ASK1 Inhibitor NQDI‑1 Decreases Oxidative Stress And Neuroapoptosis Via The ASK1/p38 And JNK Signaling Pathway in Early Brain Injury After Subarachnoid Hemorrhage in Rats Part 1

Abstract. Oxidative stress and neuroapoptosis are key pathological processes after subarachnoid hemorrhage (SAH). The present study evaluated the anti‑oxidation and anti‑apoptotic neuroprotective effects of the apoptosis signal‑regulating kinase 1 (ASK1) inhib‑  itor ethyl‑2,7‑dioxo‑2,7‑dihydro‑3H‑naphtho(1,2,3‑de)  quinoline1‑carboxylate (NQDI‑1) in early brain injury (EBI)   following SAH in a rat model. A total of 191 rats were used and the SAH model was induced using monofilament perforation. Western blotting was subsequently used to detect the endogenous expression levels of proteins. Immunofluorescence was then used to confirm the nerve cellular localization of ASK1. Short‑term neurological function was assessed using the modified Garcia scores and the beam balance test 24h after SAH, whereas long‑term neurological function was assessed using the rotarod test and the Morris water maze test. Apoptosis of neurons was assessed by TUNEL staining and oxidative stress was assessed by dihydroethidium staining 24 h after SAH. The protein expression levels of phosphorylated (p‑)ASK1 and ASK1 rose following SAH. NQDI‑1 was intracerebroventricularly injected 1h after SAH and demonstrated significant improvements in both short and long‑term neurological function and significantly reduced oxidative stress and neuronal apoptosis. Injection of NQDI‑1 caused a significant decrease in protein expression levels of p‑ASK1, p‑p38, p‑JNK, 4 hydroxynonenal, and Bax and significantly increased the protein expression levels of heme oxygenase 1 and Bcl‑2. The use of the p38 inhibitor BMS‑582949 or the JNK inhibitor SP600125 led to significant decreases in the protein expression levels of p‑p38 or p‑JNK, respectively, and a significant reduction in oxidative stress and neuronal apoptosis; however, these inhibitors did not demonstrate an effect on p‑ASK1 or ASK1 protein expression levels. In conclusion, treatment with NQDI‑1 improved neurological function and decreased oxidative stress and neuronal apoptosis in EBI following SAH in rats, possibly via inhibition of ASK1 phosphorylation and the ASK1/p38 and JNK signaling pathway.  NQDI‑1 may be considered a potential agent for the treatment of patients with SAH.

Glycoside of cistanche can also increase the activity of SOD in heart and liver tissues, and significantly reduce the content of lipofuscin and MDA in each tissue, effectively scavenging various reactive oxygen radicals (OH-, H₂O₂, etc.) and protecting against DNA damage caused by OH-radicals. Cistanche phenylethanoid glycosides have a strong scavenging ability of free radicals, a higher reducing ability than vitamin C, improve the activity of SOD in sperm suspension, reduce the content of MDA, and have a certain protective effect on sperm membrane function. Cistanche polysaccharides can enhance the activity of SOD and GSH-Px in erythrocytes and lung tissues of experimentally senescent mice caused by D-galactose, as well as reduce the content of MDA and collagen in lung and plasma, and increase the content of elastin, have a good scavenging effect on DPPH, prolong the time of hypoxia in senescent mice, improve the activity of SOD in serum, and delay the physiological degeneration of lung in experimentally senescent mice With cellular morphological degeneration, experiments have shown that Cistanche has the good antioxidant ability and has the potential to be a drug to prevent and treat skin aging diseases. At the same time, echinacoside in Cistanche has a significant ability to scavenge DPPH free radicals and can scavenge reactive oxygen species, prevent free radical-induced collagen degradation, and also has a good repair effect on thymine free radical anion damage.

Click on Anti-aging Cistanche Para Que Serve

【For more info: david.deng@wecistanche.com / WhatApp:8613632399501】

Keywords: subarachnoid hemorrhage, NQDI‑1, apoptosis signal‑regulating kinase 1, oxidative stress, apoptosis, early brain injury

Introduction

Subarachnoid hemorrhage (SAH) is a serious acute cerebrovascular disease caused by the flow of blood into the subarachnoid space of the brain (1). However, at present, there are no effective drugs available for improving brain tissue damage after SAH following the prevention of rebleeding (2).  Therefore, further studies into the mechanisms of brain injury following SAH and the exploration of appropriate treatment options for the improvement of the prognosis of SAH are urgently required.

