• <tr id="yyy80"></tr>
  • <sup id="yyy80"></sup>
  • <tfoot id="yyy80"><noscript id="yyy80"></noscript></tfoot>
  • 99热精品在线国产_美女午夜性视频免费_国产精品国产高清国产av_av欧美777_自拍偷自拍亚洲精品老妇_亚洲熟女精品中文字幕_www日本黄色视频网_国产精品野战在线观看 ?

    Family with sequence similarity 134 member B-mediated reticulophagy ameliorates hepatocyte apoptosis induced by dithiothreitol

    2022-06-29 01:46:56YiXinGuoBingHanTingYangYuSiChenYiYangJiaYaoLiQinYangRuJiaXie
    World Journal of Gastroenterology 2022年23期

    Yi-Xin Guo, Bing Han,Ting Yang, Yu-Si Chen, Yi Yang, Jia-Yao Li, Qin Yang,Ru-Jia Xie

    Abstract

    Key Words: Hepatocytes; Reticulophagy; Family with sequence similarity 134 member B; Apoptosis;Endoplasmic reticulum stress; Endoplasmic reticulum homeostasis

    INTRODUCTION

    Endoplasmic reticulum (ER) stress-related hepatocyte apoptosis participates in multiple hepatic diseases, including viral hepatitis[1], hepatic fibrosis[2], fatty liver[3,4] and cirrhosis[5]. Therefore, the alleviation of ER stress-mediated hepatocyte apoptosis is crucial in the treatment of hepatic diseases.Recent findings have indicated that endoplasmic reticulophagy (ER-phagy) promotes degradation of damaged ER fragments during ER stress. Although ER-phagy has a vital role in maintaining ER homeostasis and inhibiting cell apoptosis[6-8], the exact regulatory mechanisms behind this are largely unknown.

    Glucose-regulated protein 78 (GRP78) is a prominent ER molecular chaperone, while calnexin (CNX)is a membrane-bound lectin protein in the ER that can increase the protein folding capacity[9,10]. Even though the excessive build-up of misfolded or unfolded proteins can be alleviatedviaER stress,previous studies reported that a selective autophagic mechanism, defined as ER-phagy, can also be activated by ER stress to restore ER homeostasis[11,12]. Family with sequence similarity 134 member B(FAM134B), an ER-resident protein, may interact with CNX in the cytosol or the ER membrane[13].Since FAM134B is not predicted to have an ER lumenal domain, there is an indirect interaction between FAM134B and lumenal proteins through the lumen-resident segment, which has a chaperone activity attributed to CNX. CNX forms transient but relatively stable complexes with unfolded ER proteins until they either become folded or are degraded. Moreover, it has been reported that as with other cargo receptor molecules, FAM134B can interact directly with microtubule-associated protein 1 light chain 3(LC3) when its LIR motif is exposed. The CNX-FAM134B-LC3 complex can mediate the selective isolation of ER fragments containing misfolded proteins, which are subsequently transported to lysosomes for degradation[14-16]. Thus, FAM134B-mediated ER-phagy may play an essential role in maintaining ER homeostasis and promoting cell survival. However, it is unclear whether FAM134Bmediated ER-phagy is involved in the regulation of hepatocyte apoptosis induced by ER stress. In this study, dithiothreitol (DTT) was used to induce ER stress in buffalo rat liver 3A (BRL-3A) hepatocytes,and the expression of ER stress-related and autophagy-related proteins was assessed. In addition, small interfering RNA (siRNA) was used to knockdown the expression ofFAM134Bin hepatocytes and an apoptosis analysis followed. Our study reveals an emerging role of FAM134B-mediated ER-phagy in ER stress-mediated hepatocyte apoptosis, which may provide a novel target for the treatment of hepatic diseases.

    MATERIALS AND METHODS

    Antibodies and reagents

    Dulbecco's modified Eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco(Grand Island, NY, United States). Trypsin-EDTA solution, trypsin solution without EDTA, and penicillin-streptomycin were purchased from Biological Industries (BioInd, Israel). Bicinchoninic acid(BCA) protein assay kit, DTT, RIPA lysis buffer, and protease inhibitor were obtained from Solarbio(Beijing, China). Annexin V-FITC/PI Apoptosis Detection Kit and Cell Cycle Detection Kit were purchased from KeyGEN BioTECH (Nanjing, China). PVDF membranes were obtained from Merck Millipore. Rabbit polyclonal antibody against FAM134B was purchased from Proteintech (Wuhan,China). Rabbit polyclonal antibodies against ATG12, cytochrome c (cyt c), and cleaved caspase-3 were obtained from Cell Signaling Technology (Danvers, MA, United States). Rabbit polyclonal antibodies against β-actin, LC3, CNX, CHOP and GRP78, and the Ca2+indicator (Rhod-2 AM) were purchased from Abcam (Cambridge, United Kingdom). Dynabeads protein G immunoprecipitation kit and lipofectamine 3000 reagent were purchased from Thermo Fisher Scientific, Inc. HRP-labeled Goat Anti-Rabbit IgG (H + L), Mito-Tracker Green, Lyso-Tracker Green, ER-Tracker Red, and immunofluorescencerelated reagents were purchased from Beyotime Institute of Biotechnology (Nanjing, China).

    Cell culture and experiment protocol

    BRL-3A cells, bought from Cell Bank of the Chinese Academy of Sciences (Shanghai, China), were cultivated and maintained in DMEM culture media supplemented with 1% penicillin-streptomycin and 10% FBS. BRL-3A cells were seeded at 37 °C and 5% CO2in a constant temperature and humid atmosphere, pre-cultured every 3 d, and further passaged until the density reached approximately 80%.To induce the ER stress, BRL-3A cells were treated with DTT (2.0 mmol/L based on previous studies[17]) for 0, 3, 6, 12, 24, or 48 h.

    Apoptosis assessment

    Cells were cultured to 80% confluency and treated with 2.0 mmol/L DTT for the specified point-in-time intervals. To determine the efficacy of the different DTT treatments, a cell apoptosis analysis was evaluated with flow cytometry. Each group of cells was trypsinized without EDTA and rinsed thrice with PBS. After centrifugation at 2000 rpm for 5 min, cells were loaded with 500 μL binding buffer and labeled with 5 μL of Annexin V-FITC/PI, according to the manufacturer’s instructions. Labeled cells were detected and analyzed with flow cytometry and NovoExpress?software 1.4.1. The experiments were performed in triplicate.

