mRNA Levels Of LC3 Research Articles

Expression pattern of ATG4C and its effect on early embryonic development of porcine oocytes

Autophagy is essential for oocyte maturation and preimplantation embryo development. ATG4C, a member of the ATG4 family, plays a crucial role in the autophagy process. The effect of ATG4C on the early embryonic development in pig has not been studied. In this study, the expression patterns of ATG4C were explored using qRT-PCR and immunofluorescence staining. Different concentrations of serum were added to in vitro maturation (IVM) medium to investigate its effects on oocyte maturation and embryonic development. Finally, the developmental potential of parthenogenetic embryos was detected by downregulating ATG4C in MII stage oocytes under 0 % serum condition. The results revealed that ATG4C was highly expressed in porcine oocytes matured in vitro and in parthenogenetic embryos. Compared with the 10 % serum group, the cumulus cell expansion, first polar body (PB1) extrusion rate, and subsequent developmental competence of embryos were reduced in the 0 % and 5 % serum groups. The mRNA levels of LC3, ATG5, BECLIN1, TFAM, PGC1α, and PINK1 were significantly increased (P < 0.05) in the 0 % serum group. ATG4C was significantly upregulated in the embryos at the 1-cell, 2-cell, 8-cell, and 16-cell stages in the 0 % serum group (P < 0.05). Compared with the negative control group, downregulation of ATG4C significantly decreased the 4-cell, 8-cell, and blastocyst rates (P < 0.05), and the expression of genes related to autophagy, mitochondria, and zygotic genome activation (ZGA) was significantly decreased (P < 0.05). The relative fluorescence intensity of LC3 and mitochondrial content in the ATG4C siRNA group was significantly reduced (P < 0.05). Collectively, the results indicate that ATG4C is highly expressed in porcine oocytes matured in vitro and in early embryos, and inhibition of ATG4C effects embryonic developmental competence by decreasing autophagy, mitochondrial content, and ZGA under serum-free condition.

Acid ground nano-realgar processed product inhibits breast cancer by inducing mitophagy via the p53/BNIP3/NIX pathway.

Breast cancer is a highly prevalent malignancy with the first morbidity and the primary reason for female cancer-related deaths worldwide. Acid ground nano-realgar processed product (NRPP) could inhibit breast cancer cell proliferation and induce autophagy in our previous research; however, the underlying mechanisms are still unclear. Therefore, this research aimed to verify whether NRPP induces breast cancer mitophagy and explore the mitophagy-mediated mechanism. Primarily, rhodamine-123 assay and transmission electron microscopy were applied to detect mitochondrial membrane potential (MMP) and ultrastructural changes in the MDA-MB-435S cells, respectively. Mito-Tracker Green/Lyso-Tracker Red staining, western blot, immunofluorescence and RT-PCR were used to explore molecular mechanisms of NRPP-induced mitophagy in vitro. MDA-MB-435S breast cancer xenograft models were established to assess the activity and mechanisms of NRPP in vivo. Our results showed that NRPP decreased MMP and increased autophagosome numbers in MDA-MB-435S cells and activated mitophagy. Furthermore, mitophagy was consolidated because mitochondria and lysosomes colocalized phenomenology were observed, and the expression of LC3II/I and COXIV was upregulated. Additionally, we found the p53/BNIP3/NIX pathway was activated. Finally, NRPP inhibited tumour growth and downregulated the levels of TNF-α, IL-1β and IL-6. Necrosis, damaged mitochondria and autophagosomes were observed in xenograft tumour cells, and proteins and mRNA levels of LC3, p53, BNIP3 and NIX were increased. Overall, NRPP inhibited MDA-MB-435S cell proliferation and tumour growth by inducing mitophagy via the p53/BNIP3/NIX pathway. Thus, NRPP is a promising candidate for breast cancer treatment.

