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Regulation of autophagy by the nuclear factor κB signaling pathway in the hippocampus of rats with sepsis.

Su Y, Qu Y, Zhao F, Li H, Mu D, Li X - J Neuroinflammation (2015)

Bottom Line: Hematoxylin-eosin staining and biological signal recording was used to measure the morphological and physiological signs of hippocampal dysfunction.An electron microscope was used to observe autophagosome formation and lysosome activation in the hippocampus after CLP.Western blotting and immune histochemistry were used to detect the hippocampus levels of NF-κB and essential proteins involved in formation of the autophagosome (microtubule-associated protein light chain 3 (LC3), Beclin1, Lamp-1, and Rab7).

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, West China Second University Hospital, Sichuan University, Chengdu, 610041, China. syj412811@163.com.

ABSTRACT

Background: Sepsis with brain dysfunction has contributed to an increase risk of morbidity and mortality. In its pathophysiology, both autophagy and nuclear factor κB (NF-κB) have been suggested to play important roles. Based on the fact that crosstalk between autophagy and NF-κB, two stress-response signaling pathways, has been detected in other pathophysiological processes, this study was undertaken to explore the process of autophagy in the hippocampus of septic rats and the role NF-κB plays in the regulation of autophagy during the process.

Methods: Cecal ligation and puncture (CLP) or a sham operation was conducted on male Wistar rats. Pyrrolidine dithiocarbamate (PDTC), an inhibitor of the NF-κB signaling pathway, or a vehicle control, was used to treat with the rats 2 h before the CLP operation. Hematoxylin-eosin staining and biological signal recording was used to measure the morphological and physiological signs of hippocampal dysfunction. An electron microscope was used to observe autophagosome formation and lysosome activation in the hippocampus after CLP. Western blotting and immune histochemistry were used to detect the hippocampus levels of NF-κB and essential proteins involved in formation of the autophagosome (microtubule-associated protein light chain 3 (LC3), Beclin1, Lamp-1, and Rab7).

Results: Compared with sham-operated rats, the CLP rats showed decreasing mean arterial pressure (MAP), increasing heart rate (HR), and pathological histological changes. CLP rats exhibited not only increased vacuolization through electron micrographs but also increased LC3-II, decreased Beclin1, LAMP-1, and Rab7 through the immunofluorescence and Western blot. However, PDTC + CLP rats revealed that inhibition of the NF-κB signal axis by PDTC increased the levels of LC3-II, Beclin1, LAMP-1, and Rab7 and improved physiological function including blood pressure and heart rate.

Conclusions: The autophagy process during the hippocampus of CLP rats might be blocked by the activation of NF-κB signaling pathway. Inhibition of NF-κB signaling pathway could enhance the completion of autophagy with a neuroprotective function in septic brains.

No MeSH data available.


Related in: MedlinePlus

Electron micrographs of the hippocampus detected at 12 h following sham operation (a), CLP-12 h (b), and CLP + PDTC-12 h (c). a Sham-operated control rats showed organelles almost without pathological changes; no alteration of tissue integrity could be observed in low magnification images. Magnification: ×10,000. b A large autophagosome contains mitochondria and other organelles; endoplasmic reticulum matrix into adjacent lysosomal structures (arrow). Magnification: ×15,000. c CLP + PDTC-12 h displaying multiple double or multiple-membrane autophagic vesicles (arrows) in the cytoplasm, with loss of discernable organellar fragments; autophagosomes assume a more complex appearance, with redundant whorls of membrane-derived material. Magnification: ×10,000. CLP cecal ligation and puncture, PDTC pyrrolidine dithiocarbamate
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Fig2: Electron micrographs of the hippocampus detected at 12 h following sham operation (a), CLP-12 h (b), and CLP + PDTC-12 h (c). a Sham-operated control rats showed organelles almost without pathological changes; no alteration of tissue integrity could be observed in low magnification images. Magnification: ×10,000. b A large autophagosome contains mitochondria and other organelles; endoplasmic reticulum matrix into adjacent lysosomal structures (arrow). Magnification: ×15,000. c CLP + PDTC-12 h displaying multiple double or multiple-membrane autophagic vesicles (arrows) in the cytoplasm, with loss of discernable organellar fragments; autophagosomes assume a more complex appearance, with redundant whorls of membrane-derived material. Magnification: ×10,000. CLP cecal ligation and puncture, PDTC pyrrolidine dithiocarbamate

Mentions: Autophagic vacuoles were observed by transmission electron microscopy. As shown in Fig. 2a, the hippocampal tissue from sham-operated rats displayed nearly normal structure and proper organelle distribution. No alteration of tissue integrity was observed in low magnification images. In Fig. 2b, CLP rats demonstrated autophagic vacuolization. A number of irregularities were seen sporadically in high-power electron microscopic images, including a large autophagosome containing mitochondria and other organelles, herniation of outer membranes of endoplasmic reticulum into adjacent lysosomal structures. Other changes including prominent matrix granules and crystalline-like inclusion were seen in selected examples of septic rats. The PDTC-treated rats showed multiple double or multiple-membrane autophagic vesicles in the cytoplasm, with loss of discernable organellar fragments. Autophagosomes assume a more complex appearance, with redundant whorls of membrane-derived material. As shown in Fig. 2, there were many differences among the sham group, the CLP group, and the PDTC-treated group in the performance of the rats’ hippocampus, which indicates that intraperitoneal administration of PDTC has an impact on autophagic vacuolization.Fig. 2


Regulation of autophagy by the nuclear factor κB signaling pathway in the hippocampus of rats with sepsis.

