Several studies have demonstrated that overexpression of mutant a-synuclein in PC12 cells is related to occurrence of autophagy. The present study established mutant α-synuclein （A30P） -transfected PC12 cells and t...Several studies have demonstrated that overexpression of mutant a-synuclein in PC12 cells is related to occurrence of autophagy. The present study established mutant α-synuclein （A30P） -transfected PC12 cells and treated them with the autophagy inducer rapamycin and autophagy inhibitor wortmannin, respectively. Results demonstrated that mutant a-synuclein resulted in cell death via autophagy and involved a-synuclein accumulation, membrane lipid oxidation, and loss of plasma membrane integrity. Mutant a-synuclein （A30P） also mediated toxicity of 1-methyl-4-phenylpyridinium ion. Moreover, rapamycin inhibited a-synuclein aggregation, while wortmannin promoted α-synuclein aggregation and cell death. To further determine the role of autophagy due to mutant α-synuclein, the present study measured expression of microtubule-associated protein light chain 3. Results revealed that wortmannin and 1-methyl-4-phenylpyridinium ion inhibited expression of microtubule-associated protein light chain 3 while rapamycin promoted its expression. These findings suggested that abnormal aggregation of a-synuclein induced autophagic programmed cell death in PC12 cells.展开更多
AIM: To investigate the effects of mammalian target of rapamycin(mT OR) inhibition on liver regeneration and autophagy in a surgical resection model.METHODS: C57BL/6 mice were subjected to a 70% partial hepatectomy(PH...AIM: To investigate the effects of mammalian target of rapamycin(mT OR) inhibition on liver regeneration and autophagy in a surgical resection model.METHODS: C57BL/6 mice were subjected to a 70% partial hepatectomy(PH) and treated intraperitoneally every 24 h with a combination of the m TOR inhibitor rapamycin(2.5 mg/kg per day) and the steroid dexamethasone(2.0 mg/kg per day) in phosphate bufferedsaline(PBS) or with PBS alone as vehicle control. In the immunosuppressant group, part of the group was treated subcutaneously 4 h prior to and 24 h after PH with a combination of human recombinant interleukin 6(IL-6; 500 μg/kg per day) and hepatocyte growth factor(HGF; 100 μg/kg per day) in PBS. Animals were sacrificed 2, 3 or 5 d after PH and liver tissue and blood were collected for further analysis. Immunohistochemical staining for 5-Bromo-2'-deoxyuridine(Brd U) was used to quantify hepatocyte proliferation. Western blotting was used to detect hepatic microtubule-associated protein 1 light chain 3(LC3)-Ⅱ protein expression as a marker for autophagy. Hepatic gene expression levels of proliferation-, inflammation- and angiogenesisrelated genes were examined by real-time reverse transcription-polymerase chain reaction and serum bilirubin and transaminase levels were analyzed at the clinical chemical core facility of the Erasmus MC-University Medical Center.RESULTS: m TOR inhibition significantly suppressed regeneration, shown by decreased hepatocyte proliferation(2% vs 12% Brd U positive hepatocyte nuclei at day 2, P < 0.01; 0.8% vs 1.4% at day 5, P = 0.02) and liver weight reconstitution(63% vs 76% of initial total liver weight at day 3, P = 0.04), and furthermore increased serum transaminase levels(aspartate aminotransferase 641 U/L vs 185 U/L at day 2, P = 0.02). Expression of the autophagy marker LC3-Ⅱ, which was reduced during normal liver regeneration, increased after mT OR inhibition(46% increase at day 2, P = 0.04). Hepatic gene expression showed an increased inflammation-related response [tumor necrosis factor(TNF)-α 3.2-fold upregulation at day 2, P = 0.03; IL-1Ra 6.0-fold upregulation at day 2 and 42.3-fold upregulation at day 5, P < 0.01] and a reduced expression of cell cycle progression