Neuroprotective effect of a novel brain-derived peptide, HIBDAP, against oxygen-glucose deprivation through inhibition of apoptosis in PC12 cells

The effect of a novel brain-derived peptide, hypoxic-ischemic brain damage associated peptide (HIBDAP), on apoptosis after oxygen-glucose deprivation (OGD) in PC12 cells was investigated. The HIBDAP sequence (HSQFIGYPITLFVEKER) was coupled with the carrier peptide of the transactivator of transcription (TAT) sequence (YGRKKRRQRRR). FITC-labelled TAT-HIBDAP was observed by fluorescence microscopy. After TAT-HIBDAP treatment and OGD treatment, the PC12 cell apoptosis rate was analysed using lactate dehydrogenase (LDH) leakage and Annexin V-fluorescein isothiocyanate (FITC) assays. Mitochondrial membrane potential (ΔΨm) was examined by fluorescence microscopy. Protein expression of apoptotic factors was examined by Western blotting. FITC-labelled TAT-HIBDAP entered the PC12 cell nucleus. Compared with the OGD group, TAT-HIBDAP at low concentrations (1 µM, 5 µM, 10 µM) significantly reduced the apoptosis rate of PC12 cells (except at 20 µM); 5 µM TAT-HIBDAP had the most obvious effect. There were remarkable increases in ΔΨm at different concentrations (1 µM, 5 µM, 10 µM, 20 µM) of TAT-HIBDAP pretreatment, and 5 µM TAT-HIBDAP also had the most obvious effect. TAT-HIBDAP reversed the increased ratio of Bax/Bcl-2 and activation of Caspase-3 induced by OGD. TAT-HIBDAP is resistant to OGD-induced PC12 cell apoptosis by regulating the Bax/Bcl-2/Caspase-3 pathway, which may provide a novel therapeutic strategy for neonatal HIBD.


Introduction
Hypoxic-ischemic brain damage (HIBD) in newborns caused by perinatal asphyxia is a major cause of neonatal disability and death. Until now, there has been no specific treatment for neonatal HIBD except hypothermia therapy [1]. Despite the use of hypothermia therapy, some neonates with severe perinatal asphyxia still suffer serious dysfunctional consequences, including cerebral palsies, intellectual disabilities, cognitive disorders and other permanent nerve damage sequelae [2]. Therefore, it is urgent to continue to find new safe and effective treatments.
Recently, peptides have been widely studied because they represent an increasingly popular class of therapeutic agents [3], especially in the fields of diabetes treatment [4] and tumours [5]. Compared to traditional drugs, peptide drugs have several beneficial properties, such as less toxicity, accumulation in tissue and drug-drug interactions, and high biological and structural diversity [6]. Our previous study showed that the levels of a 2671.5 Da peptide (HSQFI-GYPITLFVEKER) were decreased in the cerebrospinal fluid of neonatal HIBD patients, and we named this peptide Hypoxic-Ischemic Brain Damage Associated Peptide (HIBDAP) [7]. HIBDAP is derived from heat shock protein Chenhong Jiang and Yina Hu have contributed equally to this work.
In this paper, we describe the effect of HIBDAP on neuronal cell apoptosis after HI in vitro, which may provide a novel therapeutic strategy for neonatal HIBD.

Materials and methods
The peptides HIBDAP consists of 17 amino acids (HSQFIGYP-ITLFVEKER) and corresponds to the 210 to 226 aa positions of HSP90α/HSP90AA1. The transactivator of transcription (TAT) sequence (YGRKKRRQRRR), a carrier peptide, was attached to the N-terminal end of HIBDAP. TAT-HIBDAP was dissolved to different concentrations (1 µM, 5 µM, 10 µM, and 20 µM) using double-distilled water. FITC-tagged TAT-HIBDAP was used at a concentration of 20 µM and coincubated with PC12 cells (American Type Culture Collection, USA) for 30 min. All peptides (purity > 95%) were synthesized by Shanghai Science Peptide Biological Technology.

Cell culture and treatment
PC12 cells were cultured in a T25 cell culture flask at a density of 2 × 10 4 /mL using RPMI 1640 (Invitrogen Life Technologies, USA) culture medium containing 10% horse serum (Invitrogen Life Technologies, USA), 5% foetal bovine serum (Invitrogen Life Technologies, USA) and appropriate antibiotics in 5% CO 2 at 37 °C. Cells were switched to RPMI 1640 medium without glucose for oxygen and glucose deprivation (OGD), and TAT-HIBDAP was added. After 1 h, the cells were placed in 93% N 2 , 5% CO 2 and 2% O 2 at 37 °C for 6 h. The controls were incubated in a normoxic incubator for the same time in glucose-containing RPMI 1640 medium.