Su Y, Qu Y, Zhao F, Li H, Mu D, Li X - J Neuroinflammation (2015)

Electron micrographs of the hippocampus detected at 12 h following sham operation (a), CLP-12 h (b), and CLP + PDTC-12 h (c). a Sham-operated control rats showed organelles almost without pathological changes; no alteration of tissue integrity could be observed in low magnification images. Magnification: ×10,000. b A large autophagosome contains mitochondria and other organelles; endoplasmic reticulum matrix into adjacent lysosomal structures (arrow). Magnification: ×15,000. c CLP + PDTC-12 h displaying multiple double or multiple-membrane autophagic vesicles (arrows) in the cytoplasm, with loss of discernable organellar fragments; autophagosomes assume a more complex appearance, with redundant whorls of membrane-derived material. Magnification: ×10,000. CLP cecal ligation and puncture, PDTC pyrrolidine dithiocarbamate
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4472259&req=5

Fig2: Electron micrographs of the hippocampus detected at 12 h following sham operation (a), CLP-12 h (b), and CLP + PDTC-12 h (c). a Sham-operated control rats showed organelles almost without pathological changes; no alteration of tissue integrity could be observed in low magnification images. Magnification: ×10,000. b A large autophagosome contains mitochondria and other organelles; endoplasmic reticulum matrix into adjacent lysosomal structures (arrow). Magnification: ×15,000. c CLP + PDTC-12 h displaying multiple double or multiple-membrane autophagic vesicles (arrows) in the cytoplasm, with loss of discernable organellar fragments; autophagosomes assume a more complex appearance, with redundant whorls of membrane-derived material. Magnification: ×10,000. CLP cecal ligation and puncture, PDTC pyrrolidine dithiocarbamate
Mentions: Autophagic vacuoles were observed by transmission electron microscopy. As shown in Fig. 2a, the hippocampal tissue from sham-operated rats displayed nearly normal structure and proper organelle distribution. No alteration of tissue integrity was observed in low magnification images. In Fig. 2b, CLP rats demonstrated autophagic vacuolization. A number of irregularities were seen sporadically in high-power electron microscopic images, including a large autophagosome containing mitochondria and other organelles, herniation of outer membranes of endoplasmic reticulum into adjacent lysosomal structures. Other changes including prominent matrix granules and crystalline-like inclusion were seen in selected examples of septic rats. The PDTC-treated rats showed multiple double or multiple-membrane autophagic vesicles in the cytoplasm, with loss of discernable organellar fragments. Autophagosomes assume a more complex appearance, with redundant whorls of membrane-derived material. As shown in Fig. 2, there were many differences among the sham group, the CLP group, and the PDTC-treated group in the performance of the rats’ hippocampus, which indicates that intraperitoneal administration of PDTC has an impact on autophagic vacuolization.Fig. 2

Bottom Line: Hematoxylin-eosin staining and biological signal recording was used to measure the morphological and physiological signs of hippocampal dysfunction.An electron microscope was used to observe autophagosome formation and lysosome activation in the hippocampus after CLP.Western blotting and immune histochemistry were used to detect the hippocampus levels of NF-κB and essential proteins involved in formation of the autophagosome (microtubule-associated protein light chain 3 (LC3), Beclin1, Lamp-1, and Rab7).

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, West China Second University Hospital, Sichuan University, Chengdu, 610041, China. syj412811@163.com.

ABSTRACT

Background: Sepsis with brain dysfunction has contributed to an increase risk of morbidity and mortality. In its pathophysiology, both autophagy and nuclear factor κB (NF-κB) have been suggested to play important roles. Based on the fact that crosstalk between autophagy and NF-κB, two stress-response signaling pathways, has been detected in other pathophysiological processes, this study was undertaken to explore the process of autophagy in the hippocampus of septic rats and the role NF-κB plays in the regulation of autophagy during the process.

Methods: Cecal ligation and puncture (CLP) or a sham operation was conducted on male Wistar rats. Pyrrolidine dithiocarbamate (PDTC), an inhibitor of the NF-κB signaling pathway, or a vehicle control, was used to treat with the rats 2 h before the CLP operation. Hematoxylin-eosin staining and biological signal recording was used to measure the morphological and physiological signs of hippocampal dysfunction. An electron microscope was used to observe autophagosome formation and lysosome activation in the hippocampus after CLP. Western blotting and immune histochemistry were used to detect the hippocampus levels of NF-κB and essential proteins involved in formation of the autophagosome (microtubule-associated protein light chain 3 (LC3), Beclin1, Lamp-1, and Rab7).

Results: Compared with sham-operated rats, the CLP rats showed decreasing mean arterial pressure (MAP), increasing heart rate (HR), and pathological histological changes. CLP rats exhibited not only increased vacuolization through electron micrographs but also increased LC3-II, decreased Beclin1, LAMP-1, and Rab7 through the immunofluorescence and Western blot. However, PDTC + CLP rats revealed that inhibition of the NF-κB signal axis by PDTC increased the levels of LC3-II, Beclin1, LAMP-1, and Rab7 and improved physiological function including blood pressure and heart rate.

Conclusions: The autophagy process during the hippocampus of CLP rats might be blocked by the activation of NF-κB signaling pathway. Inhibition of NF-κB signaling pathway could enhance the completion of autophagy with a neuroprotective function in septic brains.

No MeSH data available.


Related in: MedlinePlus