Expression Of Light Chain 3 Research Articles

Effects of hypoxia/reoxygenation on apoptosis, autophagy and pyroptosis in H9C2 cardiomyocytes

Objective To investigate the effects of hypoxia/reoxygenation (HR) on apoptosis, autophagy and pyroptosis in H9C2 cardiomyocytes. Methods The cultured H9C2 cardiomyocytes were divided into two groups according to the random number table method: a normal control (Ctrl) group and an HR group. The H9C2 cardiomyocytes in the HR group were deprived of oxygen and glucose for 8 h in a hypoxic incubator, followed by reoxygenation for 12 h. Cell damage was assessed through detection of lactate dehy-drogenase (LDH) content in culture medium and determination of cell viability by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) method (n=6). Apoptosis related proteins [cysteinyl aspartate-specific proteinase (caspase)-3, B-cell lymphoma/leuke-mia 2 (Bcl-2) and Bcl-associate x protein (Bax)], autophagy related proteins [(light chain 3 (LC3) Ⅱ/Ⅰ , p62, Beclin-1, and phosphory-lated mammalian target of rapamycin (p-mTOR)]and pyroptosis related proteins [(nod-like receptor pyrin domain 3 (NLRP3), apoptosis associated speck-like protein (ASC), caspase-1p20, interleukin (IL)-1β, and IL-18]were detected by Western blot (n=9). Cell apopto-sis, autophagy and pyroptosis were further assessed by immunofluorescence staining (n=6) Results Compared with the Ctrl group, the HR group presented reduced viability of H9C2 cardiomyocytes (P<0.05), increased LDH release (P<0.05), and up-regulated expres-sion of activated caspase-3 and Bax (P<0.05), as well as decreased expression of Bcl-2 (P<0.05). TUNEL staining showed that apoptosis significantly was enhanced in the cells after HR (P<0.05). These results indicated that apoptosis and cell damage were enhanced after HR. In contrast, the expression of p-mTOR and p62 increased (P<0.05), but the expression of LC3 Ⅱ/Ⅰ and Beclin-1 protein decreased(P<0.05). The immunofluorescence staining of LC3 Ⅱ/Ⅰ showed that the number of autophagosome within the cells increased after HR(P<0.05), indicating that autophagy in H9C2 myocardial cells was significantly inhibited. Meanwhile, the expression of NLRP3 inflamma-some, ASC and caspase-1p20 protein was remarkably up-regulated (P<0.05), and the expression of activated inflammatory cellular factors IL-1β and IL-18 increased (P<0.05), suggesting that NLRP3 inflammasomes were activated and pyroptosis enhanced after HR. Conclusions Autophagy is inhibited in H9C2 cardiomyocytes during HR injury, but pyroptosis and apoptosis are enhanced. Key words: Autophagy; Apoptosis; Pyroptosis; Cardiomyocytes; Hypoxia-reoxygenation injury

Melatonin attenuates vascular calcification by activating autophagy via an AMPK/mTOR/ULK1 signaling pathway

Melatonin has been demonstrated to protect against calcification in cyclosporine nephrotoxicity. Autophagy may affect vascular calcification by inhibiting apoptosis and the transdifferentiation process. This study sought to explore whether melatonin attenuates vascular calcification by regulating autophagy via the AMP-activated protein kinase/mammalian target of rapamycin/Unc-51-like kinase 1 (AMPK/mTOR/ULK1) signaling pathway. The effects of melatonin on vascular calcification were investigated in vascular smooth muscle cells (VSMCs). Calcium deposits were visualised by Alizarin red staining, while calcium content and alkaline phosphatase (ALP) activity were used to evaluate osteogenic differentiation. Western blots were used to measure expression of runt-related transcription factor 2 (Runx2, an osteogenic transcription factor), light chain 3 (LC3) II/I, and cleaved caspase 3. Melatonin markedly reduced calcium deposition and ALP activity. Runx2 and cleaved caspase 3 were downregulated, whereas LC3 II/I was increased in response to melatonin, and was accompanied by decreased apoptosis. An immunofluorescence assay revealed that melatonin treatment markedly decreased Runx2 expression and upregulated LC3 expression. Treatment with the autophagy inhibitor 3-methyladenine reversed this phenomenon. Melatonin significantly increased expression of p-AMPK and p-ULK1, and decreased mTOR expression. Treatment with compound C (an inhibitor of AMPK) or MHY1485 (an agonist of mTOR) ablated the observed benefits of melatonin treatment. Melatonin protects VSMCs against calcification by activating autophagy via the AMPK/mTOR/ULK1 pathway.

Protective effects of autophagy inhibitor 3-methyladenine on ischemia-reperfusion-induced retinal injury.

To investigate the protective effects of autophagy inhibitor 3-methyladenine (3-MA) in a rat model of ischemic-reperfusion injury (IRI). Forty Sprague-Dawley male rats (weight 220-250g) were randomly divided into four groups: a control group (NC, n = 10), a Sham surgery group (n = 10), an IRI group (n = 10), and a 3-MA-treated IRI group [10 μL 3-MA (10mmol/L) was injected in vitreous after the injury, n = 10]. The retinal IRI was induced by elevating the intraocular pressure to 110mmHg for 60min. Hematoxylin and eosin (HE) staining was used to calculate the number of retinal ganglion cells (RGCs). The level of microtubule-associated protein 1A/1B light chain 3 (LC3), Beclin-1, and Caspase-3 in the retina was detected using the immunofluorescence staining method. The LC3, Beclin-1, B-cell lymphoma/leukemia-2 (Bcl-2), and Caspase-3 protein levels were examined by Western blotting. The number of RGCs in IRI group was significantly lower than that in NC group (P < 0.05), demonstrated by HE staining. Western blotting results indicated that the protein expression of LC3 and Beclin-1 in the IRI group was significantly elevated compared with those in the NC group (P < 0.05). However, with 3-MA treatment, the number of RCGs in 3-MA-treated IRI group was elevated and protein levels of LC3, Beclin-1 were down-regulated, compared with those in the IRI group (P < 0.05). Further immunohistochemistry staining and Western blot showed that 3-MA-treated IRI group presented down-regulated Caspase-3 and up-regulated Bcl-2 protein expression with comparison of IRI group (P < 0.05). Retina IRI-caused RGCs loss involved activated autophagy pathway and apoptosis, which could be prevented by autophagy inhibitor 3-MA. Autophagy inhibitor 3-MA may act as a potent therapeutic tool in attenuating retina IRI.