    Cell cycle analysis

    To determine the effect of DTT’s 0, 3, 6, 12, 24, and 48 h incubation on the cell cycle progression of BRL-3A, the harvested cells were trypsinized without EDTA and rinsed three times with cold PBS, followed by fixation with 70% ethanol in cold storage. After 24 h incubation at 4 °C, 500 μL PI/RNase was added to each group and maintained at 37 °C for 60 min in a dark place. Stained cells were processed using flow cytometry and further measuredviathe NovoExpress?software 1.4.1. The experiments were performed in triplicate.

    Western blot analysis

    BRL-3A cells were grown on 10 cm diameter dishes and treated with 2.0 mmol/L DTT for different times. Cells were rinsed three times with pre-cooled PBS after experimentation and collected with cell scrapers in 100 μL RIPA buffer containing 1 mmol/L PMSF. After centrifugation at 12000 rpm for 25 min at 4 °C, the concentrations of total cellular protein extracts were determined using the BCA kit(Solarbio Science, Beijing, China), and known concentrations of BSA were used as standard. The total cellular protein extracts were denatured by boiling at 100 °C using dry bath incubator (Hangzhou Miu Instruments Co., Ltd, Zhejiang, China). Protein samples (30-40 mg) were loaded onto SDS-PAGE and transferred onto PVDF membranes for immunostaining. After blocking with 5% defatted milk for 90 min, membranes were stained overnight with primary antibodies, including β-actin (1:1000), GRP78(1:1000), CNX (1:3000), ATG12 (1:1000), LC3 (1:1000), FAM134B (1:1000), CHOP (1:1000), cleaved caspase-3 (1:1000), cyt c (1:1000) in cold storage, followed by incubation with secondary antibodies(1:4000). The density of protein bands on membranes was exposed and quantifiedviafluorography using Image J software. The images shown are representative of experiments carried out at least three times.

    Co-immunoprecipitation analysis

    BRL-3A cells, treated with DTT (2.0 mmol/L for 0 h and 24 h), were lysed in RIPA lysis buffer and the lysates were centrifuged at 12000 rpm for 15 min at 4 °C. The supernatant was resuspended in ice-cold PBS to a total volume of 500 μL, and 5 μL of the designated antibody was added overnight at 4 °C. The next day, the Ab-Ag complexes were bound to Dynabeads magnetic beads on a rotary shaker for 10 min. The magnetic bead-Ab-Ag complex was washed and eluted by adding a washing buffer and elution buffer, respectively, according to the manufacturer's protocol. Immunocomplexes were heated for 5 min at 100 °C and prepared for analysis by western blot. The images shown are representative of experiments carried out at least three times.

    Calcium imaging and mitochondrial labeling

    To observe the effects of DTT treatment at 2.0 mmol/L for specified time points, mitochondrial Ca2+levels were determined using Rhod-2 AM, a specific detection dye for calcium. The treated cells were rinsed with HBSS three times and stained with a mixture of 5 μM Rhod-2 AM and 20 nM Mito-Tracker Green at 37 °C for 30 min in the dark. Finally, live cells were extensively rinsed thrice by adding HBSS without calcium, and images were visualized with Zeiss LSM Image Browser using a Zeiss LSM 900 confocal microscope. The images shown are representative of experiments carried out at least three times.

    Live imaging of ER and lysosome

    To observe the intracellular localization of the ER and lysosomes, after treatment with 2.0 mmol/L DTT for 0, 3, 6, 12, 24, and 48 h, ER and lysosomes were stained with ER-tracker and Lyso-tracker. Prior to staining, trackers were diluted appropriately in DMEM, on the basis of the manufacturer's instructions.Following dilution, cells were simultaneously incubated with the two trackers listed above, maintained for 30 min at 37 °C, and finally rinsed thrice with HBSS. Stained cells were visualized under the Zeiss LSM 900 confocal microscope. Images shown are representative of experiments carried out at least three times.

    SiRNA transfections

    Specific siRNA against buffalo ratFAM134Bwas designed and synthesized by OriGene. Product number and targeting sequence: SR510501A-rGrGrArArGrUrGrGrUrUrUrArUrCrArArArUr-UrCrUrGrATA; SR510501B-rArArArUrUrUrGrArCrUrUrArCrArGrUrGrGrArArArCrCAA;SR510501C-rArArGrUrGrGrUrUrUrArUrCrArArArUrUrCrUrGrArUrAGA. Cells were cultured in sixwell dishes until the density of cell fusion reached 60%. Briefly, 75 pmol ofFAM134BsiRNA were added to Lipofectamine 3000 Transfection Reagent and gently mixed for 15 min, then administered to BRL-3A cells, which were resuspended in DMEM. After transfection for 6 h, cells were washed, and then supplemented with fresh medium. Finally, cells were treated with DTT (2.0 mmol/L) for a further 24 h and subjected to western blot assay and apoptosis assessment.

    Statistical analysis

    GraphPad Prism 7 software was used to perform all the statistical analyses and prepare experimental graphs. Data are expressed as the mean ± SD. Shapiro-Wilk normality test was used to test the normal distribution of the data and all the data were fit to a normal followed by Tukey's post hoc test was performed, and a significant difference was considered asP< 0.05.

    RESULTS

    DTT-mediated ER stress upregulates ER-phagy-related FAM134B in BRL-3A cells

    To assess whether the drug treatments could alter the protein expression of CNX and GRP78, BRL-3A cells were subjected to short-term (3, 6, 12, 24 h) or long-term (48 h) treatment with DTT, and the protein extracts from BRL-3A cells were analyzed by western blot. We found that treatment of BRL-3A cells with 2.0 mmol/L DTT resulted in a prominent increase in CNX and GRP78 levels, both in a timedependent manner (Figure 1A and B). Moreover, CHOP is a specific and stress-responsive transcription factor during ER stress and its protein expression was significantly increased in the 12, 24, and 48 h groups (Figure 1A and B). However, the expression of CHOP in BRL-3A cells treated with DTT for 48 h was lower than that after DTT treatment for 24 h. These alterations in CNX, GRP78, and CHOP confirm that ER stress in BRL-3A was activated.

