SA4503 plays a protective role in post-resuscitation injury by reducing apoptosis caused by mitochondrial dysfunction and endoplasmic reticulum stress through the activation of sigma-1 receptor

Cell culture and OGD/reperfusion (OGD/R) model preparation

HT22 cells [Shanghai Cell Stock Platform (SCSP)-5419, Shanghai, China] were obtained from the Chinese Academy of Sciences cell bank (mycoplasma test results were negative, and short tandem repeats (STR) identification was correct). The cells were cultured in complete Dulbecco’s modified eagle medium (DMEM) (11965092, Gibco, Grand, USA) supplemented with 10% fetal bovine serum (FBS; SV30208.02, HyClone, Utah, USA). The cells were seeded into either 96-well or 6-well plates. On achieving an 80% to 90% confluence attachment rate on the well surface, the cells were deemed ready for experimental procedures. Next, serum-free, glucose-free DMEM (11966025, Gibco, Grand, USA) was preconditioned by rinsing with 95% N₂ for 30 min. This preconditioned medium was then replaced with standard high-glucose DMEM supplemented with 10% FBS. The environmental parameters within the three-gas incubator (RS Biotech, UK) were meticulously adjusted to maintain a hypoxic atmosphere consisting of 0.3% O₂, 95% N2, and 5% CO₂. The cells were subsequently placed in this incubator for 6 h under hypoxic conditions. After the 6-h OGD period was completed, the glucose-free, serum-free DMEM was carefully replaced with high-glucose DMEM supplemented with 10% FBS in a sterile environment. The cells were then transferred to a standard incubator set at 37°C in a 5% CO₂ atmosphere, where they were allowed to recover for 10 h in the presence of oxygen and glucose.

Cell counting kit-8 (CCK-8) assay

HT22 cells were seeded into 96-well plates and randomly allocated into six groups. One group was maintained under standard culture conditions for 16 h as the control group (Control). The remaining five groups were subjected to an OGD/R protocol. Among these, one group received pure water treatment at the onset of OGD and was designated the OGD/R group (vehicle). The other four groups were treated with various concentrations of SA4503 (HY-14813, MCE, New Jersey, USA) solution – 10 μM, 50 μM, 100 μM, or 150 μM – immediately before OGD initiation. After 16 h, the 96-well plates were removed from the cell incubator. In addition to the cell-containing wells, negative control wells filled with high-glucose DMEM supplemented with 10% FBS were prepared. Then, 10 μL of CCK-8 solution (C0038, Beyotime, Shanghai, China) was added to each well, and the plates were returned to the cell culture incubator for 2 h. Following incubation, the optical density (OD) value of each well was measured at a wavelength of 450 nm using an enzyme labeling instrument (Multiskan MK3, Thermo, USA). The OD values of the negative control wells were subtracted to normalize the readings. The cell viability (%) for each group was then calculated using the following formula: OD value of the experimental group – OD value of the negative control group/OD value of the control group – OD value of the negative control group × 100%.

Lactate dehydrogenase (LDH) activity assay

A LDH cytotoxicity assay kit (C0017, Beyotime, Shanghai, China) was used for measurements. Normal HT22 cells in a subset of wells served as the control group. One hour before the scheduled LDH activity assessment, LDH release reagent (10% of the original culture medium volume) was added to the wells, and the plate was returned to the incubator for further cultivation. The cell-free culture wells were supplemented with high-glucose DMEM supplemented with 10% FBS to serve as background negative control wells. On reaching the predetermined time point for each experimental group, the cell culture plate was centrifuged at 500 × g for 5 min using a multiwell plate centrifuge (Shanghai Medical Analysis Instrument Factory, China). Next, 120 μL of the supernatant from each culture well was carefully aspirated and transferred into a new set of 96-well plates, after which 60 μL of the LDH assay working solution was added to each well and thoroughly mixed. The plate was incubated at room temperature in the dark for 30 min. After the incubation period, the 96-well plate was placed in an enzyme-labeled instrument, and the OD was measured at 490 nm. The relative degree of cell damage was calculated using: OD value of assay tube – OD value of background tube/OD value of maximum enzyme activity of cells – OD value of background tube × 100%.

