RETRACTED ARTICLE: Regulation of autophagy by the nuclear factor κB signaling pathway in the hippocampus of rats with sepsis

Thirty-day-old male Wistar rats (100 to 120 g) were purchased from Sichuan Jianyang Dashuo Animal Science and Technology Co., Ltd. All rats were housed with free access to food and water in a 22–25 °C, 55–58 % relative humidity environment on a 12-h light/dark cycle. Every effort was made to minimize the number and suffering of animals used.

In the experiments, Wistar rats were randomly divided into four groups: cecal ligation and puncture group (CLP), sham-operated group, PDTC-treated group (PDTC + CLP), and vehicle-treated group (Veh + CLP). Each group was randomly divided into 3-, 6-, 12-, 24-, and 48-h groups. For the CLP group, sepsis was induced by CLP, as described previously by Rittirsch et al. [24]. Firstly, anesthesia was conducted with 10 % (w/v) chloral hydrate (1 ml/300 mg, i.p.), followed by a 2-cm midline abdominal incision. Secondly, the cecum was exteriorized and ligated immediately distal to the ileocecal valve. Thirdly, the cecum was punctured twice with an 18-gauge hollow-bore needle. Finally, the abdominal wall was closed in two layers. For the sham-operated group, rats were treated identically, except that the cecum was neither ligated nor punctured. The PDTC-treated group rats were injected intraperitoneally with PDTC (100 mg/kg, BioVision, USA) 2 h before the operation [25]. For the vehicle-treated group, mice received the same volume of vehicle (DMSO, 6.7 ml/kg, Solarbio, USA) intraperitoneally. Each animal received a subcutaneous injection of 5 ml 0.9 % (w/v) saline to compensate for the blood volume lost during the surgery. All surgical procedures were performed in an aseptic manner and were approved by the Research Animal Care Committee of Sichuan University.

Anesthesia was completed with 10 % (w/v) chloral hydrate by intraperitoneal injection. An indwelling tube was placed in the femoral artery to provide a venous channel for connecting the iWorx biological signal recorder, which was used to monitor the blood pressure and heart rate continually.

Rats were deeply anesthetized by an intraperitoneal injection of 10 % (w/v) chloral hydrate then transcardially perfused with 200 ml of 5 mM sodium phosphate-buffered 0.9 % (w/v) saline (PBS; pH 7.3), followed by 200 ml of 4 % (w/v) paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.3). After the brains were harvested and fixed in 4 % (w/v) paraformaldehyde at 4 °C for 24–48 h, they were cut in a coronal plane—from the optic chiasma through to the back (1 cm long) with the segment of the brain tissue paraffin-embedded. Then, brain samples were cut (6 mm thick) and routinely stained with hematoxylin and eosin. Finally, the Leica inverted optical microscope was used to capture images (Leica, USA).

The tissue samples for electron microscopy were obtained 2 mm posterior to the optic chiasma in a coronal plane (1 mm long) and were then fixed in a buffer containing 2 % (w/v) paraformaldehyde and 2.5 % (w/v) glutaraldehyde in 0.1 M PBS at 4 °C for 24–36 h. Afterwards, they were post-fixed in 3 % (v/v) glutaraldehyde and 1 % (w/v) phosphate-buffered osmium tetroxide and embedded in Epon812. Then, sections were cut at 0.12 μm thickness and stained with 0.2 % (w/v) lead citrate and 1 % (w/v) uranyl acetate, which were subsequently observed under an H-600IV transmission electron microscope (Hitachi, Japan).