    Figure 1 Impact of the endoplasmic reticulum stressor, dithiothreitol, on endoplasmic reticulophagy mediated by family with sequence similarity 134 member B in buffalo rat liver 3A cells. A and B: Buffalo rat liver 3A (BRL-3A) cells were treated with 2.0 mmol/L dithiothreitol (DTT) for the time intervals (0, 3, 6, 12, 24, 48 h); Western blot showed the effect of endoplasmic reticulum (ER) stressor, DTT, on expression of the ER stress-related proteins glucose-regulated protein 78 (GRP78), calnexin (CNX), and C/EBP homologous protein (CHOP); β-actin was used as a control for normalization; C and D: Analysis of autophagy related gene 12 (ATG12), family with sequence similarity 134 member B (FAM134B), and microtubule-associated protein 1 light chain 3 (LC3) protein expression by western blot. Protein levels were normalized to β-actin; E: BRL-3A cells were treated with 2.0 mmol/L DTT for 0 and 24 h; co-immunoprecipitation analysis detected the presence of CNX-FAM134B-LC3 complex in BRL-3A cells. Values are represented as mean ± SD (n = 3), aP < 0.05 vs 0 h group; bP < 0.05 vs 48 h group.

    To determine the effects of ER stress on FAM134B-mediated ER-phagy, alterations in FAM134B,ATG12, and LC3 expression were detected by western blot. As expected, DTT treatment for 3, 6, 12, 24,and 48 h increased the conversion ratio of LC3-I to LC3-II and the FAM134B and ATG12 expression levels compared to those in the 0 h group (Figure 1C and D). Thus, our results revealed that the expression of FAM134B is induced in response to ER stress.

    Furthermore, we used an anti-CNX antibody to immunoprecipitate the CNX-FAM134B-LC3 complex,confirming the hypothesis that FAM134B forms a complex with CNX and LC3, exerting a positive influence on ER-phagy (Figure 1E).

    Long-term DTT treatment relieved the gradually blocked ER autolysosome delivery in BRL-3A cells

    Typically, ER is delivered to lysosomes and finally degraded. To analyze whether ER autolysosomes are formed, we examined the subcellular location of the ER and lysosomes using cell organelle markers. As shown in Figure 2, the treatment groups of 3, 6, 12, 24, and 48 h DTT incubation significantly alleviated the co-localization of the ER with lysosomes, compared to that in the 0 h group. Notably, the colocalization of ER and lysosomes in BRL-3A cells treated with DTT for 48 h was increased compared to those treated for 24 h (Figure 2).

    Short-term DTT treatment induces mitochondrial calcium uptake while prolonged DTT treatment reduces it

    Calcium in the ER can be released and transferred to the mitochondria owing to an imbalance of ER homeostasis. To explore the altered localization of calcium, collected cells were co-loaded with Rhod-2 AM and Mito-Tracker Green. In response to DTT treatment for 3, 6, 12, 24, and 48 h, the co-localized fluorescence increased considerably (Figure 3). However, the distribution of the co-localized signal was weaker in the 48 h group, compared to that in the 24 h group (Figure 3). These results strongly suggest that mitochondrial calcium accumulation is related to DTT treatment.

    Figure 2 Impact of dithiothreitol treatment on the formation of autolysosomes in buffalo rat liver 3A cells. After dithiothreitol treatment for 0, 3, 6,12, 24, and 48 h, the buffalo rat liver 3A cells labeled with endoplasmic reticulum (ER)-Tracker Red and Lyso-Tracker Green were observed and captured under confocal fluorescence microscopy (200 ×) in a live cell imaging experiment. Insets show the magnification of the pictures. Scale bars indicate 100 μm. Arrows head to indicate ER-localized lysosomes. Values are represented as mean ± SD (n = 3), aP < 0.05 vs 0 h group; bP < 0.05 vs 48 h group.

    Figure 3 Impact of dithiothreitol treatment on mitochondrial calcium uptake in buffalo rat liver 3A cells. Buffalo rat liver 3A cells were treated for 0, 3, 6, 24, and 48 h with 2.0 mM dithiothreitol, followed by co-incubating with Mitochondria-Tracker Green and Rhod-2 AM, and visualized by confocal microscopy(400 ×). Scale bars indicate 100 μm. Values are represented as mean ± SD (n = 3), aP < 0.05 vs 0 h group; bP < 0.05 vs 48 h group.

    DTT treatment induces cell cycle arrest and apoptosis in BRL-3A cells, which is relieved at 48 h

    To further validate that DTT treatment leads to apoptosis in BRL-3A cells, we quantitatively measured the number of apoptotic cells using the Annexin V-FITC/PI double staining assay. As shown in Figure 4A and B, the ratio of apoptotic cells treated with DTT for 0, 3, 6, 12, and 24 h exhibited a timedependent increase. Interestingly, the apoptotic percentage in the 48 h group was significantly lower than that in the 24 h group (Figure 4A and B). Subsequently, we sought to use flow cytometry to determine the impact of DTT treatment on the cell cycle progression, and the data suggests that the proportion of BRL-3A cells in G1 phase after DTT treatment was noticeably higher than that of the 0 h group (Figure 4C and D and Table 1). Moreover, the number of cells in G1 phase in the 48 h group was smaller than that of the 24 h group.

    BRL-3A cells undergo apoptosis upon FAM134B knockdown

    We further verified whetherFAM134Bknockdown could alter DTT-induced apoptosis. We first investigated the transfection efficiency of siRNA with three different siRNAs targetingFAM134B(siRNA 1, 2,and 3) and found that theFAM134BsiRNA2 was the most effective (Figure 5A and B). Next, we investigated FAM134B protein levels by performing a western blot on already transfected samples, which were treated with DTT for 24 h. As shown in Figure 5C and D, FAM134B and β-actin expression levels were determined, and it was found that FAM134B protein levels were down-regulated compared with the control and control siRNA groups.