The Prognostic Value of Autophagy-Related Markers Bclin-1 and LC-3 in Colorectal Cancers: A Systematic Review and Meta-analysis.

Objective At present, the relationship between autophagosomes and the prognosis of various cancers has become a subject of active investigation. A series of studies have demonstrated the correlation between autophagy microtubule-associated protein light chain 3 (LC-3), Beclin-1, and colorectal cancer (CRC). Since autophagy has dual regulatory roles in tumors, the results of this correlation are also uncertain. Hence, we summarized the relationship between Beclin-1, LC-3, and CRC using systematic reviews and meta-analysis to clarify their prognostic significance in it. Methods PubMed, EMBASE, Cochrane Library, and Web of Science databases were searched online up to April 1, 2019. The quality of the involving studies was assessed against the Newcastle-Ottawa Scale (NOS). Pooled hazard ratio (HR) and 95% confidence interval (CI) in a fixed or random effects model were used to assess the strength of correlation between Beclin-1, LC-3, and CRC. Results A total of 9 articles were collected, involving 2,297 patients. Most literatures scored more than 6 points, suggesting that the quality of our including research was acceptable. Our finding suggested that the expression of Beclin-1 was not associated with overall survival (HR = 0.68, 95% CI (0.31–1.52), P=0.351). Nonetheless, LC-3 expression exerted significant impact on OS (HR = 0.51, 95% CI (0.35–0.74), P < 0.05). Subgroup analysis exhibited that Beclin-1 expression was associated with OS at TNM stage III (HR = 0.04, 95% CI = 0.02–0.08, P < 0.05), surgical treatment (HR = 1.53, 95% CI (1.15–2.02), P=0.003), and comprehensive treatment (HR = 0.27 95% CI (0.08–0.92), P=0.036), respectively. Similarly, the results showed the increased LC-3 expression in CRC was related to OS in multivariate analyses (HR = 0.44, 95% CI (0.34–0.57), P < 0.05), stages (HR = 0.51, 95% CI (0.35–0.74), P < 0.05), and comprehensive treatment (HR = 0.44, 95% CI (0.34–0.57), P < 0.05). Conclusions Autophagy-related proteins of LC-3 might be an important marker of CRC progression. However, since the number of the original studies was limited, more well-designed, large-scale, high-quality studies are warranted to provide more convincing and reliable information.

Role of Autophagy in Acquired Cholesteatoma.

Autophagy and its enhancement may have a role in the pathogenesis of acquired cholesteatoma. The etiopathogenesis of acquired cholesteatoma remains unclear. Some clinical features of cholesteatoma are similar to those of cancer. The study of autophagy in cancer has indicated that enhanced autophagy enables tumor cell survival and growth. Cholesteatoma epithelium and normal external auditory canal (EAC) epithelium were obtained from patients with acquired cholesteatoma, and marginal epithelium of the tympanic membrane perforation was obtained from patients with chronic otitis media (COM). Immunohistochemistry (IHC) was performed to detect the expression of light chain 3 (LC3) in cholesteatoma and EAC epithelium. Western blotting (WB) was performed to detect the expression of LC3, Beclin-1, or the PI3K/AKT pathway in cholesteatoma, EAC, and COM epithelium. LC3 staining of IHC was stronger in cholesteatoma epithelium compared with normal EAC epithelium. The ratios of LC3-II/I and Beclin-1 expression on WB were significantly higher in cholesteatoma epithelium compared with EAC epithelium or COM epithelium, and there was a significantly higher ratio of p-PI3K/PI3K and p-AKT/AKT in cholesteatoma epithelium compared with EAC epithelium. Enhanced autophagy might play a role in the pathogenesis of acquired cholesteatoma. PI3Ks might have different regulatory functions on autophagy in the cholesteatoma epithelium.

MIR-190B Alleviates Cell Autophagy and Burn-Induced Skeletal Muscle Wasting via Modulating PHLPP1/Akt/FoxO3A Signaling Pathway.