Early brain injury (EBI) refers to secondary brain tissue damage caused by multiple physiological disturbances and a range of pathological changes that occur within 72 h of the onset of SAH (3). In the acute response phase following SAH,   the injury begins with the entry of large amounts of blood into the subarachnoid space and this is followed by a series of pathological injuries (4). As mitochondria are particularly susceptible to hypoxia and ischemic injury, mitochondrial dysfunction is one of the primary pathological injuries suffered after SAH (5). Large numbers of reactive oxygen species (ROS) are released, which leads to the enhancement of oxidative stress and activation of the mitochondrial apoptotic pathway, causing mitochondrial injury and dysfunction (6). Therefore, therapeutic modalities that reduce oxidative stress and apoptosis are key for the improvement of the prognosis of SAH.

Apoptosis signal‑regulating kinase 1 (ASK1), a member of the mitogen‑activated protein 3 kinase (MAP3K) family, is activated by numerous different types of cellular stress and has an important role in oxidative stress and apoptosis (7).  In primary cultures of mouse hippocampal neurons, the Rho kinase inhibitor fasudil reverses β‑amyloid‑induced elevation of phosphorylated (p‑)ASK1, which decreases neuronal apoptosis in Alzheimer's disease (8). To the best of our knowledge,   however, therapeutic options targeting ASK1 following SAH   have not been evaluated.

Ethyl‑2,7‑dioxo‑2,7‑dihydro-3H-naphtho(1,2,3-de)quinoline1-carboxylate (NQDI‑1) has been reported to be a specific inhibitor of ASK1 and has been validated using in vitro kinase assays (9,10). Previous studies have reported that NQDI‑1 may be effective in the treatment of numerous diseases through inhibition of ASK1 activity (11,12). NQDI‑1 alleviates microcystin‑LR‑induced mitochondrial damage and apoptosis in ovarian granulosa cells via inhibition of the activation of  ASK1 (13). Furthermore, indomethacin can induce apoptotic cascade responses in glial cells via ASK1 activation of endoplasmic reticulum stress (14). Therefore, the ASK1‑specific   inhibitor NQDI‑1 may exert neuroprotective effects following  SAH through inhibition of ASK1 activity. p38 and JNK are considered to form the downstream signaling pathways of ASK1; activated p38 and JNK can further activate a variety of substrates, including nuclear transcription factors as well as protein kinases and other functional proteins, which leads to the onset of oxidative stress and apoptosis (15).

It was hypothesized that ASK1 inhibitor NQDI‑1 exerts neuroprotective effects and decreases oxidative stress and neuroapoptosis via the ASK1/p38 and JNK signaling pathway in EBI following SAH in rats.

Materials and methods

Experimental animals. The animal experiments were approved by the Ethics Committee for Experimental Animals of Central  South University (approval no. 2019sydw0104). A total of 191 male Sprague‑Dawley (SD) rats weighing 280‑320 g were housed in a constant temperature (21‑23˚C) and humidity  (45‑50%) environment with a 12/12‑h light/dark cycle and had free access to water and food. All animal experiments were performed according to the Animal Research: Reporting  In Vivo Experiments guidelines (16) and were performed by the National Institutes of Health (NIH) Guide for the Care and Use of Animals guidelines (17). When rats reached the designated preset time point of the experiment or met certain criteria for euthanasia [complete loss of appetite for 24 h or poor appetite (50% below normal) for 3 days,   inability to eat or drink, or they were assessed as being close to death (self‑injurious behavior, abnormal posture, respiratory distress, etc.)], the rats were placed in a carbon dioxide euthanasia device with CO2 volume displacement rate set at 30%/min (18). Death was confirmed by cessation of breathing and heartbeat and pupil dilation. The carcasses of all rats were bagged and frozen for environmentally sound disposal.

SAH model. The rat SAH model was induced using monofil‑  ment perforation (19). After anesthetizing the rats (induction,  3% isoflurane; maintenance, 2% isoflurane), the left common,   internal and external carotid arteries of the rats were exposed.  The distal external carotid artery was clipped using cauterize‑ to form the stump of the external carotid artery. A 3‑cm   sharpened model 4‑0 proline wire was inserted ~2 cm through the external carotid artery until a slight resistance was felt. The puncture line was slowly advanced by ~3 mm to puncture the arterial wall. Brain tissue was removed at the corresponding time points predetermined for the experiment. If a blood clot was found on the ventral side of the brain tissue, it indicated that the rat had SAH. Rats with a score >8 by assessment of the  SAH grading score were considered to meet the experimental requirements and to be a successful model. The procedure for the sham group was similar to that of the SAH group, with the exception that the 4‑0 proline wire did not pierce the arterial wall. The respiration, heart rate, skin color, and pedal reflex were monitored every 5 min throughout the surgical and anesthetic recovery stages. Of these, 23 rats died and 8 rats were excluded, and these deaths and exclusions were supplemented by new SAH-modeled rats.