    It has been reported that cyt c and cleaved caspase-3 are apoptosis-related proteins and important hallmarks of apoptosis activation involved in mitochondrial dysfunction. Consequently, siRNAmediated silencing ofFAM134Bcaused a high level of cleaved caspase-3 and cyt c in BRL-3A cells treated with DTT for 24 h (Figure 5E and F). We examined the rates of apoptotic cells using Annexin-VFITC/PI staining assays, which revealed that the apoptotic rates also increased in theFAM134BsiRNAgroup, compared with those in the control and control siRNA groups (Figure 5G and H). These results suggest that ER-phagy mediated by FAM134B is likely to serve a cytoprotective function in response to DTT treatment in BRL-3A cells.

    Table 1 The cell cycle distribution of buffalo rat liver 3A cells treated with dithiothreitol for different times was detected by flow cytometry

    DISCUSSION

    Hepatic injury caused by multiple harmful factors is closely associated with ER stress-induced hepatocyte apoptosis[18-20]. The ER is responsible for proper protein folding, intracellular calcium storage, and lipid biosynthesis[21,22]. Various stressors, including unfolded protein aggregation in the ER, intracellular Ca2+disturbance, and pharmacological inducers, such as DTT, can disrupt ER homeostasis and lead to ER stress in hepatocytes. If the ER stress cannot be alleviated, aberrant ER stress can trigger cell apoptosis[23]. In the present study, we found that the protein levels of GRP78 and CNX,which are ER stress biomarkers, were upregulated in BRL-3A cells during ER stress. GRP78 and CNX are ER chaperone proteins and accelerate the proper folding of the accumulated unfolded proteins in the ER, which engages effector mechanisms to rebalance ER homeostasis[24,25]. A series of studies have revealed that ER-phagy is an ER selective autophagy mechanism that can promote the clearance of damaged ER lumens containing the unfolded proteins, and helps restore ER homeostasis[26-28]. ERphagy is a critical quality control mechanism for the ER in multiple cell types. Defects in ER-phagy pathways are associated with multiple human pathologies, including infectious and neurodegenerative diseases, aging and cancer. However, whether ER-phagy is involved in the regulation of ER homeostasis in hepatocytes under ER stress remains elusive. In this study, we assessed the levels of reticulophagyrelated proteins in BRL-3A cells treated with DTT. We found that the levels of FAM134B and ATG12 were markedly elevated, and the ratio of LC3II/LC3I also increased. These data indicate that DTTinduced ER stress increases the level of reticulophagy-associated proteins.

    Recent findings have indicated that receptor proteins of ER-phagy play crucial roles in driving the sequestration of isolated ER fragments into autophagosomes[29]. FAM134B, an ER-anchored protein,was recently proposed as a major mammalian receptor for reticulophagy[30,31]. FAM134B contains an LC3-interacting region that can interact with LC3 protein to form autophagosomal membranes, leading to efficient ER sequestration into an autophagosomal lumen[32-34]. In a previous report, the authors found that CNX serves as a co-receptor that recognizes misfolded proteins within the ER lumen and interacts with FAM134B[35,36]. In turn, the CNX-FAM134B complex binds with LC3, the autophagosome membrane-related protein, which delivers ER lumens containing misfolded proteins to the lysosome for degradation. To investigate how FAM134B modulates ER-phagy in BRL-3A cells, immunoprecipitation was performed to detect the interaction between CNX, FAM134B, and LC3. The results confirmed that CNX interacted with FAM134B, and FAM134B interacted with LC3 after DTT treatment.Thus, the formation of the CNX-FAM134B-LC3 complex allows for the selective delivery of ER lumens containing misfolded proteins to the lysosome for eventual degradation. Complete ER-phagy indicates that autophagosomes fuse to form autolysosomes[37,38], hence, we detected the number of autolysosomes in BRL-3A cells treated with DTT. We found that the formation of autolysosomes decreased in the early stages of ER stress, whereas autolysosomes were elevated in later stages. As it has been reported that CHOP can suppress autolysosome formation[39], we speculated that decreased autolysosomes in the early stages of ER stress were associated with increased CHOP expression.

    Figure 4 Impact of dithiothreitol treatment on cell cycle and apoptosis of buffalo rat liver 3A cells. A and B: Buffalo rat liver 3A (BRL-3A) cells were treated with 2.0 mmol/L dithiothreitol (DTT) for 0, 3, 6, 12, 24 and 48 h. The population of apoptotic cells was detected by flow cytometry. The lower right quadrant represents the early apoptotic cells, and the upper right quadrant represents the late apoptotic cells; C and D: BRL-3A cells were treated with 2.0 mmol/L DTT for 0, 3, 6, 12, 24 and 48 h. The analysis of the cell cycle was assessed by flow cytometry. aP < 0.05 vs 0 h group; bP < 0.05 vs 48 h group.

    The ER is the main pool for Ca2+storage, and ER dysfunction leads to Ca2+efflux from the ER[40,41].In the early stages of ER stress, the suppression of the autophagosomes’ fusion with lysosomes may lead to calcium release and subsequent Ca2+overload in mitochondria[42-44]. As expected, we found that DTT treatment dramatically elevated the levels of mitochondrial Ca2+, the apoptotic rate, and G1 arrest in BRL-3A cells. Nevertheless, these trends were relieved after treatment with DTT for 48 h. Our results reveal that hepatocytes initiate adaptive mechanisms in response to DTT-induced ER stress;consequently, apoptosis in BRL-3A cells treated with DTT for 48 h was lower than that in cells treated with DTT for 24 h.

    To clarify whether FAM134B is involved in the regulation of cellular homeostasis during ER stress,we used a small interference RNA technique to knockdownFAM134Bexpression in hepatocytes. We found thatFAM134Bsilencing not only significantly attenuated the DTT-upregulated FAM134B expression, but also accelerated the activation of the mitochondrial apoptotic pathway and aggravated DTT-triggered hepatocyte apoptosis.