Cell autophagy is an important material recycling process and is involved in regulating many vital activities under both physiological and pathological conditions. However, the mechanism of autophagy regulating burn-induced skeletal muscle wasting still needs to be elucidated. The rat burn model with 30% total body surface area and L6 cell line were used in this study. An immunofluorescence assay was used to detect autophagic levels. MicroRNA array and real-time PCR were employed to measure miR-190b levels, and its influence on PH domain and leucine-rich repeat protein phosphatase 1 (PHLPP1) protein translation was estimated using luciferase reporter assay. The expression levels of autophagy-related proteins were analyzed by Western blot. Skeletal muscle wasting was evaluated by the ratio of tibias anterior muscle weight to body weight. Our study demonstrates that burn injury promotes expression of the autophagy-related proteins light chain 3 (LC3) and Beclin-1, suppresses expression of Akt and Forkhead box O (FoxO) 3a protein phosphorylation, and increases PHLPP1 protein level which is required for Akt dephosphorylation. miR-190b, the regulator of PHLPP1 protein translation, also significantly decreases after burn injury. Ectopic expression of miR-190b in L6 myoblast cell downregulates PHLPP1 protein expression, elevates Akt and FoxO3a phosphorylation, and subsequently reduces cell autophagy. Finally, suppressing autophagy with 3-methyladenine represses the protein expression of LC3 and Beclin-1 and mitigates burn-induced skeletal muscle wasting. Burn injury induced skeletal muscle cell autophagy and subsequently resulted in skeletal muscle wasting via regulating miR-190b/PHLPP1/Akt/FoxO3a signaling pathway.

Conditioned medium obtained from human amniotic mesenchymal stem cells attenuates focal cerebral ischemia/reperfusion injury in rats by targeting mTOR pathway

Conditioned medium obtained from human amniotic mesenchymal stem cells (hAMSC-CM) was recently shown to have many antioxidant, antiapoptotic and proangiogenic growth factors. The present study was performed to investigate whether protective effects of hAMSC-CM against focal cerebral ischemia/ reperfusion (I/R) injury is associated with modulation of the mammalian target of rapamycin (mTOR) pathway.A rat model of middle cerebral artery occlusion (MCAO) was created and the animals were divided into three groups including sham, MCAO and MCAO + hAMSC-CM. Drug was administrated immediately after cerebral reperfusion (i.v). The expressions of mTOR, p-mTOR and LC3 were measured using Western blotting and real time-PCR, respectively. Apoptosis and neuronal loss were determined using TUNEL and Nissl staining, respectively. Infarct volume and the blood–brain barrier (BBB) damage were evaluated using 2,3,5-triphenyltetrazolium chloride (TTC) staining and Evans Blue (EB) uptake, respectively.Compared with sham, significant infarct volume, apoptotic cell death, and neuronal loss were found in MCAO rats that reversed by hAMSC-CM (P < 0.05). Likewise, MCAO rats exhibited increased mRNA level of light-chain 3 (LC3) and the LC3II/LC3I ratio as well as decreased expression level of p-mTOR that reversed by hAMSC-CM (P < 0.05). There were no significant differences in the expression of total mTOR among the experimental groups.In summary, our results demonstrate that hAMSC-CM gives rise to neuroprotection following ischemic stroke by restoring mTOR activity and inhibiting autophagy.

Characterization of in vivo autophagy during avian spermatogenesis

Spermatogenesis is a complex cellular process that includes many subcellular events that are essential for the production of healthy spermatozoa. Autophagy is a physiological process that plays a significant role in the process of spermatogenesis; however, autophagy during avian spermatogenesis has not yet been reported. In the current study, we characterized in vivo autophagy throughout the process of domestic fowl spermatogenesis. Autophagy-specific markers, including microtubule-associated protein light chain 3 (LC3), sequestosome 1 (p62), and autophagy-related 7 (Atg7), were used to confirm the occurrence of autophagy in testicular germ cells. The protein expression of Atg7, LC3, and p62 in domestic fowl testes was confirmed by Western blotting. The immunohistochemical staining indicated a strong localization of LC3 and Atg7 within spermiogenic cells (intermediate and late spermatids) and primary spermatocytes. However, poorly expressed in cells (spermatogonia) that were located near the basement membrane. The immunofluorescence staining results showed the opposite tendency for LC3 and p62. LC3 was more strongly localized within the elongated spermatids, while p62 was strongly localized within the early spermatids. Moreover, the ultrastructural components of autophagy were revealed by transmission electron microscopy. Well-developed autophagosomes and multivesicular bodies were found to be prominent in primary spermatocytes (zygotene and pachytene) and spermiogenic cells. Furthermore, other vesicular structures, such as early endosomes and amphisomes, were also observed during spermatogenesis. The above findings collectively suggest that autophagy is active during spermatogenesis and that the level of autophagy increases from the basal to the luminal regions of the seminiferous tubules of domestic fowl testes. We propose that autophagic pathways may be involved in multiple functions to sustain spermatogenesis.

WNT1-inducible signaling protein-1 mediates TGF-β1-induced renal fibrosis in tubular epithelial cells and unilateral ureteral obstruction mouse models via autophagy.

Renal fibrosis is a common pathway for the progression of all chronic kidney diseases to end-stage kidney disease. Studies show that WNT1-inducible signaling pathway protein-1 (WISP-1) is involved in the fibrosis of various organs. The aim of the study was to explore the functional role and potential mechanism of WISP-1 in renal fibrosis. We observed that overexpression of WISP-1 in rat tubular epithelial cells (TECs) enhanced transforming growth factor-β1 (TGF-β1)-induced production of fibrotic markers, including collagen I (Col I), fibronectin (FN) and TGF-β1, while inhibition of WISP-1 suppressed such production. In vivo, the messenger RNA and protein levels of Col I, FN, and α-smooth muscle actin were significantly inhibited after anti-WISP-1 antibody treatment for 7 days in unilateral ureteral obstruction mouse models. Moreover, blockade of WISP-1 by anti-WISP-1 antibody significantly reduced autophagy-related markers, including anti-microtubule-associated protein-1 light chain 3 (LC3) and beclin 1, while increasing sequestosome 1. In addition, overexpression of WISP-1 in TECs increased autophagy as evidenced by greater numbers of GFP-LC3 puncta and increased expression of LC3 and beclin 1 in response to TGF-β1. In contrast, knockdown of WISP-1 by small interfering RNA decreased the number of GFP-LC3 puncta and the expression of LC3 and beclin 1 in TGF-β1-treated TECs. Collectively, these data suggest that WISP-1, as a profibrotic protein, may mediate renal fibrosis by inducing autophagy in both obstructive nephropathy and TGF-β1-treated TECs. WISP-1 may serve as an effective therapeutic target for the treatment of renal fibrosis.