Detection of cell viability by Trypan Blue staining

At the designated time points for each experimental group, the culture medium was carefully aspirated and discarded. Each well was then treated with 1 mL of 0.25% trypsin (25200056, Gibco, Grand, USA) and incubated for approximately 1 min to facilitate cell detachment. The cells were monitored under an inverted microscope (Olympus, Japan), and once observed, 1 mL of DMEM was added to each well to neutralize the trypsin. The adherent cells were then gently dislodged by repeated pipetting to create a single-cell suspension. The cell suspension was adjusted to a concentration of approximately 1 × 10⁵ cells/mL in culture medium. A total of 100 μL of the cell suspension was transferred into 1.5 mL centrifuge tubes, followed by the addition of 100 μL of a 2 × BioReagent solution (ST2780, Beyotime, Shanghai, China). The cells were stained for 3 min. Cell viability was determined using a hemocytometer, and 500 cells were counted per sample. The cell survival rate was calculated as follows: Total number of cells – number of blue-stained cells/total number of cells × 100%.

Apoptosis assay

Apoptosis was detected using an annexin V-fluorescein isothiocyanate (V-FITC) Apoptosis Detection Kit (C1062M; Beyotime, Shanghai, China). The cells were harvested, gently resuspended in phosphate-buffered saline (PBS), and counted to adjust the concentration to 1 × 10⁶ cells/mL. A centrifugation step at 1,000 rpm for 5 min was carried out to pellet the cells, after which the supernatant was discarded. The cell pellet was then resuspended in 195 μL of Annexin V-FITC conjugate, followed by the addition of 5 μL of Annexin V-FITC. The mixture was gently vortexed to ensure uniform labeling and then incubated at room temperature in the dark for 10 min. Another round of centrifugation at 1,000 rpm for 5 min was performed to separate the cells from the excess conjugate, and the supernatant was again discarded. The cells were subsequently resuspended in 190 μL of Annexin V-FITC binding buffer, and 10 μL of propidium iodide staining solution was added. Following gentle mixing, the cells were gently mixed and incubated at room temperature in the dark for an additional 5 min. Finally, the stained cells were analyzed by flow cytometry (AccuriC6, BD Biosciences, USA) within 4 h of staining.

Cellular mitochondrial membrane potential assay

A Mitochondrial Membrane Potential Assay Kit (JC-1) (C2006, Beyotime, Shanghai, China) was used for this assay. The cells were seeded into 35 mm confocal Petri dishes and subjected to OGD for 6 h followed by OGR for 10 h. After the intervention, the culture medium was aspirated, and the cells were gently washed with PBS before being replenished with 1 mL of DMEM. Next, 1 mL of JC-1 staining working solution was added to the cells, and the mixture was thoroughly vortexed to ensure uniform staining. The dishes were incubated at 37°C for 20 min in a cell culture incubator. After incubation, the supernatant was carefully removed, and the cells were washed twice with 1 × JC-1 staining buffer to remove any unbound dye. Subsequently, 1 mL of fresh cell culture medium was added to the dishes. Intracellular red and green fluorescence was subsequently visualized using a laser scanning confocal microscope (LSM) (LSM 710, Zeiss, Germany).