TYRO3 promotes chemoresistance via increased LC3 expression in pancreatic cancer

Pancreatic cancer (PC) is an aggressive malignancy with few treatment options, and improved treatment strategies are urgently required. TYRO3, a member of the TAM receptor tyrosine kinase family, is a known oncogene; however, the relationship between TYRO3 expression and PC chemoresistance remains to be elucidated. We performed gain- and loss-of-function experiments on TYRO3 to examine whether it is involved in chemoresistance in PC cells. TYRO3 knockdown decreased cell viability and enhanced apoptosis following treatment of PC cells with gemcitabine and 5-fluorouracil (5-FU). In contrast, no such effects were observed in TYRO3-overexpressing PC cells. It is known that autophagy is associated with cancer chemoresistance. We then examined effects of TYRO3 on autophagy in PC cells. TYRO3 overexpression increased LC3 mRNA levels and induced LC3 puncta in PC cells. Inhibition of autophagy by chloroquine mitigated cell resistance to gemcitabine and 5-FU. In a xenograft mouse model, TYRO3 silencing significantly increased sensitivity of the cells to gemcitabine and 5-FU. To further investigate the involvement of autophagy in patients with PC, we immunohistochemically analyzed LC3 expression in the tissues of patients who underwent pancreatectomy and compared it with disease prognosis and TYRO3 expression. LC3 expression was negatively and positively correlated with prognosis and TYRO3 expression, respectively. Furthermore, LC3- and TYRO3-positive patients had a significantly worse prognosis among patients with PC who received chemotherapy after recurrence. These results indicated that the TYRO3-autophagy signaling pathway confers PC resistance to gemcitabine and 5-FU, and could be a novel therapeutic target to resolve PC chemoresistance.

Sulforaphane Ameliorates Hepatic Lipid Metabolism via Modulating Lipophagy In Vivo and In Vitro.

Although sulforaphane (SFN) is reported to ameliorate the excessive accumulation of lipid droplets (LDs) in hepatocytes, its underlying mechanism remains unclear. This paper aims to investigate how SFN induces hepatic LD degradation via activating macroautophagy. High-fat diet and free fatty acids (FFAs) were used to induce excessive LD formation in hepatocytes in vivo and in vitro, respectively. SFN-induced macroautophagy was shown by the increased LC3 protein expression both (1.32 ± 0.18) in vivo and (2.43 ± 0.22) in vitro. The mRNA levels of Lc3 (1.99 ± 0.16), Atg4 (2.12 ± 0.23), Ulk1 (1.19 ± 0.12), Atg7 (1.25 ± 0.11), and Atg5 (0.81 ± 0.1) genes were elevated by SFN. SFN individually enhanced the localization of LC3 (0.41 ± 0.15), LAMP1 (0.66 ± 0.14), ATG7 (0.26 ± 0.08), and ATG5 (0.38 ± 0.09) with LDs, indicating the occurrence of lipophagy. In the components of LDs isolated from SFN treatment, the expressions of LC3, ATG7, and ATG5 protein were largely increased both in vivo and in vitro. LDs were visualized in autophagosomes which confirmed that the lipophagy was triggered by SFN. Moreover, SFN treatment improved the profile of FFAs which was characterized by increasing the FFAs in liver (total FFA: 261.51 ± 39.58 μM/g) and serum (total FFA: 967.59 ± 239.18 nM/mL). After silencing the nrf2 gene, ATG7 and ATG5 protein expressions were decreased and attenuated this induction by SFN. Nrf2 gene silencing inversely increased TG contents. In summary, SFN enhanced the LD degradation via stimulating lipophagy in a Nrf2-dependent manner.

Mechanism of Atractylodes macrocephala against Alzheimer's disease via regulating lysophagy based on LKB1-AMPK-TFEB pathway