Lactate dehydrogenase (LDH) leakage assay
Immediately after 6 h of OGD, the LDH leakage assay was performed using an LDH detection kit (Beyotime, China) following the manufacturer's instructions. Cells were switched into a 96-well plate at a concentration of 1 × 10 4 cells/well and then treated according to different experimental groups. Next, the supernatant (120 µL) was transferred to another 96-well plate, and 60 µL of freshly prepared reaction mixture was added. The absorbance was measured at 490 nm on a microplate reader after incubation at room temperature for 30 min in the dark.

Annexin V-fluorescein isothiocyanate (FITC) assay
Immediately after 6 h of OGD, the loss of membrane integrity was measured using the Annexin V-FITC kit (BD Biosciences, USA). Cells were resuspended in 1× binding buffer to a density of 1 × 10 6 cells/mL. Then, 100 µL of the cell mixture was stained with 5 µL propidium iodide (PI) and 5 µL Annexin V-FITC. After 15 min of incubation at room temperature in the dark, 400 µL of 1× binding buffer was added to each tube. Cells were distinguished and analysed on a FACSCalibur Flow Cytometer (BD Biosciences, USA) and with FlowJo software (Tree Star Corp, OR).

Mitochondrial membrane potential (ΔΨm) assay
Immediately after 6 h of OGD, ΔΨm was measured with a mitochondrial membrane potential assay kit (Beyotime Institute of Biotechnology, China). After resuspension in PBS, cells (5 × 10 5 ) were incubated with 10 µg/mL JC-1 for 10 min in the dark at 37 °C and immediately analysed on a FACSCalibur flow cytometer. The value of ΔΨm is expressed as the ratio of red (590 nm) to green (525 nm) fluorescence intensity.

Western blot analysis
Immediately after 6 h of OGD, cells were ultrasonically homogenized in RIPA buffer containing protease inhibitor cocktail. After protein concentration was quantified with the BCA protein assay kit (Pierce, USA), samples were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, USA). Then, the membranes were blocked and subsequently incubated with antibodies against β-actin (Cell Signaling Technology, USA), Bax (Abcam, UK), Bcl-2 (Abcam, UK), and Caspase-3 (Santa Cruz, USA). Membranes were washed and incubated with goat anti-rabbit IgG H&L (Abcam, UK) or horseradish peroxidase-conjugated anti-mouse IgG (Abcam, UK).

Statistical analysis
Statistical analysis was performed using the SPSS 22.0 statistical software package. All values are expressed as the mean ± SEM. Quantitative analysis was performed by Student's paired t test or one-way ANOVA. Statistical significance was determined based on P values < 0.05.

The subcellular localization of TAT-HIBDAP
HIBDAP is a hydrophilic peptide and does not easily enter cells. To increase the delivery efficiency of this peptide into cells, HIBDAP was coupled to the TAT protein, which is a transduction peptide. Through microscopic fluorescence, we determined that TAT-HIBDAP entered the cell nucleus ( Fig. 1).

Effects of HIBDAP on the apoptosis rate of PC12 cells under OGD
We measured the cell apoptosis rate under OGD using the LDH leakage assay and Annexin V-FITC assay. Compared to the control group (NC), the cell apoptosis rate of the OGD group was significantly upregulated in PC12 cells after 6 h of OGD, as shown by the results of the LDH leakage assay (Fig. 2) and Annexin V-FITC assay (Fig. 3). Different concentrations of TAT-HIBDAP (1 µM, 5 µM, 10 µM, 20 µM) were applied. Concentrations of 1 µM, 5 µM and 10 µM TAT-HIBDAP significantly reduced the cell apoptosis rate, and 5 µM TAT-HIBDAP had the most obvious effect. However, compared to the OGD group, 20 µM TAT-HIB-DAP significantly increased cell death due to drug toxicity (*P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001).

Effects of HIBDAP on the ΔΨm of PC12 cells under OGD
As shown in Fig. 4, the red fluorescence intensity in the OGD group was weaker than that in the control group (NC), which indicated that the ΔΨm in the OGD group was lower than that in the NC group. There were remarkable increases in ΔΨm after treatment with different concentrations of TAT-HIBDAP (1 µM, 5 µM, 10 µM, and 20 µM), and 5 µM TAT-HIBDAP again had the most obvious effect (**P < 0.01; ***P < 0.005).