Ca²⁺ concentration assay

A 5 mM stock solution of Fluo-3 AM (S1056, Beyotime, Shanghai, China) was diluted to prepare a 5 μM working solution. For the Rhod-2 (40776ES50, Yeasen, Shanghai, China) Ca²⁺ detection solution, 50 μg was dissolved in 100 μL of dimethyl sulfoxide to generate a 0.5 mM stock solution. Following 6 h of OGD and 10 h of OGR, the culture medium was aspirated, and the cells were rinsed once with PBS. The medium was then replenished with 1 mL of DMEM. The Fluo-3 AM or Rhod-2 assay mixture was added to the wells, and the mixture was thoroughly mixed before the plate was placed back into a 37°C, 5% CO₂ incubator for a 30 min incubation period. The cells were washed twice with PBS to remove any unbound dye. The cells were then digested and transferred into centrifuge tubes, followed by centrifugation at 1,000 rpm for 5 min. The cell pellet was gently washed with PBS, resuspended in 200 μL of PBS, and analyzed by flow cytometry. The fluorescence was detected at an excitation wavelength of 552 nm and an emission wavelength of 581 nm.

Mitochondrial separation

Cellular mitochondria were extracted using a mitochondrial isolation kit (C3601; Beyotime, Shanghai, China). The cells were harvested and centrifuged at 1,000 rpm for 5 min, after which the supernatant was discarded. The cell pellet was resuspended in precooled PBS, followed by a second centrifugation step at 600 × g for 5 min, with subsequent removal of the supernatant. Next, 2 mL of precooled mitochondrial isolation reagent (supplemented with 1 mM phenylmethylsulfonyl fluoride [PMSF] immediately before use) was added to gently resuspend the cells. The cell concentration was adjusted to 2 × 10⁷ cells/mL. The single-cell suspensions were transferred to a Dounce homogenizer (Kimble Konte, Fisher Scientific, USA), and vertical homogenization was performed for 20–30 strokes. To monitor homogenization efficiency, after approximately 10 strokes, a small sample of approximately 2 μL of the cell homogenate was collected and mixed with 30–50 μL of Trypan Blue staining solution. If <50% of the cells appeared blue, homogenization was continued for an additional five strokes, and the samples were reevaluated. If ≥50% of the cells were blue, homogenization was stopped. Following completion, the homogenate was centrifuged at 600 × g for 10 min at 4°C. The supernatant was carefully transferred to a new tube and centrifuged again at 11,000 × g for 10 min at 4°C to precipitate the mitochondria. Next, 150 μL of mitochondrial storage solution was added, and the mixture was set aside on ice.

Mitochondrial respiratory function assay

Mitochondrial respiratory function was assessed using a respiratory control ratio (RCR) assay kit (GMS10097, GENMED, Shanghai, China). A total of 2.5 mL of medium solution (Reagent A) was added to a glass reaction chamber. A mini magnetic rotor was activated to stir the solution thoroughly and eliminate all the air bubbles. Once the bubbles were expelled, the chamber was sealed. After the oxygen concentration curve stabilized, the baseline plateau was recorded for 1 min. Twenty microliters of fresh mitochondrial suspension (equivalent to approximately 2 mg protein) was injected into the chamber, causing a slight immediate decrease in the oxygen concentration. The curve was recorded for 1 min until stable, followed by the addition of state IV substrate solution (Reagent B) to initiate state IV respiration, which was characterized by a slow decline in the oxygen concentration. After the curve plateaued, state III substrate solution (Reagent C) was added to induce state III respiration, which was marked by a rapid decrease in oxygen within 10 seconds. The recording continued until an inflection point appeared in the linear downward slope. The state III and state IV respiration rates were calculated as the oxygen concentration decreased per unit time (min) from the average of the 4 time points. The RCR was determined as the ratio of the state III respiration rate to the state IV respiration rate.

Experimental animal and housing conditions

All animal procedures were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals formulated by the Ministry of Science and Technology of the People’s Republic of China. This study was approved by the Animal Care and Use Committee of Kunming Medical University (permit number: Kmmu20211316). Healthy male Sprague-Dawley (SD) rats, aged 8–12 weeks, with body weights ranging from 350 g to 410 g, were provided by the Experimental Animal Center of Kunming Medical University. The animals were housed in an environment with a temperature of 21°C, humidity of 40%, and a 12-h light cycle followed by a 12-h dark cycle and had ad libitum access to food and water, with five rats per cage. SA4503 was dissolved in normal saline at a concentration of 1 or 5 mg/kg (mg/kg) and administered through intravenous (IV) injection. This experiment strictly followed the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.