    CONCLUSION

    In conclusion, DTT treatment significantly upregulated the protein levels of GRP78, CNX, FAM134B,and ATG12, and also increased the ratio of LC3II/LC3I in BRL-3A cells. Moreover, FAM134B-mediated reticulophagy ameliorates DTT-induced hepatocyte apoptosisviaselective clearance of damaged ER lumens. Accordingly, knockdown of FAM134B enhanced ER stress-mediated apoptosis in BRL-3A cells.Our data show that FAM134B-mediated reticulophagy plays a key role in rebalancing ER homeostasis in hepatocytes undergoing ER stress. Therefore, FAM134B-mediated reticulophagy may be a novel therapeutic target, and our findings may provide emerging evidence to demonstrate the prominence of ER-phagy in ER stress-related hepatocyte apoptosis. Alleviation of ER stress-mediated hepatocyte apoptosisviarestoring ER homeostasis is critical in the treatment of liver diseases.

    ARTICLE HIGHLIGHTS

    Research results

    DTT treatment upregulated glucose-regulated protein 78 (GRP78), CNX, FAM134B, and autophagy related gene 12 (ATG12) protein levels and increased the ratio of LC3II/LC3I in BRL-3A cells.FAM134B-mediated reticulophagy maintains ER homeostasis in ER-stressed hepatocytesviathe clearance of damaged ER fragments. FAM134B-mediated reticulophagy ameliorates DTT-induced hepatocyte apoptosis. Knockdown ofFAM134Benhanced ER stress-mediated apoptosis in BRL-3A cells.

    Research conclusions

    FAM134B-mediated ER-phagy attenuates hepatocyte apoptosis by suppressing the mitochondrial apoptotic pathway.

    Research perspectives

    FAM134B-mediated reticulophagy may be a novel therapeutic target, and our findings provide emerging evidence demonstrating the prominence of ER-phagy in ER stress-related hepatocyte apoptosis. Alleviation of the ER stress-mediated hepatocyte apoptosisviarestoring ER homeostasis is critical in the treatment of liver diseases.

    ACKNOWLEDGEMENTS

    We thank the Basic Medical Science Research Center of Guizhou Medical University for their technical advice in using the microscope.

    FOOTNOTES

    Author contributions:Yang Q and Xie RJ designed and coordinated the study; Guo YX, Han B and Yang T performed the experiments and acquired data; Chen YS, Yang Y and Li JY analyzed and interpreted data; Guo YX and Xie RJ drafted the manuscript; all authors approved the final version of the article.

    Supported byNational Natural Science Foundation of China, No. 81560105; Science and Technology Foundation of Guizhou Province, No. Qiankehe Jichu-ZK[2021]365, and No. Qiankehe Pingtai Rencai[2019]5801; and National Natural Science Foundation Cultivation Project of Guizhou Medical University, No. 20NSP016.

    Institutional review board statement:This study did not involve human subjects or living animals.

    Institutional animal care and use committee statement:This study did not involve human subjects or living animals.

    Conflict-of-interest statement:The authors declare no conflicts of interest.

    Data sharing statement:The data used to support the findings of this study are available from the corresponding author at 592153968@qq.com upon request.

    Open-Access:This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BYNC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is noncommercial. See: https://creativecommons.org/Licenses/by-nc/4.0/

    Country/Territory of origin:China

    ORCID number:Yi-Xin Guo 0000-0001-6789-8669; Bing Han 0000-0002-9577-293X; Ting Yang 0000-0001-5174-7575; Yu-Si Chen 0000-0003-2566-8878; Yi Yang 0000-0003-2756-6955; Jia-Yao Li 0000-0003-2880-4978; Qin Yang 0000-0003-1479-6700; Ru-Jia Xie 0000-0001-5991-2678.