Myloid beta(Aβ) is produced by cleavage of amyloid precursor protein(APP), which is a main reason for Alzheimer's disease(AD) occurrence and development. This study preliminarily investigated the mechanism of Atractylodes macrocephala(AM) against AD based on LKB1-AMPK-TFEB pathway. The effect of AM on memory ability of AD transgenic Caenorhabditis elegans CL2241 was detected, and then the APP plasmid was transiently transferred to mouse neuroblastoma(N2 a) cells in vitro. The mice were divided into the blank control group, APP group(model group), positive control group(100 μmol·L~(-1) rapamycin), and AM low-, medium-and high-dose groups(100, 200 and 300 μg·mL~(-1)). The content of Aβ_(1-42) in cell medium, the protein level of APP, the fluorescence intensity of APP, the transcriptional activity of transcription factor EB(TFEB), the activity of lysosomes in autophagy, and autophagy flux were determined by enzyme-linked immunosorbent assay(ELISA), Western blot, fluorescence microscope, luciferase reporter gene assay, RLuc-LC3 wt/RLuc-LC3 G120 A, and mRFP-GFP-LC3, respectively. The protein expression of TFEB, LC3Ⅱ, LC3Ⅰ, LAMP2, Beclin1, LKB1, p-AMPK and p-ACC was detected by Western blot. Immunofluorescence and reverse transcription-polymerase chain reaction(RT-PCR) were used to detect the fluorescence intensity of TFEB and the mRNA expression of TFEB and downstream target genes, respectively. The results showed that AM reduced the chemotactic index of transgenic C. elegans CL2241, and decreased the content of Aβ in the supernatant of cell culture medium at different concentrations. In addition, AM lowered the protein level of APP and the fluorescence intensity of APP in a dose-dependent manner. Transcriptional activity of TFEB and fluorescence intensity of mRFP-GFP-LC3 plasmid were enhanced after AM treatment, and the value of RLuc-LC3 wt/RLuc-LC3 G120 A was reduced. AM promoted the protein levels of TFEB, LAMP2 and Beclin1 at different concentrations, and increased the protein expression ratio of LC3Ⅱ/LC3Ⅰ in a dose-dependent manner. Immunofluorescence results revealed that AM improved the fluorescence intensity and nuclear expression of TFEB, and RT-PCR results indicated that AM of various concentrations elevated the mRNA expression of TFEB in APP transfected N2 a cells and promoted the transcription level of LAMP2 in a dose-dependent manner, and high-concentration AM also increased the mRNA levels of LC3 and P62. The protein levels of LKB1, p-AMPK and p-ACC were elevated by AM of different concentrations. In summary, AM regulating lysophagy and degrading APP are related to the activation of LKB1-AMPK-TFEB pathway.

Mitochondrial Calcium Transporters Regulate Autophagy

Autophagy is an essential cellular process regulated by intracellular calcium signals, which play an important role in autophagic activation during metabolic changes. Calcium transporters are present in mitochondria, an organelle able to uptake and release calcium ions, thus participating in cellular calcium signaling. Uptake by mitochondria is mediated by the mitochondrial calcium uniporter (MCU), while extrusion occurs through the mitochondrial sodium/lithium/calcium exchanger (NCLX). The aim of this work was to investigate how MCU and NCLX affect autophagy. To do so, we evaluated makers of autophagic activity in murine Aml‐12 hepatic cells transfected with siRNAs targeting either MCU or NCLX expression, leading to genetic knockdown (KD). Additionally, we investigated the autophagic response to serum/amino acid starvation and rapamycin (mTORC1 inhibitor) treatment in Aml‐12 cells with NCLX KD or pharmacological inhibition by CGP37157 (CGP). Using a cell line stably expressing the autophagic probe LC3‐GFP‐mCherry, we observed that NCLX KD leads to impaired autophagosome formation under basal conditions. Curiously, this effect was associated with a significant decrease in mRNA expression of LC3A and LC3B genes, while the expression of other autophagy‐related genes, such as TFEB, ATG5, ATG12, and ATG7, was upregulated or unchanged. Conversely, MCU KD led to an apparent increase in autophagosome and autolysosome numbers, indicating enhanced autophagic activity. Interestingly, MCU KD also led to decreased expression of LC3A, but not LC3B. The expression of TFEB, ATG12, ATG5, and ATG7 were decreased or unchanged by MCU KD. After autophagic stimulation by serum/amino acid starvation, the levels of LC3 II were lower in NCLX KD cells compared to negative control in the presence or absence of bafilomycin A1, which indicates a reduction of autophagic flux. The levels of LC3 I were significantly lower in NCLX KD cells under basal and stimulated conditions, corroborating the decreased LC3 mRNA levels observed. Importantly, these effects were also observed using CGP to inhibit NCLX. The reduction of autophagic flux by NCLX KD or CGP was not observed in cells treated with rapamycin. We also measured the levels of phosphorylated 4E‐BP1 as an indication of mTORC1 activity. As expected, serum/amino acid starvation and rapamycin decreased the levels of p‐4E‐BP1; however, this decrease was modulated by CGP only in starved cells, indicating that NCLX may affect mTORC1 activity in an upstream pathway. In conclusion, we show that mitochondrial calcium transporters are novel autophagy‐regulating pathways: MCU modulates autophagic activation under basal conditions, while NCLX maintains autophagic activity under basal and stimulated conditions.