Preparation of a rat model for resuscitation after cardiac arrest

All the rats were fasted overnight with free access to water before the experiment. Anesthesia was induced by intraperitoneal injection of pentobarbital sodium (45 mg/kg). In accordance with previous methods,^[9,10] the chest and inguinal regions were shaved, and a 23-gauge catheter polyethylene-50 (PE-50) was inserted into the left femoral artery to measure the mean arterial pressure (MAP). Another 23-gauge catheter (PE-50) was inserted into the left femoral vein for intravenous fluid administration. The end-tidal partial pressure of carbon dioxide (PETCO₂) was measured using a sidestream infrared CO₂ analyzer (CAPSTAR-100, CWE Inc., Ardmore, PA, USA), which was placed between the endotracheal tube and the ventilator. Hemodynamic parameters, including the MAP and lead II electrocardiogram, were continuously monitored using the WinDaq data acquisition system (DataQ, Akron, OH, USA).

The rectal core temperature was monitored with a heating lamp and maintained at 36.5 ± 0.5°C. Cardiac arrest was induced by clamping the endotracheal tube to cause asphyxial arrest, with successful induction recorded as a MAP <20 mmHg. After 4 min, precordial compression and 100% FiO₂ mechanical ventilation were initiated. Precordial compressions were performed at a rate of 250/min to achieve a compression-to-ventilation ratio of 5:1. Epinephrine (20 μg/kg) was injected 2 min after CPR onset. Return of spontaneous circulation (ROSC) was defined as the recovery of supraventricular rhythm with a MAP >60 mmHg sustained for 10 min. After 4 h of monitoring, the intravascular catheters and endotracheal tube were removed, the surgical wounds were sutured, and the animals were returned to their cages. Rats that failed to achieve ROSC were excluded from the data analysis.

Assignment of experimental groups after CPR

After ROSC was achieved, the animals were randomized into four groups. (1) CPR group (n = 10): Rats received an intravenous injection of 0.9% normal saline (1 mL/kg) within 10 min after ROSC. (2) Low-dose SA4503 group (n = 10): Rats were intravenously infused with 1 mg/kg SA4503. (3) High-dose SA4503 group (n = 10): Rats were intravenously infused with 5 mg/kg SA4503. (4) Control group (n = 8): Rats underwent the same anesthetic and surgical procedures but were not subjected to asphyxia, cardiac arrest, or CPR. Neurobehavioral assessments and magnetic resonance imaging of the rat brain were conducted 24 h after ROSC for each group of rats. The rats in each group were euthanized by the injection of sodium pentobarbital (120 mg/kg) at the end of the experiment.

Western blot

Cell lysis was performed on six-well cell culture plates by adding 70 μL of radio immunoprecipitation assay (RIPA) lysis buffer (P0013C; Beyotime, Shanghai, China) supplemented with PMSF (ST505; Beyotime, Shanghai, China) to each well. The cells were lysed for 30 min on ice, followed by centrifugation at 4°C and 12,000 × g for 10 min. The supernatant was carefully transferred to a presterilized centrifuge tube. To quantify the protein concentration, a portion of the supernatant was assayed using a bicinchoninic acid (BCA) protein concentration assay kit (P0010S, Beyotime, Shanghai, China). Subsequently, 50 μg of protein was mixed with 4 × loading buffer (PR20003, Beyotime, Shanghai, China) and heated at 95°C for 10 min for denaturation. The protein samples were then separated by SDS‒PAGE according to the Laemmli method^[11] and transferred onto nitrocellulose membranes. Immunoblotting was carried out using specific antibodies against the Sig-1R (ab307548; Abcam, Cambridge, England) and cleaved caspase-12 (ab62484; Abcam, Cambridge, England). Specific antibodies against GPR78 (11587-1-AP, Proteintech, Wuhan, China), CHOP (AC532, Beyotime, Shanghai, China), and cleaved caspase-3 (AC033, Beyotime, Shanghai, China) were used. To ensure equal protein loading, an anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (BM3874; Boster Biotech, Wuhan, China) was used as a loading control. For detection, HRP-conjugated secondary antibodies, including goat anti-rabbit (BA1054, Boster Biotech, Wuhan, China) and goat anti-mouse (BA1050, Boster Biotech, Wuhan, China) antibodies, were utilized. Immunoreactive bands were visualized using a chemiluminescence kit (P0018S, Beyotime, Shanghai, China) and captured on film. Relative protein expression was analyzed and quantified with ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Statistical analysis