    S-Editor:Fan JR

    L-Editor:A

    P-Editor:Qi WW

    欧美日韩亚洲高清精品| a级毛片免费高清观看在线播放| 国产探花极品一区二区| 久久久久精品性色| 久久久精品94久久精品| 一级毛片黄色毛片免费观看视频| 老女人水多毛片| 亚洲精品日本国产第一区| 国产极品粉嫩免费观看在线 | 免费看日本二区| 欧美性感艳星| 亚洲天堂av无毛| 精品国产一区二区三区久久久樱花| av在线app专区| av卡一久久| 亚洲欧美精品自产自拍| 午夜影院在线不卡| 熟女人妻精品中文字幕| 青春草亚洲视频在线观看| 日韩精品有码人妻一区| 久久人妻熟女aⅴ| 成人午夜精彩视频在线观看| a级毛色黄片| av卡一久久| 久久人人爽人人爽人人片va| 国产一区二区三区av在线| 内射极品少妇av片p| 自线自在国产av| 日韩欧美精品免费久久| 日本色播在线视频| 制服丝袜香蕉在线| av天堂中文字幕网| 一级毛片aaaaaa免费看小| 久久久久国产精品人妻一区二区| 亚洲av电影在线观看一区二区三区| 日本wwww免费看| 亚洲激情五月婷婷啪啪| 女人久久www免费人成看片| 久久久a久久爽久久v久久| 久久精品国产亚洲网站| 高清不卡的av网站| 高清欧美精品videossex| 熟女av电影| 纯流量卡能插随身wifi吗| 一区二区三区乱码不卡18| 免费不卡的大黄色大毛片视频在线观看| 成人毛片60女人毛片免费| 欧美激情国产日韩精品一区| 亚洲精品456在线播放app| 日韩免费高清中文字幕av| 丰满迷人的少妇在线观看| 精品久久久久久电影网| 一个人看视频在线观看www免费| 久久精品国产亚洲av天美| 午夜福利影视在线免费观看| 色哟哟·www| 日本色播在线视频| 永久免费av网站大全| 赤兔流量卡办理| 久久精品国产亚洲av天美| 国产成人aa在线观看| 日本爱情动作片www.在线观看| 一本色道久久久久久精品综合| 中文天堂在线官网| 伦理电影大哥的女人| 日韩精品免费视频一区二区三区 | 91精品国产国语对白视频| 免费不卡的大黄色大毛片视频在线观看| 午夜福利网站1000一区二区三区| 精品酒店卫生间| 国产日韩欧美亚洲二区| 曰老女人黄片| av国产精品久久久久影院| 日本猛色少妇xxxxx猛交久久| 亚洲欧美成人综合另类久久久| 日日啪夜夜撸| 永久网站在线| 日韩,欧美,国产一区二区三区| 亚洲三级黄色毛片| 三级国产精品片| 国语对白做爰xxxⅹ性视频网站| 国产高清国产精品国产三级| www.色视频.com| 亚洲精品久久久久久婷婷小说| 青春草视频在线免费观看| 久久久久久久国产电影| 亚洲欧美中文字幕日韩二区| av在线播放精品| 精品一区在线观看国产| 日日撸夜夜添| 国产极品天堂在线| 国产av码专区亚洲av| 亚洲欧美成人精品一区二区| 91成人精品电影| 啦啦啦啦在线视频资源| 我要看黄色一级片免费的| 天堂8中文在线网| 桃花免费在线播放| 亚洲天堂av无毛| 天天操日日干夜夜撸| 不卡视频在线观看欧美| 国产色婷婷99| 国产在线一区二区三区精| 亚洲精品456在线播放app| 下体分泌物呈黄色| 嫩草影院入口| 天堂8中文在线网| 2018国产大陆天天弄谢| 国产真实伦视频高清在线观看| 国产成人精品久久久久久| 国产一区二区三区综合在线观看 | 国产 精品1| 精品卡一卡二卡四卡免费| 国产亚洲午夜精品一区二区久久| 亚洲怡红院男人天堂| 深夜a级毛片| 男的添女的下面高潮视频| 国产毛片在线视频| 国产乱人偷精品视频| 一级av片app| 男男h啪啪无遮挡| 亚洲自偷自拍三级| av福利片在线| 男女边摸边吃奶| 精品一区二区三区视频在线| 高清午夜精品一区二区三区| 国产又色又爽无遮挡免| 99热全是精品| 日韩亚洲欧美综合| 91久久精品国产一区二区成人| 国产一区亚洲一区在线观看| 又大又黄又爽视频免费| 蜜臀久久99精品久久宅男| 永久网站在线| 精品国产一区二区三区久久久樱花| 日韩一区二区视频免费看| 97在线人人人人妻| 如何舔出高潮| 韩国av在线不卡| 精品人妻熟女av久视频| 亚洲欧美清纯卡通| 亚洲高清免费不卡视频| 久久久久久久精品精品| 成人影院久久| 91久久精品国产一区二区三区| 一本—道久久a久久精品蜜桃钙片| 日产精品乱码卡一卡2卡三| 肉色欧美久久久久久久蜜桃| 成人亚洲精品一区在线观看| 精品人妻熟女毛片av久久网站| 又黄又爽又刺激的免费视频.| 午夜精品国产一区二区电影| 亚洲欧美中文字幕日韩二区| 99久久精品国产国产毛片| av卡一久久| 久久 成人 亚洲| 一级二级三级毛片免费看| 国产成人精品婷婷| 一级毛片 在线播放| 国产真实伦视频高清在线观看| 亚洲欧美精品专区久久| 男的添女的下面高潮视频| 亚洲av日韩在线播放| 高清午夜精品一区二区三区| 国产黄片美女视频| 精品酒店卫生间| 九草在线视频观看| 亚洲久久久国产精品| 尾随美女入室| 尾随美女入室| 久久毛片免费看一区二区三区| 一级毛片aaaaaa免费看小| 欧美丝袜亚洲另类| 久久人人爽人人片av| 最近2019中文字幕mv第一页| av女优亚洲男人天堂| 亚洲精品国产av成人精品| 免费观看性生交大片5| 亚洲情色 制服丝袜| 久久久久久久久大av| 草草在线视频免费看| 日韩中字成人| 亚洲天堂av无毛| 欧美日韩一区二区视频在线观看视频在线| 人妻制服诱惑在线中文字幕| 极品教师在线视频| 一级av片app| 欧美日韩亚洲高清精品| 亚洲成人一二三区av| 麻豆乱淫一区二区| 丝袜脚勾引网站| 国产老妇伦熟女老妇高清| a 毛片基地| 成年人午夜在线观看视频| 99久久精品国产国产毛片| 插阴视频在线观看视频| 好男人视频免费观看在线| 少妇裸体淫交视频免费看高清| 91久久精品国产一区二区三区| 精品久久国产蜜桃| 国产亚洲最大av| 亚州av有码| 国产亚洲5aaaaa淫片| 在线观看免费日韩欧美大片 | 久久6这里有精品| 99九九线精品视频在线观看视频| 在线观看国产h片| 国国产精品蜜臀av免费| 成人漫画全彩无遮挡| 亚洲av福利一区| 在线观看免费高清a一片| 狂野欧美白嫩少妇大欣赏| av在线app专区| 99re6热这里在线精品视频| 亚洲第一区二区三区不卡| 99热国产这里只有精品6| 高清黄色对白视频在线免费看 | 观看美女的网站| av在线播放精品| 久久国产亚洲av麻豆专区| 一二三四中文在线观看免费高清| 日本爱情动作片www.