Dihydromyricetin reduces lipid accumulation in LO2 cells via AMPK/mTOR-mediated lipophagy pathway and inhibits HepG2 cell proliferation in vitro

To explore the mechanism underlying the hepatoprotective effect of dihydromyricetin (DMY) against lipid accumulation in light of the lipophagy pathway and the inhibitory effect of DMY on HepG2 cell proliferation. LO2 cells were cultured in the presence of 10% FBS for 24 h and treated with 100 μg/mL DMY, or exposed to 50% FBS for 24 h followed by treatment with 50, 100, or 200 μg/mL DMY; the cells in recovery group were cultured in 50% FBS for 24 h and then in 10% FBS for another 24 h. Oil red O staining was used to observe the accumulation of lipid droplets in the cells, and the levels of TC, TG, and LDL and activities of AST, ALT and LDH were measured. The expression of LC3 protein was detected using Western blotting. AO staining and transmission electron microscopy were used to determine the numbers of autophagolysosomes and autophagosomes, respectively. The formation of autophagosomes was observed with MDC staining, and the mRNA expression levels of LC3, ATG7, AMPK, mTOR, p62 and Beclin1 were determined with q-PCR. Flow cytometry was performed to analyze the effect of 50, 100, and 200 μg/mL DMY on cell cycle and apoptosis of HepG2 cells; DNA integrity in the treated cells was examined with cell DNA fragmentation test. DMY treatment and pretreatment obviously inhibited lipid accumulation and reduced the levels of TC, TG, LDL and enzyme activities of AST, ALT and LDH in LO2 cells (P < 0.05). In routinely cultured LO2 cells, DMY significantly promoted the formation of autophagosomes and autophagolysosomes and upregulated the expression of LC3 protein. DMY obviously attenuated high FBS-induced inhibition of autophagosome formation in LO2 cells, up- regulated the mRNA levels of LC3, ATG7, Beclin1 and AMPK, and downregulated p62 and mTOR mRNA levels (P < 0.05 or 0.01). In HepG2 cells, DMY caused obvious cell cycle arrest, inhibited cell proliferation, and induced late apoptosis and DNA fragmentation. DMY reduces lipid accumulation in LO2 cells by regulating the AMPK/ mTOR-mediated lipophagy pathway and inhibits the proliferation of HepG2 by causing cell cycle arrest and promoting apoptosis.

APOE4 expression is associated with impaired autophagy and mitophagy in astrocytes.

APOE4 expression is associated with impaired autophagy and mitophagy in astrocytes.

Denatured Collagen Could Increase the Autophagy Level and Inhibit Apoptosis of Fibroblasts to Help Cell Survival and Influence Wound Healing.

When exposed to thermal factors, collagen in the dermis denatures, which could affect the biological behavior of cells. Previous studies have demonstrated that denatured collagen could influence the activity of fibroblasts and induce fibroblasts differentiate into myofibroblasts. However, information on the regulation of fibroblasts by denatured collagen-modulated autophagy and apoptosis during the wound healing process is limited. In this article, we researched the effect of denatured collagen-modulated autophagy and apoptosis on fibroblasts. An in vitro model comprising fibroblasts and denatured collagen was established to identify the potential ability of denatured collagen to modulate autophagy and apoptosis. Western blotting, quantitative polymerase chain reaction, transmission electron microscopy, TUNEL assay, and immunofluorescence staining were used to examine the changes induced by denatured collage. Protein and mRNA levels of LC3 and beclin-1 were significantly increased after stimulated by denatured collagen, while those of caspase-3 were reduced. Unlike stimulation with normal collagen, denatured collagen enhanced autophagy and inhibited apoptosis of fibroblasts. After blocking autophagy using 3-methyladenine, the apoptotic function was increased. Denatured collagen could increase autophagy and inhibit apoptosis of the fibroblasts to promote cell survival and influence wound healing.

Acrylamide impairs ovarian function by promoting apoptosis and affecting reproductive hormone release, steroidogenesis and autophagy-related genes: An in vivo study.

Acrylamide (ACR) toxicity is quite common due to its widespread use in industry and due to the Maillard browning reaction that occurs in foods containing high concentrations of hydrocarbons subjected to high temperatures. This study aimed to elucidate the female reproductive toxicity of ACR in vivo. Fifty-day-old Wistar-Albino female rats were treated with different dosages of ACR (2.5, 10, and 50mg/kg/day). After treatment, the animals were sacrificed, and serum and ovary samples were collected for histological examination, hormone analysis, TUNEL analysis, and RT-PCR studies. We found that ACR acts by significantly reducing ovarian weight and serum progesterone and estradiol concentrations. In addition, ACR treatment led to pyknotic, heterochromatic characteristics and nuclear fragmentation, as evidenced by hematoxylin staining. The TUNEL assay revealed that granulosa cells were affected after the oral administration of ACR, leading to the apoptosis of follicles at different stages of growth. Compared with the control condition, high doses of ACR (50mg/kg/day) significantly induced the overexpression of INSL3, CYP17a, IGF1, ESR1, ESR2, ATG5, ATG12 and LC3 in the ovary. Moreover, LC3 mRNA levels significantly increased with increasing doses of ACR (2.5, 10 and 50mg/kg/day), suggesting that ACR treatment induced autophagy. In conclusion, ACR induced ovarian dysfunction by affecting steroid hormone release, increasing apoptosis and mRNA levels of autophagy-related genes. The eventual correlation between apoptotic granulosa cell death and autophagy needs to be further explored.