Statistical analyses were conducted using the Statistical Package for the Social Sciences (SPSS) 20.0 statistical software (SPSS Inc., Chicago, IL). Continuous variables are reported as the means ± standard deviations (means ± SDs), whereas non-normally distributed data are presented as medians with interquartile ranges. For multiple group comparisons, one-way analysis of variance was used, and for pairwise comparisons between two groups, the t-test was used. Statistical significance was set at P < 0.05.

Physiological parameters and hemodynamic changes in rats after CPR

Following successful modeling in SD rats according to the experimental protocol, hemodynamic levels and various physiological parameters were monitored within 4 h post-resuscitation for the control group, CPR group, low-dose SA4503-treated CPR group, and high-dose SA4503-treated CPR group. The experimental results revealed that there were no statistically significant differences in heart rate, body temperature, MAP, PETCO₂, arterial blood partial pressure of oxygen (PaO₂), PaCO₂, pH, or lactate levels between the CPR group and the SA4503-treated groups compared with the control group. After CPR, a significant decrease in MAP was observed in the SA4503-treated groups compared with both the control and CPR groups. Notably, during the 4-h post-resuscitation monitoring period, the MAP in the SA4503-treated rats was maintained above 100 mmHg, and there was no statistically significant difference in the MAP between the SA4503 low-dose and SA4503 high-dose groups [Table 1]. These results indicate that SA4503 treatment does not have a significant adverse effect on physiological stability or hemodynamics after CPR in rats.

Treatment with SA4503 reduces neurological impairment in rats that have been resuscitated from cardiac arrest

Neurologic deficit scores (NDSs) were evaluated using the scale developed by Guo et al.^[12] to evaluate post-resuscitation neurologic function in rats 24 h after ROSC. The scoring was performed by trained professionals. The NDS criteria primarily include consciousness, motor function, brainstem function, somatosensory function, balance response, and the occurrence of seizures. The scoring range is from 0 to 80, where 0 corresponds to brain death and 80 corresponds to normal brain function. The detailed scoring is presented in Table 2.

Compared with those in the control group, the brain function of the rats in the CPR group, low-dose SA4503 group, and high-dose SA4503 group was significantly impaired. At 24 h post-resuscitation, the NDS scores of the rats treated with high-dose SA4503 (5 mg/kg) significantly improved, reflecting enhanced neurological function relative to those of the CPR group. At 24 h post-resuscitation, there was no significant difference in the NDS score between the SA4503 low-dose treatment group and the CPR group [Figure 1a].

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Figure 1:

SA4503 mitigates neurological deficits in rats following cardiopulmonary resuscitation (CPR). (a), NDS statistics of the rats in each group after CPR. (b), The levels of S-100B in the serum were quantified by ELISA. (c), MR image of the rat brain. n=6, ^✶indicates a comparison with the control group, P<0.05; # indicates a comparison with the CPR group, P<0.05. Control: Rats without asphyxiation, cardiac arrest or CPR; CPR after cardiac arrest in rats; SA4503 low-dose: Rats were treated with low doses of SA4503 after CPR; SA4503 high-dose: Rats were treated with high doses of SA4503 after CPR. NDS: Neurologic deficit score, ELISA: Enzyme-linked immunosorbent assay.