Experimental and control animals were deeply anesthetized by injection of 10 % (w/v) chloral hydrate (1 ml/300 mg, i.p.) and then rapidly sacrificed at 3, 6, 12, 24, and 48 h post-operation. The brains were rapidly dissected out on a bed of ice. The hippocampus was dissected out from each hemisphere and deposited in ice-cold lysis buffer (50 mmol/l Tris–HCl pH 7.4, 150 mmol/l NaCl, 10 mg/l NP-40, 0.1 % protease inhibitor cocktail (Roche, USA)). Tissue samples were homogenized on ice with tweezers. Homogenates were centrifuged at 12,000 g for 30 min at 4 °C. The supernatant was collected and stored at −80 °C. All samples were analyzed for protein content with the BCA assay (Sigma, USA) to ensure equal protein loading. Proteins were separated by 12 % SDS-PAGE and transferred to nitrocellulose. For immunoblotting, membranes were blocked with 5 % (w/v) milk in TBS-T at 4 °C overnight; incubated with the following primary antibodies: rabbit anti- microtubule-associated protein light chain polyclonal antibody (LC3; 1:2000, Novus, USA), rabbit anti-Beclin1 polyclonal antibody (1:2000, CellSignaling, USA), rabbit anti-Rab7 monoclonal antibody (1:1000, Abcam, USA), rabbit anti-lysosome-associated membrane protein-1 polyclonal antibody (LAMP-1; 1:1000, Abcam, USA), mouse anti-NF-κB monoclonal antibody (1:2000, Millipore, USA), and mouse anti-actin monoclonal antibody (1:3000, Cell Signaling, USA) in 5 % (w/v) milk in TBS-T at room temperature for 2 h; and washed and incubated with a 1:2000 dilution of HRP-conjugated anti-rabbit or anti-mouse secondary antibody (1:3000, ZSGB- BIO, China) in 3–5 % (w/v) milk in TBS-T for 1 h at room temperature. Bands were visualized with enhanced chemiluminescence (ECL, Millipore, USA) by using the gel imaging analysis system (Bio-RAD, USA). In addition, Western blot analysis was performed with a beta-actin antibody (1:3000) as a loading control. The intensity of each band was quantitatively determined by Gel-Pro Image Analyzer Software. The density ratio represented the relative intensity of each band against actin. The intensity of blots was quantified with densitometry by people who were blinded to the treatments.

At the time points of 3, 6, 12, 24, and 48 h after CLP surgery, the rats were deeply anesthetized by injection of 10 % (w/v) chloral hydrate (1 ml/300 mg, i.p.) and transcardially perfused with 200 ml of 5 mM sodium phosphate-buffered 0.9 % (w/v) saline (PBS; pH 7.3), followed by 200 ml of 4 % (w/v) paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.3). The brains were harvested and fixed in 4 % (w/v) paraformaldehyde at 4 °C for 24–48 h. Brain samples were embedded in 4 % (w/v) agarose and were cut (40 μm thick) with an oscillate slicer. The sections were washed three times in PBS and blocked for 1 h at room temperature in PBS containing 2 % (v/v) fetal calf serum and 0.2 % (v/v) Triton X-100. Primary antibodies rabbit anti-LC3 polyclonal antibody (1:200, Novus, USA) or mouse anti-NeuN monoclonal antibody (1:200, Millipore, USA) in PBS containing 1 % (v/v) fetal calf serum and 0.1 % (v/v) Triton X-100 were added and incubated at 4 °C overnight. Next, the sections were washed three times in PBS (10 min each) and incubated with a mixture of DyLight 488-conjugated donkey anti-rabbit IgG (1:800, Jackson ImmunoResearch, USA) and Cy3-conjugated donkey anti-mouse IgG (1:800, Jackson ImmunoResearch, USA) for 1 h in PBS containing 1 % (v/v) fetal calf serum and 0.1 % (v/v) Triton X-100 at room temperature. Afterwards, the sections were washed for another three times in PBS (10 min each). Then, 4,6-diamidino-2-phenylindole (DAPI, 1:5000, Beyotime, China) nuclear stain was applied (1 μg/ml in PBS) for 10 min at room temperature. After staining, all of the sections were mounted onto glass slides and cover-slipped with antifade mounting medium (Beyotime, China). Using a confocal laser scanning microscope (Olympus, Japan), the sections were observed with the appropriate laser beams and filter settings for green-emitting Dylight 488 (excitation peak 493 nm, emission peak 518 nm) or red-emitting Cy3 (excitation peak 550 nm, emission peak 570 nm). The digital images were captured with FV10-ASW-3.1 software (Olympus, Japan).

The mean arterial pressure (MAP) was significantly (P < 0.05) lower in the CLP group than in the sham-operated group at each time point. The MAP began to decrease at 3 h, hit rock bottom at 12 or 24 h, and gradually rose by 48 h (Table 1). Conversely, the HR was significantly higher (P < 0.05) in the CLP group than in the sham-operated group at each time point (Table 1). In the PDTC + CLP groups, the vital signs were much higher (P < 0.05) than those in both the CLP groups and the vehicle control groups. It can be concluded that the MAP level was strikingly higher (P < 0.05) in the PDTC + CLP groups than in the CLP groups and that the HR level was notably lower (P < 0.05) in the PDTC + CLP groups than in the CLP groups (Table 2).