在线观看| 91久久精品国产一区二区成人| 六月丁香七月| 一级二级三级毛片免费看| 日韩成人av中文字幕在线观看| 黄色怎么调成土黄色| av国产精品久久久久影院| 久久国产亚洲av麻豆专区| videossex国产| 亚洲国产精品成人久久小说| 午夜激情福利司机影院| 婷婷色av中文字幕| 亚洲av综合色区一区| 欧美最新免费一区二区三区| 色94色欧美一区二区| 日韩熟女老妇一区二区性免费视频| 亚洲成色77777| 亚洲电影在线观看av| 交换朋友夫妻互换小说| 汤姆久久久久久久影院中文字幕| 日本色播在线视频| 色婷婷av一区二区三区视频| 色吧在线观看| 日本猛色少妇xxxxx猛交久久| 国产亚洲最大av| av国产精品久久久久影院| 18禁在线无遮挡免费观看视频| 热re99久久国产66热| 午夜老司机福利剧场| av不卡在线播放| 日韩 亚洲 欧美在线| 亚洲av综合色区一区| 亚洲成人一二三区av| 免费看日本二区| 国产欧美日韩一区二区三区在线 | 国产色婷婷99| 大又大粗又爽又黄少妇毛片口| av黄色大香蕉| 国产色婷婷99| 另类亚洲欧美激情| 内射极品少妇av片p| av在线app专区| 在线观看免费视频网站a站| 秋霞伦理黄片| 伦精品一区二区三区| 国产av码专区亚洲av| 天天操日日干夜夜撸| 伊人亚洲综合成人网| 亚洲精品日韩在线中文字幕| 只有这里有精品99| 99热国产这里只有精品6| 日本vs欧美在线观看视频 | 国产精品秋霞免费鲁丝片| 欧美亚洲 丝袜 人妻 在线| 成人黄色视频免费在线看| 亚洲欧美成人综合另类久久久| 亚洲av.av天堂| av又黄又爽大尺度在线免费看| 婷婷色综合大香蕉| 精品国产一区二区久久| 久久久久久伊人网av| 在线观看三级黄色| 精品久久久久久久久av| 一级二级三级毛片免费看| 日本爱情动作片www.在线观看| 久久ye,这里只有精品| 亚洲精品久久午夜乱码| av国产精品久久久久影院| 久久韩国三级中文字幕| 国产亚洲精品久久久com| 99热6这里只有精品| 亚洲精品国产av蜜桃| 日韩视频在线欧美| 亚洲不卡免费看| 男女无遮挡免费网站观看| 亚洲三级黄色毛片| 一个人看视频在线观看www免费| 国产免费又黄又爽又色| 久久精品国产亚洲网站| 日韩精品免费视频一区二区三区 | 婷婷色麻豆天堂久久| 少妇丰满av| 国产av国产精品国产| 国产精品久久久久久久久免| 国产成人午夜福利电影在线观看| 午夜久久久在线观看| 国产免费一区二区三区四区乱码| 亚洲av在线观看美女高潮| 午夜福利,免费看| 欧美日韩在线观看h| 午夜免费鲁丝| 热re99久久精品国产66热6| 久久毛片免费看一区二区三区| 亚洲精品自拍成人| 亚洲欧洲精品一区二区精品久久久 | 中文欧美无线码| 天堂8中文在线网| 丝袜在线中文字幕| 黄色配什么色好看| 免费播放大片免费观看视频在线观看| 久久韩国三级中文字幕| 国产一级毛片在线| 看免费成人av毛片| 视频区图区小说| 欧美日韩国产mv在线观看视频| 国产日韩欧美亚洲二区| 欧美区成人在线视频| 十分钟在线观看高清视频www | 亚洲精品国产av蜜桃| 三级经典国产精品| 精品久久久久久久久亚洲| 毛片一级片免费看久久久久| 国产91av在线免费观看| 看十八女毛片水多多多| 亚洲av.av天堂| 性色avwww在线观看| 国产精品麻豆人妻色哟哟久久| 亚洲成人av在线免费| xxx大片免费视频| a 毛片基地| 欧美变态另类bdsm刘玥| 国产精品国产三级专区第一集| 一级毛片久久久久久久久女| 另类精品久久| 黄色一级大片看看| 亚洲国产精品专区欧美| 一本久久精品| av在线老鸭窝| 国产成人精品久久久久久| 中文精品一卡2卡3卡4更新| 欧美xxxx性猛交bbbb| 久久亚洲国产成人精品v| 久久国产乱子免费精品| 国精品久久久久久国模美| 欧美 日韩 精品 国产| 精品酒店卫生间| 国产精品秋霞免费鲁丝片| 王馨瑶露胸无遮挡在线观看| 亚洲av在线观看美女高潮| 老司机影院毛片| 日韩三级伦理在线观看| 91在线精品国自产拍蜜月| 18禁动态无遮挡网站| .国产精品久久| 又粗又硬又长又爽又黄的视频| 男人爽女人下面视频在线观看| 久久久a久久爽久久v久久| 夜夜看夜夜爽夜夜摸| 亚洲国产欧美在线一区| 欧美日韩av久久| 欧美变态另类bdsm刘玥| 亚洲av欧美aⅴ国产| 偷拍熟女少妇极品色| 色视频www国产| 亚洲无线观看免费| 免费观看a级毛片全部| 美女福利国产在线| 久久精品国产鲁丝片午夜精品| 国产欧美亚洲国产| 我的老师免费观看完整版| xxx大片免费视频| 乱系列少妇在线播放| 国产在线男女| 亚洲精品视频女| 免费大片18禁| 国产深夜福利视频在线观看| 天堂中文最新版在线下载| 精品久久久久久久久av| 久久久欧美国产精品| 国产一区二区在线观看av| 亚洲高清免费不卡视频| 伊人久久国产一区二区| 日本黄大片高清| 日韩av免费高清视频| 妹子高潮喷水视频| 成年人免费黄色播放视频 | 欧美另类一区| 麻豆精品久久久久久蜜桃| 大香蕉久久网| 菩萨蛮人人尽说江南好唐韦庄| 一本大道久久a久久精品| 人人妻人人看人人澡| 最黄视频免费看| 一区二区三区乱码不卡18| 欧美97在线视频| 亚洲av中文av极速乱| 99九九线精品视频在线观看视频| 欧美 日韩 精品 国产| 国产男女超爽视频在线观看| 亚洲怡红院男人天堂| 