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S100 calcium-binding protein B (S-100B), a calcium-binding protein secreted by astrocytes, serves as a serum biomarker for brain injury to evaluate the prognosis of cardiac arrest patients. S-100B is derived primarily from brain tissue and remains unaffected by other exogenous medications.^[13] Studies have shown that in brain injury models following CPR, elevated levels of the S-100B protein may reflect the activation of neuroglial cells and the extent of secondary brain injury.^[14] To assess the effects of SA4503 on brain tissue in CPR rats, changes in S-100B protein levels were measured. At 24 h post-resuscitation, the serum S-100B levels in the CPR group, SA4503 low-dose group, and SA4503 high-dose group were elevated compared with those in the control group. The serum S-100B levels in the SA4503 high-dose group were lower than those in the CPR group and the SA4503 low-dose group, whereas no significant difference was detected between the low-dose SA4503 group and the CPR group [Figure 1b]. Compared with the control group, the CPR group, low-dose SA4503 group, and high-dose SA4503 group presented scattered high-intensity signals in the cerebral cortex and hippocampal regions on T1-weighted images, suggesting the presence of ischemic injury foci. The high-signal intensities in the cerebral cortex and hippocampal regions on T1-weighted images were less pronounced in the SA4503 high-dose group than in the CPR group and the SA4503 low-dose group, indicating milder ischemic injury. Conversely, there was no significant difference in the high-signal intensities between the SA4503 low-dose group and the CPR group in the cerebral cortex and hippocampal regions on T1-weighted images [Figure 1c]. These results suggest that only high doses of SA4503 have a protective effect on brain damage after CPR in rats.

SA4503 attenuates OGD/R-induced neuronal cell death and apoptosis through the activation of Sig-1R

To investigate the role of Sig-1R in cerebral injury following CPR, we prepared an OGD/R-induced neuronal cell model using HT22 cells. These cells were subjected to OGD/R and treated with the Sig-1R agonist SA4503. We assessed cellular activity and damage, and the findings revealed a marked decrease in neuronal cell viability and a significant increase in cellular damage compared with those of the control group. Treatment of OGD/R-exposed neuronal cells with various concentrations of SA4503 demonstrated that SA4503 dose-dependently improved cell viability and attenuated injury, with the most robust effect observed at 100 μM SA4503 [Figure 2a and b]. To determine the role of Sig-1R in neuronal cells, we used Sig-1R agonists and antagonists for the treatment of OGD/R-induced HT22 cells. The results indicated a significant increase in cell viability after treatment with SA4503, which was subsequently reduced after antagonist treatment [Figure 2c]. Western blot analysis revealed that OGD/R decreased Sig-1R protein expression, which was restored to near-control levels by SA4503. In contrast, incubation with the antagonist BD1063 led to a marked downregulation of Sig-1R expression compared with that in the cells treated with the agonist [Figure 2d and e]. Flow cytometry analysis of apoptosis revealed that SA4503 significantly reduced OGD/R-induced apoptosis, whereas the antagonist BD1063 had the opposite effect [Figure 2f-k]. The results of these experiments confirmed that SA4503 dose-dependently exerted protective effects on cells after OGD/R in vitro, with 100 μM being the optimal SA4503 concentration. Sig-1R may be the key mediator of the protective effect of SA4503 on neuronal cells.