Autophagy and Apoptosis of Porcine Ovarian Granulosa Cells During Follicular Development

Simple SummaryGranulosa cells (GCs) provide nutrients and information for oocytes in porcine follicles. Follicular atresia is closely related to both apoptosis and autophagy of granulosa cells in ovarian follicles; however, the follicular stages of granulosa cell apoptosis or autophagy during follicular development or atresia are poorly understood. We found that autophagy and apoptosis of GCs occurred in GCs from different size follicles during follicular development, and autophagy was mainly found in GCs of medium follicles, while apoptosis was mainly found in GCs of large follicles. These data provided some useful information to understand follicular atresia which is related to the fertility of sows.Follicular atresia is closely related to both apoptosis and autophagy of granulosa cells (GCs) in ovarian follicles. In the present study, GCs were isolated from pig ovaries in small, medium and large antral follicles, and the current results showed that the proliferation of GCs was higher in medium follicles, and lower in large follicles compared to small follicles. The Bax and Caspase 3 mRNA levels were significantly higher, but the ratio of Bcl-2/Bax was lower in GCs of large follicles. The marker genes of autophagy, Atg3, Atg7 and LC3 mRNA levels were higher in GCs from medium follicles. Apoptosis- and autophagy-related proteins had a similar expression pattern to the mRNA level. Our results showed that phosphorylated ERK (p-ERK) was activated in GCs of large follicles, while phosphorylated AKT (p-AKT) and phosphorylated mTOR (p-mTOR) were inhibited in GCs of medium follicles. Labeling of autophagic vesicles with 4’,6-diamidino-2-phenylindole (DAPI) and monodansylcadaverine (MDC) confirmed the results of gene transcription and protein expression in GCs of different size follicles. We conclude that autophagy and apoptosis of GCs occurred in different size follicles during follicular development, and autophagy was mainly found in GCs of medium follicles, while apoptosis was mainly found in GCs of large follicles.

Curcumin alleviates isoproterenol-induced cardiac hypertrophy and fibrosis through inhibition of autophagy and activation of mTOR.

Curcumin has been reported to possess cardioprotective effects. However, the potential molecular mechanism of curcumin is still not clear. The aim of the present study was to investigate the role of curcumin in regulating autophagy and mammalian target of rapamycin (mTOR) signaling in isoproterenol-induced cardiac hypertrophy and fibrosis in the rat. Rats model of cardiac hypertrophy and fibrosis was induced by isoprenaline (5 mg/kg/day, subcutaneous injection), which were treated with or without curcumin (200 mg/kg/day, intragastric administration). Masson's trichrome staining was performed to investigate the effect of curcumin on fibrosis of cardiac hypertrophy rat. The expression of hypertrophic and fibrosis markers was determined by RT-qPCR. The protein expression of autophagic markers, mTOR, and phosphorylated-mTOR (p-mTOR) was performed by Western blotting. Isoprenaline treatment significantly up-regulated the mRNA expression of hypertrophic (ANP and MYH7) and fibrotic (procollagen I and III) markers in the hearts from rats. All of these markers were reversed by curcumin treatment in isoproterenol-treated rats. Histological analysis showed that curcumin attenuated the interstitial fibrosis of heart triggered by isoproterenol. Moreover, isoproterenol significantly reduced the mRNA levels of mTOR and the protein expression of p-mTOR. However, isoprenaline caused a significant induction of the mRNA levels of LC3 and Beclin-1 and the protein expression of LC3-II and Beclin-1, as well as LC3-II/I ratio. Curcumin abolished these isoprenaline-mediated changes in mTOR/autophagy signaling pathway. Our data demonstrated that curcumin targeted mTOR/autophagy axis could attenuate cardiac hypertrophy and fibrosis in a rat model.