Effect of autophagy on expression of neutrophil programmed death ligand-1 in mice with sepsis

To investigate the effect and mechanism of autophagy on the expression of neutrophil programmed death ligand-1 (PD-L1) in mice with sepsis. (1) In vivo experiment: male C57BL/6 mice aged 6-8 weeks were divided into sham operation group (Sham group), cecum ligation and perforation (CLP) group, and rapamycin (RAP)+CLP group by random number table with 10 mice in each group. The sepsis model was reproduced by CLP, and the cecum and perforation were not ligated in Sham group, and other operations were the same as CLP group. The mice in RAP+CLP group were intraperitoneally injected with autophagy agonist RAP 4 mg×kg-1×d-1 7 days before modeling, while the mice in Sham group and CLP group were not treated. Lung, liver, spleen and pancreas tissues were harvested for immunohistochemical staining 4 days after the operation, and the infiltration of neutrophils in various organs was observed under light microscope. Meanwhile, the expressions of immunosuppressive molecule PD-L1 and autophagy marker microtubule-associated protein 1 light chain 3 (LC3) in lung neutrophils were determined by immunofluorescence staining. (2) In vitro experiment: mouse bone marrow neutrophils were extracted and re-suspended to 1×1010/L, and they were divided into blank control group (without any treatment), RAP control group (RAP 100 μmol/L), autophagy inhibitor Bafilomycin A1 (Baf) control group (Baf 10 μmol/L), lipopolysaccharide (LPS) stimulation group (LPS 1 mg /L), RAP+LPS group, and Baf+LPS group. The latter two groups were pretreated with 100 μmol/L RAP or 10 μmol/L Baf 30 minutes before LPS stimulation, respectively. The expression of PD-L1 mRNA of neutrophils was determined by reverse transcription-polymerase chain reaction (RT-PCR) at 0, 4, 12 hours after LPS stimulation. At the same time, the expressions of PD-L1, LC3 and p62 at the protein level were determined by Western Blot. (1) In vivo experiment: according to immunohistochemical experiments, a large amount of infiltration of neutrophils in lung, liver, spleen and pancreas was found at 4 hours after CLP. In the immunofluorescence, with the time extension after CLP, the positive expression of LC3 in the lung tissue showed a decreased tendency, and PD-L1 expression was significantly increased. RAP pretreatment could promote the expression of LC3 and reduce the expression of PD-L1 in CLP mice. (2) In vitro experiment: in terms of mRNA levels, with the extension of LPS stimulation time, the expression of PD-L1 mRNA in mouse neutrophils was increased continuously, and peaked at 12 hours, it was significantly higher than that in the blank control group (2-ΔΔCT: 72.2±10.0 vs. 13.0±0.8, P < 0.01). Compared with LPS stimulation group, the expression of PD-L1 mRNA in RAP+LPS group was significantly down-regulated [12-hour PD-L1 mRNA (2-ΔΔCT): 47.4±7.3 vs. 72.2±10.0, P < 0.01]. In Baf+LPS group, PD-L1 mRNA expression was significantly up-regulated as compared with that in LPS stimulation group [12-hour PD-L1 mRNA (2-ΔΔCT): 109.1±7.4 vs. 72.2±10.0, P < 0.01]. At the protein levels, at 4 hours after LPS stimulation, the positive expressions of PD-L1, LC3 and p62 were increased significantly as compared with those in the blank control group, and PD-L1 and p62 were increased continuously with time. Compared with the LPS stimulation group, the expressions of PD-L1 and p62 in the RAP+LPS group were significantly down-regulated, while the expression of LC3 was continually increased, indicating that the level of autophagy was increased, and autophagy was circulated smoothly. On the contrary, the expressions of PD-L1, LC3 and p62 in the Baf+LPS group were significantly up-regulated, indicating that the binding of autophagy and lysosome was blocked, and autophagy was not smooth. In sepsis, the infiltration of neutrophils in all organs increased, and the expression of PD-L1 of neutrophils in lungs was increased significantly, while the expression level of autophagy was decreased. The expression of PD-L1 stimulated by LPS can be inhibited by autophagy agonists, and promoted by autophagy inhibitors. PD-L1 has a negative regulatory effect on sepsis. It can reduce the expression of PD-L1 molecule in sepsis by targeting autophagy, so as to improve sepsis.