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Figure 2:

SA4503 attenuates OGD/R-induced HT22 cell death and apoptosis by activating sigma-1 receptor (Sig-1R). (a) CCK-8 assay for assessing cellular activity. (b) LDH levels were detected in cells to assess injury. (c) Cell viability was determined by Trypan Blue staining. (d and e), The protein expression of Sig-1R in HT22 cells from different treatment groups was detected by Western blotting, and the blot bands were analyzed on a grayscale using ImageJ software with GAPDH as the internal reference protein. (f-k), Apoptosis was detected using flow cytometry in various treatment groups, and the results were statistically analyzed (n=5, ^✶P<0.05, vs. the control group; ^#P<0.05, vs. the vehicle group). OGD/R: Oxygen-glucose deprivation/reperfusion, LDH: Lactate dehydrogenase, GAPDH: Glyceraldehyde-3-phosphate dehydrogenase, CCK-8: Cell counting kit-8.

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SA4503 plays a protective role in OGD/R-induced mitochondrial dysfunction and ER stress in neuronal cells

To investigate the mechanism of Sig-1R activation in neuronal cell death and apoptosis, we examined the functions of mitochondria and the ER in HT22 cells. The results revealed that OGD/R significantly decreased the mitochondrial membrane potential, but this effect was attenuated by the Sig-1R agonist SA4503. Cotreatment with the antagonist BD1063 reversed the protective effect of SA4503, causing a further marked decrease in the membrane potential [Figure 3a and b]. Flow cytometry analysis of the intracellular and mitochondrial calcium levels revealed that SA4503 modulated the release of mitochondrial calcium into the cytoplasm to reduce the intracellular calcium level. In contrast, treatment with BD1063 resulted in a significant increase in the intracellular calcium ion concentration and a significant decrease in the mitochondrial calcium level [Figure 3c-n].

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Figure 3:

SA4503 attenuates OGD/R-induced mitochondrial dysfunction in HT22 cells. (a), Representative images of the cellular mitochondrial membrane potential acquired using laser confocal microscopy. Red fluorescence represents the JC-1 polymer, and green fluorescence represents the JC-1 monomer (Scale bar = 5 μm). (b), Reduction in the mitochondrial membrane potential in each group. (c-h), Detection of the intracellular calcium ion content by flow cytometry. (i-n) Mitochondrial calcium content detected by flow cytometry (n=5, ^✶P<0.05, vs. the control group; ^#P<0.05, vs. the vehicle group). OGD/R: Oxygen-glucose deprivation/reperfusion.

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In addition, the mitochondrial respiratory chain and control rates were analyzed, revealing that SA4503 significantly enhanced mitochondrial respiratory function. However, this function was disrupted again following treatment with BD1063 [Figure 4a-c].

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Figure 4:

SA4503 improves mitochondrial respiration. (a-c), The mitochondrial state III respiration rate (a) and the mitochondrial state IV respiration rate (b) were examined, as were the respiratory control rates (c) in each group. (n=5, ^✶P<0.05, vs. the control group; # P<0.05, vs. the vehicle group).

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Next, we validated ER stress- and apoptosis-related proteins using Western blot analysis [Figure 5a]. The results indicated that OGD/R significantly increased the protein levels of the ER stress protein CHOP, as well as the active forms of apoptosis-associated caspase 12 and caspase 3. Conversely, the protein level of GPR78, which is crucial for maintaining ER homeostasis, was significantly decreased. Treatment with SA4503 reversed these changes in protein expression. In addition, cotreatment with SA4503 and BD1063 resulted in further reversal of protein expression [Figure 5b-e]. The above results reveal that the protective effect of SA4503 against OGD/R-induced neuronal cell injury may be mediated by attenuating mitochondrial dysfunction and ER stress.

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Figure 5:

Changes in proteins related to ER stress. (a) The ER stress-related proteins CHOP and GPR78 and the proapoptotic proteins cleaved caspase 12 and cleaved caspase 3 were detected by Western blotting, and GAPDH was used as an internal reference. (b-e), Relative quantitative analyses of the protein levels of CHOP, GPR78, cleaved caspase 12 and cleaved caspase 3 (n=5, ^✶P<0.05, vs. the control group; ^#P<0.05, vs. the vehicle group). ER: Endoplasmic reticulum, CHOP: C/EBP-homologous protein, GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

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