Alisol A 24-Acetate and Alisol B 23-Acetate Induced Autophagy Mediates Apoptosis and Nephrotoxicity in Human Renal Proximal Tubular Cells.

Two natural compounds alisol A 24-acetate (24A) and alisol B 23-acetate (23B) are abundant in Rhizoma alismatis. In the present study, we evaluated the induction of 24A and 23B on apoptosis and possible nephrotoxicity of human renal proximal tubular (HK-2) cells by activating autophagy and also explored its regulation on PI3K/Akt/mTOR signaling pathway. Presently, Clusterin, Kim-1, and TFF-3 were considered to be new bioindicators of nephrotoxicity. Interestingly, the protein expression and mRNA levels of Clusterin, Kim-1 and TFF-3 could be significantly increased by 23B and 24A in vivo and in vitro. Furthermore, cell apoptosis could be triggered by 23B and 24A via significantly decreasing the protein expression and mRNA levels of Bcl-2 and Bcl-xl. Autophagy of HK-2 cells could be induced by both 23B and 24A via significantly enhancing the ratio of LC3II/LC3I, the protein expression of Beclin-1 as well as the mRNA levels of LC3 and Beclin-1. Meanwhile, PI3K/Akt/mTOR signaling pathway could be inhibited by these two compounds. An autophagy inhibitor, 3-methyladenine, could partially reverse cell viability and conversely change the ratio of LC3II/LC3I and the protein expression of Bcl-2 and Kim-1. Thus this study helped to understand that 23B and 24A induced autophagy resulted in apoptosis and nephrotoxicity through inhibiting PI3K/Akt/mTOR signaling pathway, facilitating further studies for nephrotoxicity induced by these two compounds and could be beneficial for safe use of Rhizoma alismatis in clinic.

Hyperbaric Oxygenation Protects Against Ischemia-Reperfusion Injury in Transplanted Rat Kidneys by Triggering Autophagy and Inhibiting Inflammatory Response.

BACKGROUND Ischemia-reperfusion injury (IRI) is a clinically common pathologic process defined as the inability to improve neuronal function. This study aimed to investigate the pathological mechanism of IRI and to explore effects of hyperbaric oxygenation (HBO) on autophagy and inflammatory response in IRI. MATERIAL AND METHODS Ninety Sprague-Dawley (SD) rats were randomly divided into a Sham group, a kidney transplant group (Trans), and a kidney transplant plus HBO treatment group (Trans+HBO). The kidney was harvested from the donor and transplanted to recipient rats according to a previously reported study. Rats were anesthetized using pentobarbital-natrium, and the kidney was resected and fixed in 4% paraformaldehyde. Serum creatinine (Scr) was detected using an automatic biochemical analyzer. The interleukin-6 (IL-6) level was assessed using enzyme-linked immunosorbent assay (ELISA). LC-3 was examined using indirect immunofluorescence assay and immunochemistry assay. LC-3 mRNA levels were analyzed using real-time PCR (RT-PCR). RESULTS The kidney transplant IRI model was successfully established. Scr and IL-6 levels were significantly increased in the Trans group (P<0.05). HBO significantly enhanced Scr and IL-6 levels. Scr was positively correlated with IL-6 levels (r-0.607, P<0.05). HBO increased LC-3 protein and mRNA expression in kidney-transplanted rats compared to the Sham and Trans group (P<0.05). Moreover, immunofluorescence assay also showed that LC-3 protein mainly distributes along renal tubular epithelial cells in a linear manner. CONCLUSIONS Autophagy dysfunction and inflammatory response after renal transplantation play important roles in processes of IRI. HBO treatment protects against the renal injury of IRI in renal tissues at the early stage, which may be triggered by the IL-6 pathway.

The metastasis suppressor gene KISS-1 regulates osteosarcoma apoptosis and autophagy processes.