亚洲综合精品二区| 久久狼人影院| 国产精品成人在线| 国产黄片视频在线免费观看| 高清毛片免费看| 亚洲欧美一区二区三区黑人 | 一区在线观看完整版| 91久久精品国产一区二区成人| 午夜老司机福利剧场| 国产淫语在线视频| freevideosex欧美| 欧美日韩视频高清一区二区三区二| 国产无遮挡羞羞视频在线观看| 91精品一卡2卡3卡4卡| 黄片无遮挡物在线观看| 亚洲色图综合在线观看| 亚洲人成网站在线播| 中文字幕人妻丝袜制服| 中文精品一卡2卡3卡4更新| 三级国产精品片| 看十八女毛片水多多多| 丰满人妻一区二区三区视频av| 97精品久久久久久久久久精品| 内射极品少妇av片p| 最近最新中文字幕免费大全7| 在现免费观看毛片| 国产成人一区二区在线| 国产成人91sexporn| 日韩免费高清中文字幕av| 国产淫语在线视频| av网站免费在线观看视频| 国产高清有码在线观看视频| 高清在线视频一区二区三区| 我的老师免费观看完整版| av天堂久久9| 在线观看人妻少妇| 亚洲欧美精品自产自拍| 如日韩欧美国产精品一区二区三区 | 啦啦啦中文免费视频观看日本| 涩涩av久久男人的天堂| 日本黄大片高清| 国产日韩一区二区三区精品不卡 | 免费大片18禁| 极品少妇高潮喷水抽搐| a级毛片在线看网站| 国产高清有码在线观看视频| 国产一区有黄有色的免费视频| 最新的欧美精品一区二区| 一区二区三区免费毛片| 久久久精品免费免费高清| 午夜激情久久久久久久| av卡一久久| 九九爱精品视频在线观看| 欧美xxⅹ黑人| 国精品久久久久久国模美| 久久久久久久亚洲中文字幕| 五月伊人婷婷丁香| 精品一区二区三区视频在线| 欧美人与善性xxx| 啦啦啦在线观看免费高清www| 特大巨黑吊av在线直播| 在线观看av片永久免费下载| 午夜老司机福利剧场| 中国美白少妇内射xxxbb| 一边亲一边摸免费视频| 女的被弄到高潮叫床怎么办| 最新的欧美精品一区二区| 日韩免费高清中文字幕av| 国产一区二区三区综合在线观看 | 亚洲国产毛片av蜜桃av| 观看av在线不卡| 久久久久国产精品人妻一区二区| 高清午夜精品一区二区三区| 久久99热这里只频精品6学生| 日韩视频在线欧美| 亚洲欧洲国产日韩| 国产精品一二三区在线看| av.在线天堂| 免费观看无遮挡的男女| 亚洲人成网站在线观看播放| 亚洲国产精品专区欧美| 色视频www国产| 美女大奶头黄色视频| 18禁在线无遮挡免费观看视频| 国产毛片在线视频| 啦啦啦在线观看免费高清www| 午夜影院在线不卡| 视频区图区小说| 少妇人妻精品综合一区二区| av.在线天堂| 久久6这里有精品| 国产伦理片在线播放av一区| 精品午夜福利在线看| 内地一区二区视频在线| 一区在线观看完整版| 最近的中文字幕免费完整| 大话2 男鬼变身卡| 六月丁香七月| 伊人久久精品亚洲午夜| 国产亚洲午夜精品一区二区久久| 国产色爽女视频免费观看| 婷婷色综合大香蕉| 性色av一级| 边亲边吃奶的免费视频| 亚洲欧美清纯卡通| 女性生殖器流出的白浆| 一级二级三级毛片免费看| 午夜免费观看性视频| av免费在线看不卡| 国产深夜福利视频在线观看| 亚洲天堂av无毛| 人妻系列 视频| 91久久精品电影网| 国产精品熟女久久久久浪| 成人亚洲精品一区在线观看| 日本爱情动作片www.在线观看| 人人妻人人看人人澡| 一级a做视频免费观看| 成人美女网站在线观看视频| 日韩制服骚丝袜av| 狠狠精品人妻久久久久久综合| 久久热精品热| 国产片特级美女逼逼视频| av.在线天堂| 欧美日韩国产mv在线观看视频| 国产欧美亚洲国产| 黄色怎么调成土黄色| 赤兔流量卡办理| 国产亚洲欧美精品永久| tube8黄色片| 我的女老师完整版在线观看| 国产欧美日韩一区二区三区在线 | 亚洲国产精品一区二区三区在线| 日韩免费高清中文字幕av| 国产白丝娇喘喷水9色精品| 偷拍熟女少妇极品色| a级片在线免费高清观看视频| 啦啦啦在线观看免费高清www| 新久久久久国产一级毛片| freevideosex欧美| 在线免费观看不下载黄p国产| 大话2 男鬼变身卡| 婷婷色综合www| av天堂中文字幕网| 亚洲高清免费不卡视频| 岛国毛片在线播放| 丰满迷人的少妇在线观看| 免费看不卡的av| 日韩强制内射视频| kizo精华| 狂野欧美激情性xxxx在线观看| 国产伦精品一区二区三区四那| 五月玫瑰六月丁香| 在线观看一区二区三区激情| 久久久久视频综合| 精品亚洲成国产av| 日韩不卡一区二区三区视频在线| 久久久久久久大尺度免费视频| 黄片无遮挡物在线观看| 性色av一级| av在线观看视频网站免费| 97精品久久久久久久久久精品| 日韩av在线免费看完整版不卡| 黄色视频在线播放观看不卡| 22中文网久久字幕| 最近2019中文字幕mv第一页| 寂寞人妻少妇视频99o| 亚洲精品日本国产第一区| 国产女主播在线喷水免费视频网站| 女性被躁到高潮视频| 欧美精品人与动牲交sv欧美| 国内精品宾馆在线| 永久免费av网站大全| 下体分泌物呈黄色| 永久免费av网站大全| 日韩亚洲欧美综合| 国产女主播在线喷水免费视频网站| 曰老女人黄片| 在线观看av片永久免费下载| 少妇人妻精品综合一区二区| 亚洲精品自拍成人| 韩国av在线不卡| 日韩伦理黄色片| 日日啪夜夜爽| 日韩大片免费观看网站| 欧美一级a爱片免费观看看| 啦啦啦在线观看免费高清www| 日韩精品免费视频一区二区三区 | 最近手机中文字幕大全| 肉色欧美久久久久久久蜜桃| 久久国内精品自在自线图片| 免费观看av网站的网址| 中文乱码字字幕精品一区二区三区| 成人毛片a级毛片在线播放| 精品人妻熟女av久视频| 亚洲精品乱码久久久久久按摩| 久久精品夜色国产| 欧美亚洲 丝袜 人妻 在线| 色视频www国产| 街头女战士在线观看网站| 久久人人爽人人片av| av播播在线观看一区| 国产精品久久久久久久久免| 少妇丰满av|