In the hippocampus of sham-operated group rats, almost no tissue alteration was observed at both the macroscopic and light microscopic levels (Fig. 1a). CLP-3 h rats showed slight neuronal edema (Fig. 1b). In CLP-12 h rats with swollen hippocampal tissue, disordered arrangement of hippocampus and unclear structure neurons appeared changed in an acute traumatic manner (Fig. 1c).

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.

Hippocampal tissues were harvested from rats at the time points of 3, 6, 12, 24, and 48 h after CLP. LC3-II significantly increased in CLP rats at 6, 12, 24, and 48 h after surgery than in sham-operated rats (P < 0.05; Fig. 3a). The punctate form and distribution of LC3 was examined to assess the autophagosomes by fluorescence microscopy. As shown in Fig. 3b, hippocampal cells of sham-operated animals expressed ubiquitously with diffuse in both the nucleus and cytoplasm of cells for LC3. Hippocampal cells after CLP appeared like intense granular LC3 staining predominantly in the cytosol. There was no notable difference in LC3 protein level between sham-operated rats at the time points of 3, 6, 12, 24, and 48 h post-surgery.

Hippocampal tissues were harvested from rats at the time points of 3, 6, 12, 24, and 48 h after CLP. Though Beclin1 level increased considerably at 6 h, it declined from 12 h after CLP compared with that in sham-operated rats (P < 0.05; Fig. 4a). LAMP-1 level dropped fast at the time points of 3, 6, 12, 24, and 48 h after CLP compared with that in sham-operated rats (P < 0.05; Fig. 4b). Rab7 level decreased at the time points of 12, 24, and 48 h after CLP compared with that in sham-operated rats (P < 0.05; Fig. 4c). There were few differences in Beclin1, Rab7, or LAMP-1 protein levels between sham-operated rats at the time points of 3, 6, 12, 24, and 48 h post-surgery.

NF-κB expression in CLP and PDTC-treated hippocampal tissueHippocampal tissues were harvested from rats at the time points of 3, 6, 12, 24, and 48 h after CLP. NF-κB increased at 12 and 24 h after CLP compared with that in sham-operated rats (P < 0.05; Fig. 5a). Western blot analysis indicated that the NF-κB protein level in Veh + CLP and CLP-12 h rats was much higher than in both the sham-operated and PDTC + CLP groups at respective time points (P < 0.05; Fig. 5b). There were no significant differences in NF-κB protein level between sham-operated rats at the time points of 3, 6, 12, 24, and 48 h post-surgery or between vehicle-operated and CLP-12 h rats.

Compared with sham-operated and CLP-12 h rats, as described as before (Fig. 1a, c), the pathological changes in the hippocampus of PDTC-treated rats were attenuated (Fig. 6c).

The effects of PDTC on LC-3, Beclin1, Rab7, and LAMP-1 protein levels were examined. Hippocampal tissues were harvested from rats 12 h after CLP. As shown in Fig. 7a, LC3-II grew in CLP rats receiving vehicle or PDTC compared with sham-operated rats (P < 0.05). Administration of PDTC after CLP showed a further increase in LC3-II compared with vehicle-treated rats (P < 0.05). Figure 7b, c, and d demonstrated that Beclin1, Rab7, and LAMP-1 fell in CLP rats receiving vehicle or PDTC compared with sham-operated rats (P < 0.05). Administration of PDTC after CLP showed an increase in Beclin1, Rab7, and LAMP-1 compared with vehicle-treated rats (P < 0.05). There were no significant differences in LC-3, Beclin1, Rab7, and LAMP-1 protein levels between vehicle-treated and CLP-12 h rats.

The effect of PDTC on LC3 in the brain was also evaluated by fluorescence microscopy. In fluorescence microscopy experiments, autophagosomes were assessed by examining the punctate form and distribution of LC3. As shown in Fig. 8, compared with rats in sham-operated, CLP-12 h, or Veh + CLP group, the PDTC + CLP rats showed an elevated autophagy—the staining pattern changes of LC3 from largely diffuse to predominantly punctate and cytoplasmic, which was consistent with the effects of PDTC on LC3 protein level observed by Western blot on a section from the hippocampus.