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1、NEURAL REGENERATION RESEARCH August 2016,Volume 11,Issue 8 www.nrronline.org RESEARCH ARTICLE Endoplasmic reticulum stress-induced apoptosis in the penumbra aggravates secondary damage in rats with traumatic brain injury Guo-zhu Sun , Fen-fei Gao , Zong-mao Zhao , Hai Sun , Wei Xu , Li-wei Wu , Yong

2、-chang He 1 Department of Neurosurgery, Second Hospital of Hebei Medical University, Shijiazhuang, Hebei Province, China 2 Department of Pharmacology, Shantou University Medical College, Shantou, Guangdong Province, China 3 Division of Neurological Surgery, Barrow Neurological Institute, St Josephs

3、Hospital and Medical Center, Phoenix, AZ, USA How to cite this article: Sun GZ, Gao FF, Zhao ZM, Sun H, Xu W, Wu LW, He YC (2016) Endoplasmic reticulum stress-induced apoptosis in the penumbra aggravates secondary damage in rats with traumatic brain injury. Neural Regen Res 11(8):1260-1266. Funding:

4、 This study was supported by the Natural Science Foundation of Hebei Province of China, No. H2014206383; and Foundation for High-Level Personnel Projects in Hebei Prov ince of China, No. A201401041. Graphical Abstract *Correspondence to: Guo-zhu Sun, M.D., Ph.D., . #These authors contributed equally

5、 to this study. orcid: 0000-0001-6380-8793 (Guo-zhu Sun) doi: 10.4103/1673-5374.189190 Accepted: 2016-05-30 Abstract Neuronal apoptosis is mediated by intrinsic and extrinsic signaling pathways such as the membrane-mediated, mitochondrial, and endo- plasmic reticulum stress pathways. Few studies hav

6、e examined the endoplasmic reticulum-mediated apoptosis pathway in the penumbra after traumatic brain injury, and it remains unclear whether endoplasmic reticulum stress can activate the caspase-12-dependent apoptotic pathway in the traumatic penumbra. Here, we established rat models of fuid percuss

7、ion-induced traumatic brain injury and found that protein expression of caspase-12, caspase-3 and the endoplasmic reticulum stress marker 78 kDa glucose-regulated protein increased in the traumatic penumbra 6 hours after injury and peaked at 24 hours. Furthermore, numbers of terminal deoxynucleotidy

8、l transferase-mediat- ed dUTP nick end labeling-positive cells in the traumatic penumbra also reached peak levels 24 hours after injury. These fndings suggest that caspase-12-mediated endoplasmic reticulum-related apoptosis is activated in the traumatic penumbra, and may play an important role in th

9、e pathophysiology of secondary brain injury. Key Words: nerve regeneration; endoplasmic reticulum stress; apoptosis; caspase-12; caspase-3; traumatic penumbra; traumatic brain injury; neural regeneration Introduction early stage is, to a large extent, responsible for the poor prog- lated long-term d

10、isability and death in patients under the brain tissue surrounding the primary lesion, in which sec- age of 45 years (Langlois et al., 2006). In addition to the pri- ondary brain injury develops after the primary insult (Stoffel mary insult, the irreversible deterioration of the traumatic et al., 19

11、97; Harish et al., 2015). Following TBI, brain lesions penumbra without timely intervention and treatment at the are not limited to the site of the primary neurotrauma, but 1260 Caspase-12-mediated endoplasmic reticulum (ER) apoptotic pathway is activated in traumatic penumbra after traumatic brain

12、injury Caspase-12 Caspase-3 1, * 2 1 3 1 1 1 nosis. The traumatic penumbra is the potentially salvageable Sun GZ, et al. / Neural Regeneration Research. 2016;11(8):1260-1266. expand progressively and centrifugally. There is evidence that focal necrosis increases over time and the volume of necrotic

13、tissue can reach 400% of the initial lesion 24 hours after impact (Stoffel et al., 2002). Clinical studies have also demonstrated that expansion of the penumbra impairs cere- bral blood fow and leads to edema and compromised local metabolism, resulting in clinical deterioration (Newcombe et al., 201

14、3; Wu et al., 2013; Sheriff and Hinson, 2015). Patho- physiologically, the traumatic penumbra involves a series of damage cascades such as glutamate excitotoxicity, loss of ionic homeostasis, inflammation, and oxidative stress, and eventually gives rise to neuronal apoptosis (Rosenfeld et al., 2012;

15、 Ding et al., 2015; Sheriff and Hinson, 2015). Apoptosis, or programmed cell death, is one of the main factors affecting the outcome and prognosis of TBI. Previ- ous studies have demonstrated neuronal apoptosis in the contusion site and penumbra in animal models and patients with TBI (Ang et al., 20

16、03; Zhang et al., 2005). Furthermore, approximately two-thirds of cell death might be attributable to apoptosis in traumatic penumbra (Zhang et al., 2005; Zie- bell et al., 2011). Intrinsic and extrinsic signaling pathways mediate neuronal apoptosis, including membrane-mediated, mitochondrial, and e