The correlation between CB2R and autophagy-related protein LC3 expression in the hippocampal CA1 re-gion of the model with status epilepticus

Objective The expression and correlation between cannabinoid receptor type 2(CB2R) and autophagy-related protein microtubule-associated protein 1 light chain 3(LC3) in the hippocampal CA1 region of developing rats with status epilepticus(SE) were investigated. Methods SE model was established using lithium chloride-pilocarpine intraperitoneally in Sprague-Dawley(SD) rats and all the rats were randomly divided into four groups( control group and 3h, 24h, 3d groups after SE). The expressions of CB2R and LC3 in the hippocampal CA1 region at different times were observed using double-label immunofluorescence and Western blotting.Spearman correlational analysis was used to compare the relationship between the two factors. Results Immunofluorescence showed that the expression of CB2R was up-regulated dynamically and peaked at 24h and presented parabolic changes over time.The expression of LC3 changed in accordance with CB2R and even co-expressed with CB2R partly, especially on neurons.Western blotting results furtherly showed the similarity of the expression of CB2R and LC3, and finally Spearman correlational analysis presented the significant correlation between these two factors(r=0.7161, P<0.05). Conclusion Significant correlation exists between the expression of CB2R and LC3 in the hippocampal CA1 neurons of developing rats with SE, indicating the essential role of CB2R in autophagy regulation of hippocampal CA1 neurons. Key words: Status epilepticus; Hippocampus; Cannabinoid receptor type 2; Autophagy; Microtubule-associated protein 1 light chain 3

Rapamycin attenuates articular cartilage degeneration by inhibiting β-catenin in a murine model of osteoarthritis

ABSTRACTPurpose: To investigate whether systemic injection of rapamycin attenuates articular cartilage degeneration by inhibiting β-catenin in a murine model of osteoarthritis (OA).Materials and methods: Ten-week-old male C57BL/6j wild-type (WT) mice and SOST-knockout (SOST−/−) mice were randomized to a sham group, a vehicle-treated group, and a rapamycin-treated group. Mice in the vehicle-treated group underwent destabilizing of the medial meniscus (DMM) in the right knee, and were then treated with vehicle. Mice in the rapamycin treatment group underwent DMM and were treated with rapamycin. Safranin O-Fast green staining and Osteoarthritis Research Society International (OARSI) modified Mankin score were used to evaluate the histopathological features of the articular cartilage in the knee. The expression of light chain 3 (LC3) was evaluated by immunofluorescence, whereas the expression of ATG5, matrix metallopeptidase 13 (MMP-13), vascular endothelial growth factor (VEGF), sclerostin, and β-catenin were evaluated by immunohistochemistry. TUNEL staining was used to determine apoptosis of chondrocytes.Results: In vehicle-treated mice when compared with mice in the sham group, the OARSI scores, expression of MMP-13, VEGF, sclerostin, β-catenin, and chondrocyte apoptosis were significantly increased, whereas the expression of LC3 and ATG5 were significantly decreased. A systemic injection of rapamycin activated chondrocyte autophagy, which increased the expression of LC3 and ATG-5, and reduced OARSI scores, the expression of β-catenin, MMP-13, and VEGF, and chondrocyte apoptosis in rapamycin treated mice when compared with vehicle-treated mice.Conclusions: Systemic injection of rapamycin attenuated articular cartilage degeneration by inhibiting β-catenin in a murine model of OA.

Autophagy suppresses radiation damage by activating PARP-1 and attenuating reactive oxygen species in hepatoma cells

Purpose: To investigate the relationship between autophagy and radiation damage of human hepatoma cells and to explore the role of reactive oxygen species (ROS).Materials and methods: HepG2 cells were exposed to X-rays, then the protein expressions of microtubule-associated protein 1 light chain 3 (LC3) and poly ADP-ribose polymerase-1 (PARP-1) were measured by Western blot assay, the formation of autophagosomes was detected by an autophagy detection kit, the intracellular ROS level was measured by flow cytometer, and DNA damage was evaluated by the incidence of micronuclei (MN). A CCK-8 kit was used to measure the proliferation ability of irradiated cells with or without N-acetyl-l-cysteine (NAC) treatment. In some experiments, the hepatoma cells were transferred with LC3 siRNA or PARP-1 siRNA before irradiation.Results: The protein expressions of LC3 and PARP-1 and the inductions of autophagosomes and intracellular ROS were increased in the irradiated HepG2 cells. Pretreatment of cells with NAC relieved the irradiation-induced inhibition of cell proliferation. When HepG2 cells were transfected with the LC3 siRNA, the over-expression of PARP-1 was diminished in the irradiated cells. Compared with the control group, the inhibitions of LC3 and PARP-1 increased ROS level in the irradiated HepG2 cells and hence sensitized radiation responses of both proliferation inhibition and MN induction.Conclusion: Autophagy upregulates the expression of PARP-1 and relieves radiation damage by reducing the generation of ROS.

Role of autophagy induced by arecoline in angiogenesis of oral submucous fibrosis

ObjectivesTo detect the expression of protein light chain 3 (LC3) and p62-SQSTM1 (p62) in the lamina propria of oral submucous fibrosis (OSF) and to determine the association of autophagy with OSF. To investigate the role of autophagy in angiogenesis of human umbilical vein endothelial cells (HUVECs) and to assess whether this effect was induced by arecoline. MethodsLC3 and p62 expression was detected in OSF tissue through immunohistochemistry (IHC). Transmission electron microscopy (TEM) and Western blot were used to investigate the expression of autophagy in HUVECs. The role of autophagy in angiogenesis in HUVECs was investigated using the Matrigel assay. Results1: LC3 expression was upregulated in OSF samples. In contrast, p62 was downregulated in early and intermediate stages but upregulated in advanced stages of OSF. 2: HUVECs treated with arecoline exhibited increased autophagosomes, LC3 expression and reduced p62 expression, when co-treated with chloroquine (CQ), which is a specific autophagy inhibitor, revealed the opposite trend. 3: Autophagy inhibited angiogenesis in HUVECs. ConclusionsOur findings suggest that arecoline induces autophagy in HUVECs. The high level of autophagy could reduce cell viability and inhibit angiogenesis in HUVECs, potentially promoting the development of OSF.