The expression of the metastasis suppressor gene KISS-1 in osteosarcoma cells during apoptosis and autophagy was evaluated. MG-63 osteosarcoma cells were transfected with either KISS-1 overexpression or KISS-1 knockdown expression vector in vitro, and compared with cell lines transfected with empty vector. After 12, 24, 48 and 72 h of cell culture, the cell proliferation was examined. The MTT method was used to detect apoptosis by flow cytometry, and the mRNA levels of apoptosis and autophagy markers caspase-3, Bcl-2, Bax, LC3 and Beclin1 were assessed by RT-PCR. Our results showed that cells in the control and low expression group kept proliferating during the cell culture period of 72 h, while the cells in the overexpression group progressively decreased in number. Also, the proliferation rate of the low expression group was significantly higher than that of the control group. The relative mRNA expression levels of caspase-3 and Bax mRNA in the control and low expression group showed no change (the expression was lowest in the low expression group). Moreover, the mRNA level of Bcl-2 increased in both cell groups. The mRNA expression levels of caspase-3 and Bax in the overexpression group were increased, and the level of Bcl-2 was reduced significantly. At the same time, the relative expression level of LC3 and Beclin1 mRNA in the control and low expression groups remained the same, and that of the overexpression group increased. The mRNA levels of LC3 and Beclin1 in the overexpression group were the highest, and that of the low expression group the lowest. The differences were statistically significant (P<0.05). Based on these results, we showed that KISS-1 inhibited the proliferation of osteosarcoma in vitro, probably by accelerating the processes of apoptosis and autophagy in the cells.

Autophagy regulation in human skeletal muscle during exercise.

Autophagy regulation in human skeletal muscle during exercise.

Detection of autophagy in Hirschsprung's disease: implication for its role in aganglionosis.

Hirschsprung's disease (HD) is a common congenital gastrointestinal malformation, characterized by the lack of ganglion cells from the distal rectum to the proximal bowel, but the pathogenesis is not well understood. This paper evaluates the effects of autophagy in HD. Using electron microscopy, the autophagosomes were detected in three segments: narrow segment (NS), transitional segment (TS), and dilated segment (DS). Typical autophagosome structures are found in the Auerbach plexus of both NS and TS. Real-time PCR results showed that Beclin1 (NS vs. TS, P<0.01) and LC3 (NS vs. TS, P<0.05) mRNA were the highest in the NS, but p75 (NS vs. TS, P<0.01) was the highest in the DS. Correlation analysis results showed a positive correlation between Beclin1 and LC3 mRNA levels (R=0.736, P=0.000), whereas inverse correlations were found between p75 and Beclin1/LC3 mRNA levels (p75 vs. Beclin1: R=-0.714, P=0.000; p75 vs. LC3: R=-0.619, P=0.000). Immunohistochemistry analyses indicated a consistent result with mRNA levels, by increased Beclin1-positive and LC3-positive neurons, but reduced p75-positive neurons in the Auerbach plexus of TS compared with DS. These findings indicated that autophagy exists in the bowel of patients with HD. On the basis of the detection of the highest expression of the autophagy genes in NS, autophagy may additionally cause the lack of neurons.

Melamine negatively affects oocyte architecture, oocyte development and fertility in mice

Does melamine have a toxic effect on oocyte development and fertility in vivo? Melamine had toxic effects on oocyte quality and fertility due to its effects on the oocyte cytoskeleton, apoptosis and autophagy induction, and epigenetic modifications in an in vivo mouse model. Melamine is a chemical compound that is widely used during the manufacture of amino resins and plastics. In 2008, melamine was reported to adulterate milk and infant formulas in China, which sparked food safety concerns worldwide. Ingesting melamine may result in reproductive damage, and bladder or kidney stones, which can lead to bladder cancer. Mice were randomly assigned to three groups and fed a diet that included melamine (0, 10 and 50 mg/kg/day) for 8 weeks. The in vivo effect of melamine on female reproduction was examined. We used immunofluorescent staining, western blotting and qRT-PCR to examine the effect of melamine on oocyte quality. Our results showed the following effects of this melamine-containing diet. (i) Ovary weights were reduced in melamine fed mice. Oocyte developmental competence was also reduced, as shown by reduced polar body extrusion rates. (ii) Melamine feeding resulted in abnormal oocyte cytoskeletons, as shown by increased rates of aberrant spindles and reduced actin microfilament expression. (iii) Melamine exposed oocytes had higher rates of abnormal mitochondrial distributions and early stage apoptosis/autophagy, which were shown by increased microtubule-associated protein 1 light chain 3 (LC3) protein expression level and caspase 9, autophagy-related protein 14 (atg14), and lc3 mRNA levels. (iv) Fluorescence intensity analysis showed that DNA methylation levels were reduced in the oocytes of melamine fed mice. Histone methylation levels were also altered, as Di-methyl-Histone H3 (Lys4) (H3K4me2) level was increased and Tri-methyl-Histone H3 (Lys9) (H3K9me3), Di-methyl-Histone H3 (Lys9) (H3K9me2), and Tri-methyl-Histone H3 (Lys27) (H3K27me3) levels were reduced in oocytes from melamine fed mice. (v) The litter sizes of melamine fed mice were significantly reduced when compared with those of controls. Although we examined the possible effects of melamine on oocyte quality and fertility, we did not determine the effect of melamine on offspring development. Our findings indicate that melamine plays a major role in oocyte quality and fertility. This information could contribute to a better understanding of melamine toxicity in female reproduction. This study was supported by the National Basic Research Program of China (2014CB138503) and the Natural Science Foundation of Jiangsu Province (BK20140030). The authors have no conflict of interest to disclose.