and angiogenesis-related factors(HGF 40% reduction at day 2; vascular endothelial growth factor receptor 2 50% reduction at days 2 and 5; angiopoietin 1 60% reduction at day 2, all P ≤ 0.01). Treatmentwith the regeneration stimulating cytokine IL-6 and growth factor HGF could overcome the inhibitory effect on liver weight(75% of initial total liver weight at day 3, P = 0.02 vs immunosuppression alone and P = 0.90 vs controls) and partially reversed gene expression changes caused by rapamycin(TNF-α and IL-1Ra levels at day 2 were restored to control levels). However, no significant changes in hepatocyte proliferation, serum injury markers or autophagy were found.CONCLUSION: mT OR inhibition severely impairs liver regeneration and increases autophagy after PH. These effects are partly reversed by stimulation of the IL-6 and HGF pathways.展开更多
Autophagy,a ubiquitous cellular biological behavior that features a lysosome-dependent degradation pathway,is an important mechanism for cellular self-protection in eukaryotes.Autophagy plays essential roles in cell s...Autophagy,a ubiquitous cellular biological behavior that features a lysosome-dependent degradation pathway,is an important mechanism for cellular self-protection in eukaryotes.Autophagy plays essential roles in cell survival,renewal,material reuse and the maintenance of homeostasis.This paper reviews recent advances in understanding the physiological function of autophagy and its possible roles in auditory diseases.We focused our review on original publications on animal models,drug models,and molecular mechanisms of hearing impairment involved in the dysregulation of autophagy.As research on the mechanisms of autophagy has deepened,it has become obvious that autophagy plays essential roles not only in cell survival,but the occurrence and development of a variety of auditory-related disorder,including aminoglycoside-induced hearing loss,age-related hearing loss,and noise-induced hearing loss.While clinical treatment of such conditions via regulation of the development of autophagy is a novel idea,more time is needed to fully elucidate the specific regulatory pathways and modes of autophagy in auditory diseases.The continued study of the mechanisms and regulation of autophagy in auditory diseases will be of great significance for the future treatment and prevention of these conditions.展开更多
基金the National Natural Science Foundation of China,No. 30970869a grant from Board of Health of Shanghai,China,No. 2008086+1 种基金Youth Key Project in College of Medicine of Fudan University,No. 09-L37a grant from the Project of Shanghai Key Laboratory of Diabetes Mellitus,No. 08DZ2230200
文摘Several studies have demonstrated that overexpression of mutant a-synuclein in PC12 cells is related to occurrence of autophagy. The present study established mutant α-synuclein （A30P） -transfected PC12 cells and treated them with the autophagy inducer rapamycin and autophagy inhibitor wortmannin, respectively. Results demonstrated that mutant a-synuclein resulted in cell death via autophagy and involved a-synuclein accumulation, membrane lipid oxidation, and loss of plasma membrane integrity. Mutant a-synuclein （A30P） also mediated toxicity of 1-methyl-4-phenylpyridinium ion. Moreover, rapamycin inhibited a-synuclein aggregation, while wortmannin promoted α-synuclein aggregation and cell death. To further determine the role of autophagy due to mutant α-synuclein, the present study measured expression of microtubule-associated protein light chain 3. Results revealed that wortmannin and 1-methyl-4-phenylpyridinium ion inhibited expression of microtubule-associated protein light chain 3 while rapamycin promoted its expression. These findings suggested that abnormal aggregation of a-synuclein induced autophagic programmed cell death in PC12 cells.