In vitro study of effects of transient receptor potential vanilloid 1 on autophagy in early hypoxic mouse cardiomyocytes and the mechanism

Objective: To explore the effects of transient receptor potential vanilloid 1 (TRPV1) on autophagy in early hypoxic mouse cardiomyocytes and the mechanism in vitro. Methods: The hearts of 120 C57BL/6 mice aged 1-2 days, no matter male or female, were isolated, and then primary cardiomyocytes were cultured and used for the following experiments, the random number table was used for grouping. (1) The cells were divided into normoxia group and hypoxia 3, 6, and 9 h groups, with one well in each group. The cells in normoxia group were routinely cultured (the same below), the cells in hypoxia 3, 6, and 9 h groups were treated with fetal bovine serum-free and glucose-free Dulbecco' s modified Eagle medium under low oxygen condition in a volume fraction of 1% oxygen, 5% carbon dioxide, and 94% nitrogen for 3, 6, and 9 h, respectively. The protein expressions of microtubule-associated protein 1 light chain 3 (LC3), Beclin-1, TRPV1 were determined with Western botting. (2) The cells were divided into normoxia group and hypoxia group, with two coverslips in each group. The cells in hypoxia group were treated with hypoxia for 6 h as above. The positive expression of TRPV1 was detected by immunofluorescence assay. (3) The cells were divided into 4 groups, with one well in each group. The cells in simple hypoxia group were treated with hypoxia for 6 h as above, and the cells in hypoxia+ 0.1 μmol/L capsaicin group, hypoxia+ 1.0 μmol/L capsaicin group, and hypoxia+ 10.0 μmol/L capsaicin group were respectively treated with 0.1, 1.0, 10.0 μmol/L capsaicin for 30 min before hypoxia for 6 h. The protein expressions of LC3, Beclin-1, and TRPV1 were detected by Western blotting. (4) The cells were divided into 5 groups, with 5 wells in each group. The cells in hypoxia group were treated with hypoxia for 6 h as above, the cells in hypoxia+ chloroquine group, hypoxia+ capsaicin group, and hypoxia+ capsaicin+ chloroquine group were treated with hypoxia for 6 h after being cultured with 50 μmol/L chloroquine, 10.0 μmol/L capsaicin, and 50 μmol/L chloroquine+ 10.0 μmol/L capsaicin for 30 min, respectively. Viability of cells was detected by cell counting kit 8 assay. (5) The cells were divided into simple hypoxia group and hypoxia+ 10.0 μmol/L capsaicin group, with one well in each group. The cells in hypoxia group were treated with hypoxia for 6 h as above, the cells in hypoxia+ 10.0 μmol/L capsaicin group were treated with 10.0 μmol/L capsaicin for 30 minutes and then with hypoxia for 6 h. The protein expressions of lysosomal associated membrane protein 1 (LAMP-1) and LAMP-2 were detected by Western blotting. Each experiment was repeated for 3 or 5 times. Data were processed with one-way analysis of variance, least significant difference t test, and Bonferroni correction. Results: (1) Compared with those of normoxia group, the protein expressions of LC3, Beclin-1, and TRPV1 were significantly increased in cardiomyocytes of hypoxia 3, 6, and 9 h groups (t(3 h)=4.891, 5.890, 4.928; t(6 h)=9.790, 6.750, 10.590; t(9 h)=6.948, 6.764, 5.049, P<0.05 or P<0.01), which of hypoxia 6 h group were the highest (1.08±0.05, 1.12±0.10, 0.953±0.071, respectively). (2) The density of TRPV1 in cell membrane and inside the cardiomyocytes in hypoxia group was significantly increased with lump-like distribution, and the expression of TRPV1 was higher than that in normoxia group. (3) Compared with those of simple hypoxia group, the protein expression of Beclin-1 in cardiomyocytes of hypoxia+ 0.1 μmol/L capsaicin group was increased (t=10.488, P<0.01), while the protein expressions of LC3 and TRPV1 were increased without statistically significant differences (t=4.372, 3.026, P>0.05); the protein expressions of LC3, TRPV1, and Beclin-1 in cardiomyocytes of hypoxia+ 1.0 μmol/L capsaicin group and hypoxia+ 10.0 μmol/L capsaicin group were significantly increased (t=15.505, 5.773, 13.430; 20.915, 8.054, 16.384; P<0.05 or P<0.01), which of hypoxia+ 10.0 μmol/L capsaicin group were the highest (2.33±0.09, 1.34±0.07, 1.246±0.053, respectively). (4) Compared with 0.585±0.045 in normoxia group, the cardiomyocyte viability in hypoxia group was significantly decreased (0.471±0.037, t=4.365, P<0.05). Compared with that in hypoxia group, the cardiomyocyte viability in hypoxia+ chloroquine group was further decreased (0.350±0.023, t=6.216, P<0.01), while 0.564±0.047 in hypoxia+ capsaicin group was significantly increased (t=3.489, P<0.05). Compared with that in hypoxia+ chloroquine group, the cardiomyocyte viability in hypoxia+ capsaicin+ chloroquine group did not significantly change (0.364±0.050, t=0.545, P>0.05). (5) Compared with 0.99±0.04 and 0.54±0.04 in simple hypoxia group, the protein expressions of LAMP-1 and LAMP-2 in hypoxia+ 10.0 μmol/L capsaicin group were significantly increased (1.49±0.06, 0.81±0.05, t=12.550, 7.442, P<0.01). Conclusions: TRPV1 can further promote the expression of autophagy-related proteins in hypoxic cardiomyocytes through autophagy-lysosomal pathway, enhance autophagy activity, and improve autophagic flow for alleviating early hypoxic cardiomyocyte injury.