Time Course of Mitophagy‐Related Signals in Skeletal Muscle Following a Single Bout of Aerobic Exercise in Mice

This study aimed to investigate mitophagy‐related signals in skeletal muscle following a single bout of aerobic exercise in mice. C57BL6 mice were submitted to a single bout of treadmill aerobic exercise for 80 minutes at 60% of maximal speed achieved in a graded treadmill test. Skeletal muscles were harvested at rest, immediately after the bout of aerobic exercise and after 3, 6, 12 and 24h of recovery. One hindlimb was used for fresh isolation of skeletal muscle mitochondrial‐enriched fraction (MF), and contralateral muscles were immediately frozen. Immunoblotting for LC3, p62 and Bnip3 were performed either in quadriceps muscles or MF. qPCR were performed to evaluate mRNA levels of LC3, p62 and Bnip3 in quadriceps muscles. No protein was differentially expressed in total lysate of quadriceps muscle. However, LC3‐II levels were significantly increased in MF immediately after the bout of aerobic exercise (72.5%) and after 12h of recovery (63.3%), whereas p62 protein levels were increased at 6 (30%) and 12h (62.5%) of recovery. Bnip3 protein levels displayed a trend toward to increase (p=0.06) after 3h of recovery (36.4%). No difference was found in mRNA levels of LC3, p62 and Bnip3 at any time point. Collectively, these results suggest that mitophagy signaling is increased during aerobic exercise and decreases after its cessation. Interestingly, the retake of mitophagy signaling after 12h of recovery suggests that mitophagy plays an important role in skeletal muscle mitochondrial remodeling to acute aerobic exercise stimulus, independently of transcriptional regulation. Thus, these results provide new insights into aerobic exercise‐induced mitophagy and mitochondrial remodeling.Financial support: FAPESP 2013/21065‐3

The expression and significance of microtubule-associated protein 1 light chain 3-related protein-1 (LC3) mRNA in peripheral blood mononuclear cells in patients with systemic lupus erythematosus

Increasing evidence supports the involvement of autophagy in the etiopathology of autoimmune diseases. Systemic lupus erythematosus (SLE) is a potentially fatal autoimmune disease characterized by production of multiple autoantibodies through poorly understood mechanism. In order to explore the role of autophagy in the development of SLE, the expression of autophagy related gene microtubule-associated protein 1 light chain 3 (MAPLC3) in peripheral blood mononuclear cells (PBMCs) was measured in patients with SLE. The mRNA levels of LC3 in PBMCs from 56 SLE patients and 45 healthy individuals were detected by real-time quantitative polymerase chain reaction (qPCR) technique. Autophagy in PBMCs was also determined by flow cytometry (FACs) in 20 SLE patients and 15 healthy controls. The correlation between LC3 mRNA expression and disease activity of SLE (SLEDAI) was then analyzed. The mRNA level of LC3 (RQ) in SLE patients was obviously downregulated compared with that in healthy population (1.30 ± 0.10 vs 1.35 ± 0.09; P = 0.029), paralleled with the decreased autophagy rate detected by flow cytometry in PBMCs of SLE patients [(2.21 ± 1.07) % vs (9.91 ± 4.01) %;P = 0.047]. Moreover, LC3 mRNA expression level was negatively correlated with SLEDAI (r = -0.337, P = 0.023). However, when the clinical features of 27 SLE patients with decreased LC3 mRNA expression (RQ<1.351) were compared with those of other 29 SLE patients with normal or high LC3 mRNA expression (RQ>1.351), increasing rates of arthritis, serositis, hematological abnormalities were noted in patients with decreased LC3 mRNA expression yet without statistically significance. However, there was a significant difference between two groups in the incidence of renal involvement (P = 0.028). The impaired autophagy due to downregulated LC3 mRNA level in SLE patients indicates that autophagy plays a role in mediating the occurrence and development of SLE.