基金Supported by Erasmus MC Grant and the Liver Research Foundation(SLO) Rotterdam
文摘AIM: To investigate the effects of mammalian target of rapamycin(mT OR) inhibition on liver regeneration and autophagy in a surgical resection model.METHODS: C57BL/6 mice were subjected to a 70% partial hepatectomy(PH) and treated intraperitoneally every 24 h with a combination of the m TOR inhibitor rapamycin(2.5 mg/kg per day) and the steroid dexamethasone(2.0 mg/kg per day) in phosphate bufferedsaline(PBS) or with PBS alone as vehicle control. In the immunosuppressant group, part of the group was treated subcutaneously 4 h prior to and 24 h after PH with a combination of human recombinant interleukin 6(IL-6; 500 μg/kg per day) and hepatocyte growth factor(HGF; 100 μg/kg per day) in PBS. Animals were sacrificed 2, 3 or 5 d after PH and liver tissue and blood were collected for further analysis. Immunohistochemical staining for 5-Bromo-2'-deoxyuridine(Brd U) was used to quantify hepatocyte proliferation. Western blotting was used to detect hepatic microtubule-associated protein 1 light chain 3(LC3)-Ⅱ protein expression as a marker for autophagy. Hepatic gene expression levels of proliferation-, inflammation- and angiogenesisrelated genes were examined by real-time reverse transcription-polymerase chain reaction and serum bilirubin and transaminase levels were analyzed at the clinical chemical core facility of the Erasmus MC-University Medical Center.RESULTS: m TOR inhibition significantly suppressed regeneration, shown by decreased hepatocyte proliferation(2% vs 12% Brd U positive hepatocyte nuclei at day 2, P < 0.01; 0.8% vs 1.4% at day 5, P = 0.02) and liver weight reconstitution(63% vs 76% of initial total liver weight at day 3, P = 0.04), and furthermore increased serum transaminase levels(aspartate aminotransferase 641 U/L vs 185 U/L at day 2, P = 0.02). Expression of the autophagy marker LC3-Ⅱ, which was reduced during normal liver regeneration, increased after mT OR inhibition(46% increase at day 2, P = 0.04). Hepatic gene expression showed an increased inflammation-related response [tumor necrosis factor(TNF)-α 3.2-fold upregulation at day 2, P = 0.03; IL-1Ra 6.0-fold upregulation at day 2 and 42.3-fold upregulation at day 5, P < 0.01] and a reduced expression of cell cycle progression and angiogenesis-related factors(HGF 40% reduction at day 2; vascular endothelial growth factor receptor 2 50% reduction at days 2 and 5; angiopoietin 1 60% reduction at day 2, all P ≤ 0.01). Treatmentwith the regeneration stimulating cytokine IL-6 and growth factor HGF could overcome the inhibitory effect on liver weight(75% of initial total liver weight at day 3, P = 0.02 vs immunosuppression alone and P = 0.90 vs controls) and partially reversed gene expression changes caused by rapamycin(TNF-α and IL-1Ra levels at day 2 were restored to control levels). However, no significant changes in hepatocyte proliferation, serum injury markers or autophagy were found.CONCLUSION: mT OR inhibition severely impairs liver regeneration and increases autophagy after PH. These effects are partly reversed by stimulation of the IL-6 and HGF pathways.
文摘Autophagy,a ubiquitous cellular biological behavior that features a lysosome-dependent degradation pathway,is an important mechanism for cellular self-protection in eukaryotes.Autophagy plays essential roles in cell survival,renewal,material reuse and the maintenance of homeostasis.This paper reviews recent advances in understanding the physiological function of autophagy and its possible roles in auditory diseases.We focused our review on original publications on animal models,drug models,and molecular mechanisms of hearing impairment involved in the dysregulation of autophagy.As research on the mechanisms of autophagy has deepened,it has become obvious that autophagy plays essential roles not only in cell survival,but the occurrence and development of a variety of auditory-related disorder,including aminoglycoside-induced hearing loss,age-related hearing loss,and noise-induced hearing loss.While clinical treatment of such conditions via regulation of the development of autophagy is a novel idea,more time is needed to fully elucidate the specific regulatory pathways and modes of autophagy in auditory diseases.The continued study of the mechanisms and regulation of autophagy in auditory diseases will be of great significance for the future treatment and prevention of these conditions.
