Am J Transl Res 2021;13(2):732-742 www.ajtr.org /ISSN:1943-8141/AJTR0110812 Original Article Possible mechanisms of the PERK pathway on neuronal apoptosis in a rat model of surgical brain injury Mu-Yao Wu1*, Fan Gao1*, Jia-Feng Tang1, Jin-Chao Shen2, Rong Gao3, Bao-Qi Dang1, Gang Chen4 1Department of Rehabilitation, Zhangjiagang TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Suzhou, China; 2Anesthesiology Department, Zhangjiagang TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Suzhou, China; 3Department of Neurosurgery, Zhangjiagang TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Suzhou, China; 4Department of Neurosurgery and Brain and Nerve Research Laboratory, The First Affiliated Hospital of Soochow University, Suzhou, China. *Equal contributors. Received March 13, 2020; Accepted December 11, 2020; Epub February 15, 2021; Published February 28, 2021 Abstract: Protein kinase R-like endoplasmic reticulum kinase (PERK) is an important transmembrane protein in the endoplasmic reticulum (ER). PERK signaling has a critical function in neuronal apoptosis. This work aimed to assess PERK signaling for its function in surgical brain injury (SBI) and to explore the underlying mechanisms. Totally 120 male Sprague Dawley (SD) rats were assessed in an SBI model. The effects of the PERK inhibitor GSK2606414 were examined by Western-blot, immunofluorescent staining, TUNEL staining, fluoro-jade C (FJC) staining and neu- rological assays in rats with SBI. In this study, p-PERK and p-eIF2α protein amounts were increased upon SBI establishment, peaking at 24 h. Meanwhile, administration of GSK2606414 reversed these effects and prevented neuronal apoptosis. The PERK pathway has a significant function in neuronal apoptosis, and its suppression after SBI promotes the alleviation of brain injury. This suggests that targeting the PERK signaling pathway may represent an efficient therapeutic option for improving prognosis in SBI patients. Keywords: ER stress, UPR, PERK, eIF2α, apoptosis, SBI Introduction tion and reduce perioperative costs. However, few studies have focused on the pathophysiol- Surgical brain injury (SBI) is caused by neuro- ogy of SBI or the development of specific treat- surgery and often occurs at the edge of the ments. Research into SBI pathology has made area upon which the surgery is performed [1]. It some progress, but the specific mechanisms may lead to multiple postoperative complica- underlying its occurrence and development are tions such as cerebral edema, ischemia, intra- very complex and remain unclear. Studies have cranial hematoma, and neurological impair- shown that SBI can lead to a series of patho- ment and behavioral degeneration [2]. SBI is logical processes such as brain edema, BBB thought to be inevitable, and is often associat- damage, inflammatory reactions, and apopto- ed with postoperative neurologic deficits [3]. sis around the surgical site [5]. Recent reports Studies have confirmed that brain edema revealed that endoplasmic reticulum (ER) stress occurs several hours after SBI, which may is substantially involved in the pathological pro- impair blood-brain barrier (BBB) integrity and cess of secondary brain damage following SBI. aggravate craniocerebral injury, including sec- ondary inflammatory reactions [4, 5]. SBI- The ER is an important membranous organelle induced nerve injury and cerebral edema may in mammalian cells, mostly contributing to pro- prolong hospital stay. Therefore, it is very im- tein folding and modification and Ca2+ storage portant to reduce secondary brain injury ca- and release. As a site of protein maturation in used by SBI, maintain neurological function cells, the ER is sensitive to changes in the inter- and reduce perioperative morbidity related to nal and external cellular environments. When intracranial neurosurgery [6]. All these actions the internal environment of the ER changes, have significant impacts on patients’ rehabilita- stress response is activated to attempt to res- Inhibiting PERK pathway alleviates apoptosis tore homeostasis; these changes in the ER are that PERK is activated after brain injury wh- called ER stress (ERs) [7]. The unfolded protein en phosphorylated at ser51. Phosphorylated response (UPR) is one of the cell response eIF2α (p-eIF2α) can also increase apoptosis by mechanisms involved in defense activation of regulating AFT4 expression [15]. Yan et al. stud- adaptation. The UPR restores the ER’s capabil- ied a rat subarachnoid hemorrhage model and ity of controlling protein folding, processing and showed that inhibiting PERK may alleviate early secretion [8]. Through the expansion of the brain damage upon subarachnoid hemorrhage membrane, the ER reduces the rate of synthe- via an Akt-related