17、ndoplasmic reticulum (ER) stress path- ways (Jin et al., 2015; Li et al., 2015; Chuang et al., 2016). Previous studies have focused on investigating the former two apoptotic pathways in the traumatic penumbra, but few reports have addressed the ER stress-induced pathway (Yoneda et al., 2001; Long et

18、 al., 2013; Han et al., 2014; Ka- badi and Faden, 2014). The caspase family of cysteine proteases plays a critical role in the caspase-12-dependent apoptotic pathway, in which caspase-12 and caspase-3 function as an upstream ini- tiator and downstream effector, respectively, of the apoptotic pathway

19、. When cells are exposed to apoptotic stimuli such as oxidative stress, caspase-12 is activated by autoproteolytic modifcation, and leads directly or indirectly to the cleavage of caspase-3 (Zhao et al., 2013). During late-stage apoptosis, caspase-3 executes the dismantling of the cell and the forma

20、- tion of apoptotic bodies. The ER- localized molecular chaperone 78 k Da glu- cose-regulated protei n (GRP78) is a marker of the ER, and its upregulation indicates ER stress (Ma et al., 2 008; Martnez-Pizar ro et al., 2016). It is well known that oxi- dative stress, including ER stress, is involved

21、 in neural cell death. However, the involvement of the ER stress-induced caspase-1 2-mediated apoptotic pathway in the traumatic penumbra is not completely clear. We hypothesized that this pathway is activated in the traumatic penumbra after TBI. To test this hypothesis, we measured neuronal apoptos

22、is and protein expression of caspase-12, caspase-3 and GRP78 in rats at various time points after TBI. Materials and Methods Ethics statement Animal studies were approved by the Institutional Animal Care and Use Committee of Hebei Medical University (ap- proval No. HbMUEC-130306) and performed in ac

23、cordance with the National Institutes of Health Guide f or the Care and Use of Laboratory Animals. Precautions were taken to minimize suffering and the number of animals used in each experiment. Exper imental animals Sixty male healthy Sprague-Dawley rats, aged 910 weeks and weighing 300350 g, were

24、purchased from the Laborato- ry Animal Center of Hebei Medical University, China (license No. SCXK (Ji) 2013-1-003). Rats were housed in separate cages at 2225C and 5070% humidity under a 12-hour reversed light/dark cycle with food and water ad libitum. The animals were habituated under these condit

25、ions for at least 1 week bef ore the start of the experiment. Rats were randomly divided into sham-operated (n = 12) and TBI (n = 48) groups. The TBI group was further divided into four subgroups (n = 12) to be sacrifced at different time points (6, 12, 24 and 72 hours after TBI). Establishment of T

26、BI models All sur gical procedures w ere perf ormed under aseptic conditions. TBI models were established using a fluid per- cussion device (HPD-1700, Dragonfly R&D, Silver Spring, MD, USA). Rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (100 mg/kg) and fxed in a st

27、ereotaxic frame in the prone position. A 5 mm craniot- omy was performed over the right parietal cortex (3.8 mm posterior to and 2.5 mm lateral to the bregma), keeping the dura intact (Tomura et al., 2011). A plastic Luer Lock hub (BD, Franklin Lakes, NJ, USA) was secured over the skull window with

28、dental acrylic cement. All rats in the TBI group were subjected to fuid percussion injury (FPI) of high grade severity (263304 kPa, 16 ms in duration). The Luer Lock was removed immediately after FPI. Sham-operated animals were subjected to all surgical procedures except FPI. Follow- ing scalp sutur

29、ing, rats were returned to their home cages with food and water available ad libitum. Brain tissue preparation Rats were anesthetized (100 mg/kg sodium pentobarbital in- traperitoneally) at 6, 12, 24 or 72 hours after TBI, according to group allocation. For terminal deoxynucleotidyl transfer- ase-me

30、diated dUTP nick end labeling (TUNEL) and immu- nohistochemistry, rats (n = 6 per subgroup) were perfused via the left cardiac ventricle with saline at 4C followed by 4% neutral-buffered formalin solution. Brains were quickly harvested and placed on a frozen plate. Tissue samples were rapidly taken

31、from the region adjacent to the site of injury (712 mm from frontal polar region), preserved overnight in 4% neutral-buffered formalin solution, and embedded in paraffn. For western blot analysis, rats (n = 6 per subgroup) were exsanguinated via cardiac puncture. Brain tissue was harvested as descri

32、bed above, and immediately stored at 80C in liquid nitrogen until western blot analysis. Immunohistochemistry Paraffin-embedded samples were placed in a microtome, 1261 Sun GZ, et al. / Neural Regeneration Research. 2016;11(8):1260-1266. serially sliced into 4 m thick sections, and routinely depa- e