Effects of HIBDAP on Bax/Bcl-2/Caspase-3 expression in PC12 cells under OGD
Western blotting was used to detect the protein expression of proteins involved in the apoptotic signalling pathway (Bax/ Bcl-2/Caspase-3) in PC12 cells treated with 5 µM TAT-HIB-DAP under OGD. Compared to the control group, Bax and Caspase-3 were upregulated and Bcl-2 was downregulated  assay. After 6 h of OGD treatment, the cell apoptosis rate of the OGD group was significantly increased compared to that of the control group (NC). Compared to the OGD group, the cell apoptosis rate was significantly decreased in PC12 cells treated with 1 µM, 5 µM, and 10 µM TAT-HIBDAP; however, that of the 20 µM group was significantly increased (****P < 0.001). All experiments were independently repeated three times in the OGD group. However, after pretreatment with TAT-HIBDAP, the expression levels of Bax and Caspase-3 were decreased, and the expression level of Bcl-2 was increased. In addition, the ratio of Bax/Bcl-2 was significantly increased in the OGD group, and TAT-HIBDAP pretreatment significantly decreased this ratio (Fig. 5).

Discussion
In this study, we highlighted HIBDAP as a promising focus and therapeutic target for neonatal HIBD by inhibiting cell apoptotic pathways. Our work provided several new findings, as follows: (1)  Most peptides are unable to enter cells, and the delivery of peptides into cells can be achieved by using TAT. TAT is a carrier molecule that delivers neuroprotective peptides into cells. For instance, in a model of HIE, the NBD peptide (TALDWSWLQTE) fused to TAT (TAT-NBD) was neuroprotective until 6 weeks after HI by upregulating p53 and activating Caspase-3 [10] and significantly improved neurobehavioural development until 14 weeks after HI [11]. TAT-mGluR1 (YGRKKRRQRRR-VIKPLTKSYQGSGK) administration significantly reduced cerebral infarction in P7 rats with HIE [12]. In another study using the P7 rat model of HIE, TAT-BH4 (YGRKKRRQRRR-RTGYDNREIVM-KYIHYKLSQRGYEW) peptide administered intracerebroventricularly 30 min before hypoxia significantly reduced Caspase-3 activation and cerebral tissue damage [13]. Similarly, HIBDAP is a hydrophilic peptide and must be coupled to cell-penetrating peptides for delivery into cells. Therefore, we coupled HIBDAP to the TAT protein sequence, and the microscopic fluorescence results confirmed that TAT-HIB-DAP successfully entered cells.
Cell apoptosis is more common in immature brains than in adult brains [14]. In neonatal HIBD patients, neuronal apoptosis is an important pathological trigger of cerebral injury [15,16]. HIBD often leads to increased neuronal apoptosis in neonates. Therefore, one of the crucial processes to induce neuronal rescue is to alleviate apoptosis. Inhibiting neuronal apoptosis is a promising therapeutic target for neuronal function restoration in neonatal HIBD patients [17]. Some studies on peptides have focused on apoptosis as a therapeutic strategy for neonatal HIBD. Intraperitoneal injection with D-JNKi, which is a small peptide c-Jun N-terminal kinase (JNK) MAP kinase inhibitor, strongly reduced the levels of apoptotic markers and increased the levels of antiapoptotic proteins in a neonatal HIBD rat model [18]. Intracerebroventricular infusion of the TAT-BH4 fusion peptide enabled it to enter neuron bodies and protect neuronal cells from apoptosis in immature HIBD rats [13]. The results of the LDH leakage assay (Fig. 2) and Annexin V-FITC assay (Fig. 3) demonstrated that TAT-HIBDAP significantly reduced the apoptosis rate of PC12 cells under OGD. The maintenance of intact ΔΨm is critical for cell survival, and the loss of ΔΨm triggers a Fig. 3 Annexin V-FITC assay. After 6 h of OGD treatment, the cell apoptosis rate of the OGD group was significantly increased compared to that of the NC group. Compared to the OGD group, TAT-HIBDAP at moderate concentrations (1 µM, 5 µM, 10 µM) significantly reduced the apoptosis rate of PC12 cells, while 20 µM significantly increased the apoptosis rate of PC12 cells (*P < 0.05; **P < 0.01; ***P < 0.005). All experiments were independently repeated three times series of reactions leading to apoptosis [19]. The decline in ΔΨm is an early indicator of apoptosis [20,21]. When the mitochondrion is damaged, the numbers of J-aggregates showing red fluorescence are reduced, and the numbers of JC-1 monomers showing green fluorescence are increased. Hence, the fluorescence intensity ratio (red/green) is negatively correlated with mitochondrial damage. The mitochondria were significantly damaged under OGD, and all concentrations of TAT-HIBDAP (1 µM, 5 µM, 10 µM, 20 µM) were effective in stabilizing ΔΨm, especially 5 µM (Fig. 4). Thus, it is reasonable to assume that TAT-HIB-DAP may have a neuroprotective effect against neonatal HIBD by reducing neuronal apoptosis and could provide a novel intervention strategy for HI-induced brain injury.