Clinicopathological Significance of Autophagy-related Proteins and its Association With Genetic Alterations in Gliomas.

To investigate clinicopathological significance of autophagy and its association with genetic alterations in gliomas. The expression of three autophagy-related proteins, light chain-3 (LC3), beclin 1, and p62 was immunohistochemically analyzed in 32 low-grade gliomas and 65 high-grade gliomas. LC3, beclin 1, and p62 expression was positive in 70/94 (74%), 51/94 (54%) and 55/96 (57%) gliomas, respectively. High expression of LC3, beclin 1 and p62 was significantly more frequent in high-grade gliomas than in low-grade. Positive expression of LC3, beclin 1 and p62 were significantly positively correlated with overall survival, methylation of O6-methylyguanine-DNA methyltransferase (MGMT) promoter, mutations of isocitrate dehydrogenase 1 (IDH1) and telomerase reverse transcriptase (TERT) promoter, and 1p/19q co-deletion. Kaplan-Meier analyses revealed that LC3, p62 and autophagy status (positivity for at least two of the three proteins) were significantly associated with poorer survival. Autophagy might be associated with the progression of glioma, particularly high-grade, and thus might be a useful prognostic factor in patients with glioma.

Changes of autophagy in brain tissue after asphyxial cardiac arrest and cardiopuimonary resuscitation

Objective To establish a simple rat model of asphyxia cardiac arrest (CA) by clamping endotracheal tube and observe the changes of autophagy after cardiopuimonary resuscitation (CPR). Methods A total of 30 clean male Sprague Dawley rats, weighing 200-300 g, were randomly divided into sham operation group (n=6) and CA/CPR group (n=24), then the CA/CPR group was subdivided into 4 subgroups: 3 h group, 6 h group, 24 h group and 72 h group after resuscation of spontaneous circulation (ROSC), with 6 rats in each group. Neurological deficit scores (NDS) were evaluated at different time points after ROSC. Morphological changes of rat brain were observed by hematoxylin and eosin (HE) staining. The expression of tubulin light chain 3 (LC3), Beclin-1 and Parkin in the hippocampus was detected by Western blotting. The immunofluorescence was used to observe the expression of LC3. Results Compared with the sham operation group, the NDS scores in the CA/CPR group were significantly reduced at each time point, and the brain tissue damage was significantly aggravated. The ratio of LC3Ⅱ/Ⅰ in the 6, 24 and 72 hsubgroups after ROSC was (0.443±0.058), (0.588±0.073), and (0.286±0.036), respectively. Compared with the sham operation group (0.127±0.386), the differences were statistically significant (P<0.01). The expression levels of Beclin-1 in the 6, 24 and72 hsubgroups after ROSC were (0.531±0.033), (0.640±0.039), and (0.435±0.036), respectively. Compared with the sham operation group (0.216±0.028), the expression levels were significantly higher (P<0.01). The expression levels of Parkin in the 6, 24 and 72 h subgroups after ROSC were (0.621±0.021), (0.706±0.020), and (0.588±0.014), respectively. Compared with the sham operation group (0.329±0.032), the expression levels were significantly higher. The difference was statistically significant (P<0.01). The average absorbance of the immunofluorescence in the CA/CPR group was (8.66±0.99)%, which was significantly higher than that in the sham operation group (3.87±0.62)% (P<0.01), suggesting that the proportion of LC3 particles in the CA/CPR group increased. Conclusion Five min of CA in rats causes significant damage to brain tissue. Autophagy (including mitophagy) was activated at 6 h after ROSC, which was the highest at 24 h, decreased at 72 h but was still activated by autophagy. Key words: Cardiac arrest; Cardiopulmonary resuscitation; Autophagy; Mitophagy; Rat

Effect of electroacupuncture with different frequencies on hindlimb locomotor function and expression of LC3, Beclin 1 and cleaved Caspase-3 proteins in spinal cord injury rats

To investigate the effect of different frequencies of electroacupuncture (EA) on motor function and expression of autophagy-related proteins microtubule-associated protein light chain 3 (LC3), etc. in spinal cord injury (SCI) rats, so as to reveal its underlying mechanisms and provide a reference for clinical application. A total of 100 male SD rats were randomly divided into sham control, model, 2 Hz-EA, 100 Hz-EA and 2 Hz/100 Hz-EA groups(n=20 in each group). EA (2 Hz, 100 Hz or 2 Hz/100 Hz, 0.4-0.5-0.6 mA increased by 0.1 mA every10 min) was applied to "HuatuoJiaji" (EX-HN1, T9 and T11) for 30 min, once a day for 7 days. The rat's hindlimb motor function was assessed by using Basso, Beattie and Bresnahan(BBB) locomotor rating scale, inclined plane test and plantar imprinting test, separately. The histological changes and neuronal apoptosis of the spinal cord tissue were observed by Fluoro-Jade B (FJB) and Nissl staining, respectively. The expression of LC3, Beclin 1 and cleaved Caspase-3 proteins in the spinal cord was detected by Western blot. After modeling, the BBB scores and the angles of inclined plane on day 1, 3 and 7 were significantly decreased (P<0.001), and the number of FJB positive cells, and the expression levels of Beclin 1 and cleaved Caspase-3 proteins were considerably increased in the model group compared with the sham control group (P<0.001, P<0.05). After EA intervention, the BBB score and the angles of inclined plate on day 3 in the 2 Hz/100 Hz-EA group (rather than in the 2 Hz- and 100 Hz-EA groups), the BBB score and the angles of inclined plate on day 7 in both 2 Hz/100 Hz and 100 Hz-EA groups(rather than in the 2 Hz-EA group), and the expression levels of Beclin 1 and LC3-Ⅱ/Ⅰ in both 2 Hz/100 Hz and 100 Hz-EA groups (rather than in the 2 Hz-EA group) on day 7 were obviously increased (P<0.001, P<0.01, P<0.05), while the number of FJB-positive neurons in the 3 EA groups, and the expression of cleaved Caspase-3 protein in both 2 Hz/100 Hz and 100 Hz-EA groups and the LC3-Ⅱ/Ⅰ in the 2 Hz-EA group were obviously decreased relevant to the model group (P<0.001, P<0.05). The expression of Beclin 1 in the 100 Hz-EA group was obviously decreased relevant to the 2 Hz/100 Hz-EA group (P<0.05) .Nissl staining displayed appearance of cavities and fuzzy shape of Nissl bodies with light coloration in the injured spinal cord of model group, which was milder in both 2 Hz/100 Hz-EA and 100 Hz-EA groups. Plantar imprinting tests showed dragging gait prints in the model group due to disability in movement, and relatively distinct foot imprints in both 2 Hz/100 Hz and 100 Hz-EA groups. Both 100 Hz and 2 Hz/100 Hz-EA can effectively promote the recovery of hindlimb locomotor function of SCI rats, which may be related to its function in promoting autophagy of damaged nerve cells and in reducing neuronal apoptosis.

