to observe the pathological changes of ischemic penumbra; TUNEL was performed to assess cell apoptosis; Western blot was performed to assess the expression of endoplasmic reticulum stress marker protein GRP78 and autophagy marker proteins LC3 and p62; Immunohistochemistry and real-time PCR were applied to examine the protein and mRNA expressions of GADD34. <i>Results</i>: Compared with the model group, neurological function score and volume of cerebral infarction were decreased, reperfusion injury of ischemic penumbra was improved with varying degrees, number of apoptotic cells was decreased, expression of GRP78 and p62 protein were down-regulated while ratio of LC3-II/ LC3 –I was up-regulated, and expression of GADD34 protein and mRNA were up-regulated in each <i>Buyang Huanwu Decoction</i> group, with statistical significance (<i>P</i><0.01). <i>Conclusion Buyang Huanwu Decoction</i> can significantly improve the damaged neurological function, cerebral infarction volume and histopathological damage of ischemic penumbra, as well as reduce the apoptosis cells, which plays a neuroprotective role in mice with CIRI. Its mechanism may be related to up regulating the expression of GADD34, regulating the steady-state balance of endoplasmic reticulum stress autophagy response in ischemic penumbra.

Plasmodium berghei sporozoites in nonreplicative vacuole are eliminated by a PI3P-mediated autophagy-independent pathway.

The protozoan parasite Plasmodium, causative agent of malaria, invades hepatocytes by invaginating the host cell plasma membrane and forming a parasitophorous vacuole membrane (PVM). Surrounded by this PVM, the parasite undergoes extensive replication. Parasites inside a PVM provoke the Plasmodium‐associated autophagy‐related (PAAR) response. This is characterised by a long‐lasting association of the autophagy marker protein LC3 with the PVM, which is not preceded by phosphatidylinositol 3‐phosphate (PI3P)‐labelling. Prior to productive invasion, sporozoites transmigrate several cells and here we describe that a proportion of traversing sporozoites become trapped in a transient traversal vacuole, provoking a host cell response that clearly differs from the PAAR response. These trapped sporozoites provoke PI3P‐labelling of the surrounding vacuolar membrane immediately after cell entry, followed by transient LC3‐labelling and elimination of the parasite by lysosomal acidification. Our data suggest that this PI3P response is not only restricted to sporozoites trapped during transmigration but also affects invaded parasites residing in a compromised vacuole. Thus, host cells can employ a pathway distinct from the previously described PAAR response to efficiently recognise and eliminate Plasmodium parasites.

Effects of All-&lt;i&gt;Trans&lt;/i&gt; Retinoic Acid on the Optimization of Synovial Explant Induced by Tumor Necrosis Factor Alpha

Current studies focused on the effects of all-trans-retinoic acid (ATRA) on synovial explants from rats with rheumatoid arthritis (RA) induced by lipopolysaccharides (LPS). In our study, synovial membranes were extracted aseptically from the quadriceps femoris of the knee joint of rats, and then incubated in medium containing 10% neonate bovine serum for 24 h adaptive culture. We first measured variations of correlation factors in synovium at 24, 48, 72, 96 and 120 h in control medium or in medium containing 20 ng/mL tumor necrosis factor alpha (TNF-α) (TNF-α-experiment). Then, we investigated the synovium exposed to three ATRA concentrations after 48 h incubation (ATRA-experiment). The effects of ATRA on synovitis were evaluated by observing the expression of inflammatory cytokines, angiogenic factors and the production of proteases in nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway and apoptosis and autophagy. In TNF-α-experiment, the secretion of nitric oxide (NO), interleukin-6 (IL-6), vascular endothelial growth factor (VEGF), and matrix metalloproteinase-9 (MMP-9) increased significantly after TNF-α stimulation without pathological damage to the synovium. Hence, we successfully obtained the synovial explants model, which had longer inflammatory response time. In the ATRA-experiment, ATRA suppressed the secretion of IL-6 and NO, downregulated the NF-κB P65 and Bcl-2, increased levels of autophagy marker protein LC3, but different doses of ATRA showed inconsistent regulatory effects on VEGF and MMP-9. In short, ATRA inhibited TNF-α induced synovitis by the regulation of inflammatory cytokines and inhibiting NF-κB signal transduction and potentially promoting autophagy, apoptosis and angiogenesis, displaying its role in alleviating synovial inflammation in patients with RA.

Staphylococcus aureus Avoids Autophagy Clearance of Bovine Mammary Epithelial Cells by Impairing Lysosomal Function.

In dairy herds, mastitis caused by Staphylococcus aureus is difficult to completely cure on the account that S. aureus can invade bovine mammary epithelial cells (BMECs) and result in persistent infection in the mammary gland. Recent studies have demonstrated that autophagy can participate in cell homeostasis by eliminating intracellular microorganisms. The aim of the study was to investigate why S. aureus can evade autophagy clearance and survive in BMECs. The intracellular infection model was first constructed; then, the bacteria in autophagosome was detected by transmission electron microscopy. The autophagy flux induced by the S. aureus was also evaluated by immunoblot analysis and fluorescent labeling method for autophagy marker protein LC3. In addition, lysosomal alkalization and degradation ability were assessed using confocal microscopy. Results showed that, after infection, a double-layer membrane structure around the S. aureus was observed in BMECs, indicating that autophagy occurred. The change in autophagy marker protein and fluorescent labeling of autophagosome also confirmed autophagy. However, as time prolonged, the autophagy flux was markedly inhibited, leading to obvious autophagosome accumulation. At the same time, the lysosomal alkalization and degradation ability of BMECs were impaired. Collectively, these results indicated that S. aureus could escape autophagic degradation by inhibiting autophagy flux and damaging lysosomal function after invading BMECs.

MTOR Inhibitor rapamycin can reverses the combination antiretroviral therapy induced central nervous system disorder

BackgroundAccumulating evidences indicate that the combined antiretroviral therapy (cART) has dramatically increased the life expectancy of HIV infected individuals. However, existing HIV population on cART show several complications including liver or pancreas damage, cardiovascular diseases and central nervous system disorders. Unexpectedly the prevalence of HIV+ associated neurotoxicity is on the rise. Hence, the current study is undertaken to determining the potential therapeutic effects of rapamycin against anti‐retroviral therapy induced neuronal dysfunction.ObjectiveThis study is aimed at exploring the therapeutic potential of rapamycin on cART induced central nervous system disorder.MethodologyPrimary midbrain neuronal cells (PMNC) were treated with antiretroviral drugs like Ritonavir (protease inhibitor) and Abacavir (reverse transcriptase inhibitor) in the combined form.ResultsAfter 24 hours treatment, immunoblot analysis shows that there is upregulation of autophagy marker proteins LC3 II, P62 levels in cART treated primary midbrain neuronal cells compared to control cells. Similarly, cART treated cells shows upregulation of endoplasmic reticulum (ER) resident proteins expression with mitochondrial dysfunction. These results are suggesting that neuronal cells treated with cART have dysregulated autophagy, higher ER stress and mitochondrial dysfunction. The beneficial effects of rapamycin are well described; though, it is unknown whether rapamycin ameliorates the harmful effects of cART‐induced toxicity in neuronal cells. Hence, in this study, we intend to investigate the effects of rapamycin on PMNC treated with cART. Our results indicate that rapamycin treatment ameliorates the autophagy dysfunction as well as, it normalizes the mitochondrial function and reduces the ER stress.ConclusionThis study shows that rapamycin have a good potential in reversing cART‐induced autophagy and mitochondrial dysfunction in primary midbrain neuronal cells.Schematic representation of therapetic effects of rapamycinFigure 1