anti-apoptotic signaling path- sis of new proteins, increases partner molecule way [16]. Our previous report confirmed that synthesis and promotes the degradation of inhibiting PERK signaling in intracerebral hem- misfolded proteins to maintain its own steady orrhage can reduce secondary brain injury fol- state, thereby restoring the normal function of lowing intracerebral hemorrhage and promote cells [9]. However, when the damage exceeds neuron survival by inhibiting apoptosis [17]. the system’s repair capacity, excessive accu- However, whether PERK signaling has a func- mulation of misfolded proteins leads to pro- tion in SBI remains undefined. grammed cell death or apoptosis [10]. In this way, the intensity and duration of stimulation Therefore, the present work aimed to assess determine whether UPR promotes cell survival PERK levels and PERK-eIF2α signaling in a rat or induces apoptosis. SBI model, and to explore the possible mecha- nism of the PERK signaling pathway in SBI. The UPR has three parallel signal branches, including protein kinase R-like endoplasmic Materials and methods reticulum kinase-eukaryotic translation initia- tion factor 2α (PERK-eIF2α), inositol requiring Experimental design protein 1-x box binding protein 1 (IRE1-XBP1), and activating transcription factor 6 (ATF6) In Experiment 1, animal weights, feed intake, pathways. Each branch can regulate a series and motor function were similar in all animals. of downstream transcription factors to partici- To examine PERK, p-PERK, eIF2α and p-eIF2α pate in the survival or death of cells [2]. Under amounts following SBI, 36 rats (36 survivors normal conditions, the ER’s chaperone glu- in totally 40) were randomized to six groups cose-regulated protein 78 (GRP78/BIP) inter- using computer-based randomization, includ- acts with the transmembrane proteins PERK, ing the Sham, SBI 6 h, SBI 12 h, SBI 24 h, SBI IRE1, and ATF6, respectively, preventing signal 48 h and SBI 72 h groups. Brain tissues sur- transduction. GRP78 can promote the folding rounding the damaged area were sampled for and assembly of nascent polypeptides, prevent Western blot (WB) and double immunofluores- their misfolding and aggregation, induce prote- cence analysis. asomal degradation of misfolded proteins, and control the initiation of each UPR pathway [11]. In Experiment 2, in order to assess the func- When unfolded and misfolded proteins in the tion of PERK signaling in SBI, 72 rats (72 survi- ER increase in aggregated amounts, GRP78 vors in totally 80 animals) were randomized to dissociates from these three proteins, which four groups using computer-based randomiza- activates them and increases folding capacity tion, including the Sham, SBI, SBI+DMSO, and [12]. Increased GRP78 level has a protective SBI+GSK groups. Euthanasia was performed effect on ischemic nerves [13]. at 24 h post-SBI (according to Experiment 1), and the damaged brain tissues were collected. More and more animal model evidence sug- Neurological tests were carried out in all ani- gests that the UPR is involved in many diffe- mals prior to euthanasia. Tissues inside of the rent diseases. The UPR is gradually becoming injury were evaluated by WB, those obtained an attractive new therapeutic drug target. Di- from the back were used to prepare paraffin seases in which UPR is involved include can- sections. TUNEL and Fluoro-jade C (FJC) stain- cer, metabolic diseases, and cerebral ischemia ing were performed for detecting neuronal [14]. Because it is one of the three main path- apoptosis and necrosis. All assays were strictly ways of the UPR, the PERK-eIF2α pathway has blinded, with specimens coded by an indepen- seen increasingly studied. It has been reported dent investigator. 733 Am J Transl Res 2021;13(2):732-742 Inhibiting PERK pathway alleviates apoptosis Animals (1:500, Abcam), p-eIF2α (1:500, Abcam), ATF4 (1:1000, Abcam) and GAPDH (1:10000, Sigma, Here, totally 120 male Sprague Dawley (SD) USA). The membranes were then incubated rats (300-350 g) were obtained from JOINN with HRP-linked goat anti-rabbit IgG (Invitro- Laboratories, Suzhou, China. Of these, 108 gen, USA) secondary antibodies for 2 h at 4°C. were analyzed. Animal housing was carried out Detection used chemiluminescent substrate under a 12 h/12 h light/dark cycle at 25°C and (Millipore) and a LUMINESCENT IMAGE ANAL- controlled relative humidity, with rodent chow YZER (GE Healthcare Bio-Sciences AB, Swe- and water ad libitum. den). ImageJ (National Institutes of Health, USA) was used to analyze immunoreactive The SBI model bands. SBI modelling was based on a previous report Immunofluorescence [1]. The rats underwent fixation in a stereotac- tic instrument upon intraperitoneal injection of Double immunofluorescence (IF) was carried