Penetrative needling improves neurological function by up-regulating expression of autophagy related protein LC3 in rats with hemorrhagic stroke

To observe the effect of penetrative needling from "Baihui" (GV20) to "Qubin" (GB7) on neurological function and expression of autophagy related protein microtubule-associated protein, light chain 3 (LC3) in rats with hemorrhagic stroke, so as to explore its mechanism underlying improvement of hemorrhagic stroke. A total of 120 male SD rats were randomly divided into sham operation, model, non-acupoint, acupuncture and medication (Rapamycin) groups which were further divided into two time-point subgroups (3, 7 days after modeling, n=12/subgroup). The intracerebral hemorrhage model was established by injection of the rat's auto-blood (50 μL) into the putaman region. Penetrative needling from GV20 to GB7 or sham acupoints (about 1 cm beside GV20 and GB7) was conducted for 30 min, twice daily for 7 days. For rats of the medication group, Rapamycin solution (7 μmol/L) was injected into the right lateral ventricle. The neurological functions (locomotor and balance deficits) were evaluated according to suspended wire test (0-6 points) and horizontal board walking test (0-6 points). Immunohistochemistry and Western blot were used to detect the expression of total LC3 and expression of LC3-Ⅰ and LC3-Ⅱ proteins in the ischemic penumbra region of brain tissue, respectively. After modeling, the neurological function scores were significantly decreased on day 3 and 7, the levels of LC3-Ⅱ/Ⅰ and LC3 protein on day 3 and 7 were significantly increased in the model group relevant to the sham operation group (P<0.05). Following the interventions, the neurological function scores as well as LC3-Ⅱ/Ⅰ and LC3 protein expression were significantly increased in both acupuncture and medication groups compared with the model group (P<0.05). The effect of Rapamycin was obviously stronger than that of penetrative needling in up-regulating the expression of LC3-Ⅱ/Ⅰ and LC3 protein (P<0.05). Penetrative needling can improve neurologic function in hemorrhagic stroke rats, which is related with its effect in up-regulating the expression of autophagy-related protein LC3.

Panax Notoginseng Saponins Attenuate Myocardial Ischemia-Reperfusion Injury Through the HIF-1α/BNIP3 Pathway of Autophagy.

Panax Notoginseng Saponins (PNS) is a formula of Chinese medicine commonly used for treating ischemia myocardial in China. However, its mechanism of action is yet unclear. This study investigated the effect and the mechanism of PNS on myocardial ischemia-reperfusion injury (MIRI) through the hypoxia-inducible factor 1α (HIF-1α)/bcl-2/adenovirus E1B19kDa-interacting protein3 (BNIP3) pathway of autophagy. We constructed a rat model of myocardial injury and compared among 4 groups (n = 10, each): the sham-operated group (Sham), the ischemia-reperfusion group (IR), the PNS low-dose group, and the PNS high-dose group were pretreated with PNS (30 and 60 mg/kg, respectively). Serum creatine kinase, malonaldehyde (MDA), lactate dehydrogenase, myocardial tissue superoxide dismutase, and reactive oxygen species were detected in rats with myocardial ischemia-reperfusion after the intervention of PNS. The rat myocardial tissue was examined using hematoxylin and eosin (H&E) staining, and the mitochondria of myocardial cells were observed using transmission electron microscopy. The expressions of microtubule-associated protein light chain 3 (LC3), HIF-1α, BNIP3, Beclin-1, and autophagy-related gene-5 (Atg5) in rat myocardial tissue were detected using Western blotting. The results showed that PNS was significantly protected against MIRI, as evidenced by the decreasing in the concentration of serum CK, MDA, lactate dehydrogenase, and myocardial tissue superoxide dismutase, reactive oxygen species, the attenuation of myocardial tissue histopathological changes and the mitochondrial damages of myocardial cells, and the increase of mitochondria autophagosome in myocardial cells. In addition, PNS significantly increased the expression of LC3 and the ratio of LC3II/LC3I in rat myocardial tissue. Moreover, PNS significantly increased the expression of HIF-1α, BNIP3, Atg5, and Beclin-1 in rat myocardial tissue. The protective effect of PNS on MIRI was mainly due to its ability to enhance the mitochondrial autophagy of myocardial tissue through the HIF-1α/BNIP3 pathway.