33、nous peroxidase activity. PBS containing 10% normal goat serum was used to block nonspecifc antibody binding. Sec- tions were incubated overnight at 4C with anti-caspase-12 (1:100; Cell Signaling, Beverly, MA, USA) and anti-caspase-3 (1:100; Cell Signaling) rabbit polyclonal antibodies. The sections

34、 were washed three times in PBS for 5 minutes each time, and then incubated with horseradish peroxidase-con- jugated goat anti-rabbit IgG antibody (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 60 minutes. Final- ize caspase-12 and caspase-3 expression. The sections were counterstained w

35、ith hematoxylin for 4 minutes to visualize the nuclei, and then mounted. Optical density of immuno- reactive cells was calculated using Image-Pro Plus 6.0 (Media Cybernetics, Rockville, MD, USA). Western blot analysis Frozen brain samples were thawed and homogenized in seven volumes of ice-cold lysa

36、te buffer (200 mM HEPES pH 7.5, 250 mM sucrose, 1 mM dithiothreitol, 1.5 mM phenylmethylsulfonyl fluoride, 10 g/mL leupeptin, 5 g/ mL pepstatin, 2 g/mL aprotinin). The homogenates were centrifuged at 3,000 g for 10 minutes at 4C and the su- pernatant was then centrifuged at 8,000 g for 20 minutes at

37、 the same temperature. The resulting supernatant was col- lected and further centrifuged at 54,000 g for 60 minutes at 4C. The pellet was identifed as the microsome fraction, which is enriched in ER, and the supernatant as the cyto- solic fraction. The Bradf ord assay (Bio-Rad Laboratories, Hercules

38、, CA, USA) was used to determine the protein con- centration of each sample against albumin standards. For the microsomal samples, aliquots (60 g) of each sample were subjected to 10% or 12.5% sodium dodecyl- sulfate-polyacrylamide gel electrophoresis. The separated protein was then transf erred f r

39、om the gel to a polyvi - nylidene dif luor ide membr ane (Gi bco, New York , NY, USA) at 120 V for 50 minutes at room temperature. For immunoblotting, nonspecifc protein binding to the mem- brane was blocked with 5% non-f at dried milk for 2 hours at room temperature, then incubated with the followi

40、ng primary antibodies, all raised in rabbit: anti-mouse GRP78 monoclonal antibody (1:500; Santa Cruz Biotechnology), anti-caspase-12 polyclonal antibody (1:500; Santa Cruz Bio- technology) and anti-caspase-3 polyclonal antibody (1:750; Santa Cruz Biotechnology). The secondary antibody was horseradis

41、h peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:2,000; Cell Signaling, Beverly, MA, USA), and rabbit monoclonal anti-actin antibody (1:10,000; Cell Signal- ing) was used as an endogenous control for all samples. SeeBlue Plus2 Pre-Stained Standard (Life Technologies, Carlsbad, CA, USA) was used

42、 to determine molecular weights. The reaction was visualized using LumiGLO che- miluminescent substrate (Cell Signaling) according to the manufacturers instructions. The bands were scanned and 1262 analyzed usi ng optical density values with background subtraction and normalized to -actin using Imag

43、eJ soft- ware (NIH). TUNEL analysis Paraffn-embedded brain tissues were sliced into 4 m thick sections in a microtome (Leica Biosystems, Nussloch, Ger- many). Apoptotic cells were identified using a TUNEL de- tection kit (DeadEnd TUNEL system, Promega, Madison WI, USA) according to the manufacturers

44、 instructions. In brief, the sections were deparaffnized, rehydrated, and then digested in proteinase K (20 g/mL) at room temperature for 15 minutes. After being washed with PBS for 5 minutes, sections were incorporated with biotinylated nucleotides at the 30-OH DNA end using terminal deoxyribonucle

45、otide transferase (TdT) for 1 hour at 37C. Negative controls were incubated with label solution not containing TdT. Sections were then incubated with FITC-conjugated avidin, counter- stained for nuclei with DAPI, and positive cells were identi- fed, counted, and analyzed under a fuorescence microsco

46、pe (Olympus, Tokyo, Japan) by an investigator blinded to the grouping. The apoptotic index was defined as the average percentage of TUNEL-positive cells in each section counted in 10 randomly selected cortical microscopic felds (at 400 magnifcation). Statistical analysis All experimental data are ex

47、pressed as the mean SD. Sta- tistical differences between sham-operated and TBI groups w ere determi ned by one-w ay analysis of variance w ith Tukeys test (SPSS 13.0 software, SPSS, Chicago, IL, USA). P 0.05 was considered statistically signifcant. Results GRP78 expression in traumatic penumbra aft

48、er TBI The expression of GRP78 increased markedly in the penum- bra from 6 hours after TBI, and peaked at 24 hours (P 0.05); GRP78 expression was signifcantly elevated compared with the sham value at all time points examined after injury (P 0.05) (Figure 1). Caspase-12 expression in penumbra after TBI Western blot assay showed that caspase-12 protein expres- sion in