The balance of pro-and anti-apoptotic proteins (Bax/ Bcl-2) is normally metastable in organisms and is a critical factor in cell apoptosis [22][23][24][25][26][27]. Our results showed that the expression of Bax was increased, the expression of Bcl-2 was decreased, and the Bax/Bcl-2 ratio was obviously upregulated after OGD. After TAT-HIBDAP treatment, the Fig. 4 Mitochondrial membrane potential assay. After 6 h of OGD treatment, the mean fluorescence intensity ratio (red/green fluorescence) was significantly decreased in the OGD group compared to the NC group. Compared to the OGD group, the mean fluorescence intensity ratio was significantly increased in cells treated with dif-ferent concentrations (1 µM, 5 µM, 10 µM, and 20 µM) of TAT-HIBDAP, and 5 µM TAT-HIBDAP had the most obvious effect (**P < 0.01; ***P < 0.005). All experiments were independently repeated three times Fig. 5 Western blotting. After 6 h of OGD treatment, compared to the NC group, Bax expression, Caspase-3 expression and the Bax/ Bcl-2 expression ratio were significantly increased and Bcl-2 expression was significantly decreased in the OGD group. TAT-HIBDAP treatment (OGD + 5 µM) significantly reversed their expression patterns (**P < 0.01; ***P < 0.005). All experiments were independently repeated three times expression of Bax was decreased, the expression of Bcl-2 was increased, and the ratio of Bax/Bcl-2 was significantly decreased. Caspase-3 induces apoptosis and participates in the final execution phase of apoptosis [28,29]. In this study, the expression of Caspase-3 was significantly increased after OGD and significantly decreased after TAT-HIBDAP treatment compared to the control. Our results demonstrated that TAT-HIBDAP could rescue the increased Bax/Bcl-2 ratio and Caspase-3 activation caused by OGD, which suggests that TAT-HIBDAP is resistant to OGD-induced PC12 cell apoptosis through the Bax/Bcl-2/Caspase-3 pathway.
Normally, the toxicity of HIBDAP increases with concentrations similar to those of other peptide drugs. Our results demonstrated that TAT-HIBDAP at moderate concentrations (1 µM, 5 µM, 10 µM) has therapeutic efficacy, while the higher apoptosis rate observed after treatment with 20 µM (relative to that of the OGD group) may be due to its toxicity. Thus, for its effective clinical application, further animal studies and preclinical and clinical trials are needed to determine a moderate concentration of TAT-HIBDAP with lower toxicity and higher efficacy.
Moreover, TAT-HIBDAP is an arginine-rich cationic peptide (CARP), which has been increasingly applied in perinatal HIBD in recent years [30]. CARPs are an expanding and relatively novel class of compounds that possess intrinsic neuroprotective properties. TAT-HIBDAP may also have intrinsic neuroprotective properties, and some other CARPs may as well [31]. CARPs may act via multiple, potentially simultaneous mechanisms, such as traversing cell membranes, entering the CNS, antagonizing calcium influx, targeting mitochondria, stabilizing proteins, inhibiting proteolytic enzymes, inducing pro-survival signalling, scavenging toxic molecules, and reducing oxidative stress [32]. Whether TAT-HIBDAP exerts a neuroprotective effect through one of these mechanisms needs further investigation.
Here, we conducted a preliminary functional study on a novel peptide to provide new ideas for the development of drugs to treat neonatal HIBD. Our study showed that TAT-HIBDAP may protect against OGD-induced PC12 cell apoptosis through the Bax/Bcl-2/Caspase-3 pathway. TAT-HIBDAP could be a potential drug and provide a guide for future fundamental research and possible clinical therapeutic strategies in neonatal HIBD. All these routes have great potential in the future, although there are still many problems and challenges that need to be overcome.