Severity of SAH. The severity of SAH was assessed using the SAH   grading score 24 h after SAH (20). The ventral side of the brain tissue was divided into 6 regions, each rated 0‑3 depending on the amount of blood clot and how well the blood vessels were obscured: A score of 0 indicates no clot, 1 indicates a small amount of clot, 2 indicates a moderate amount of clot but large vessels at the skull base are still identifiable, and 3 indicates a   large amount of clot and unidentifiable vessels at the skull base.  The six regional scores were summed to calculate a total score  (0‑18), where higher scores indicated more severe hemorrhage.  Rats with a total SAH grade score of ≤8 were considered to have mild SAH, excluded from subsequent experiments, and replaced with newly modeled rats.

Drug administration. To enable the drug concentrations in the brain tissue to be more precisely regulated independently of the liver and other factors, the siRNAs and NQDI‑1 were administered by intracerebroventricularly injection (21).  Following anesthetization (induction, 3% isoflurane; maintenance, 2% isoflurane), the rats were fixed on a brain stereotaxic apparatus. The microinjector was fixed to the bregma at  1.0 posterior, 1.5 right, and 3.3 mm deep. The ASK1 siRNA (cat. no. 4390771; Thermo Fisher Scientific, Inc.) or Scr siRNA control (cat. no. 4390843; Thermo Fisher Scientific, Inc.) were dissolved in 5 µl nuclease‑free sterile deionized water at a   concentration of 500 pmol and administered 48 h before SAH.  The sequences of ASK1 siRNA were as follows: Sense 5'‑CGG CAGACAUUGUUAUCAAtt‑3' and antisense 5'‑UUGAUA ACAAUGUCUGCCGtc‑3'. Drug concentrations and doses reported in a similar study (22) and the solubility of NQDI‑1   were used to determine the concentrations used in the present study. Three doses of NQDI‑1 (1, 3, and 10 µg/kg; Selleck  Chemicals) or vehicle solution in a total volume of 5 µl/rat was injected 1 h after SAH. The BMS‑582949 (100 mg/kg;  Selleck Chemicals), SP600125 (30 mg/kg; Selleck Chemicals), and vehicle solutions could cross the blood‑brain barrier and were administered intraperitoneally 1 h after SAH (23,24).

Short‑term neurological function. Modified Garcia score and the beam balance score were used to assess the short‑term neurological function 24 h after SAH. The modified Garcia score was divided into six categories as follows: Voluntary movement, voluntary limb movement, forepaw extension,   climbing ability, somatosensory responses, and tentacle response, each of which was scored individually and summed to give a total score ranging from 3‑18 (25). Higher scores were considered to indicate better neurological function. For the beam balance score, rats were placed on a beam for 1 min and a score ranging from 0‑4 was assessed based on the distance walked (26). Higher scores were considered to indicate better neurological function.

Long‑term neurological function. Rotarod pre‑adaptation experiments were performed on rats using the same initial velocity and acceleration before SAH modeling. The rats were placed on a Rotamex rotating bar tester (Columbus Instruments)   for the Rotarod test on days 7, 14, and 21 after SAH (27). First,   the initial Rotarod speed was set to 5 revolutions per min and the acceleration to 2 revolutions per 5 sec. After putting on the rats, the fall latency of the rats was recorded. The rats were then allowed to rest for 1 h. Finally, the fall latency of the rats was tested again at the initial speed of 5 revolutions per min and an acceleration of 2 revolutions per 5 sec. Longer fall latency was considered to indicate better motor coordination in the rat.  At week 4 after SAH modeling, the Morris water maze test was used to assess the learning memory and spatial orientation of the rats (27). The learning exercise of swimming and finding a platform located below the water surface was performed from day 1 to day 5 on week 4 (days 28‑33 post‑SAH) and the swimming trajectory, escape latency, and swimming distance were recorded. On the final day, the platform was removed for testing, and swimming trajectory, swimming distance, and probe quadrant duration were recorded for 60 sec.

IF staining. After sequential perfusion of 4˚C saline and  4˚C, 4% paraformaldehyde from the heart to remove blood from the brain tissue, intact brain tissue was obtained. After sucrose gradient dehydration, the brain tissue was then cut into  10‑µm sections in a coronal position and was stored at ‑20˚C.  IF staining was used to assess the co‑localization of ASK1 with nerve cells. Frozen tissue sections were blocked using  5% donkey serum (cat. no. G1217‑5ML; Wuhan Service Technology Co., Ltd.) for 2 h at room temperature and incubated overnight at 4˚C with primary antibodies as follows:  Anti‑ASK1 (mouse; 1:200; cat. no. 67072‑1‑Ig; ProteinTech  Group, Inc.), anti‑NeuN (rabbit; 1:200; cat. no. 26975‑1‑AP;  ProteinTech Group, Inc.), anti‑ionized calcium‑binding adapter molecule‑1 (Iba‑1; rabbit; 1:200; cat. no. 10904‑1‑AP;  ProteinTech Group, Inc.), and anti‑glial fibrillary acidic protein  (GFAP; chicken; 1:200; cat. no. ab4674; Abcam). The tissue sections were then incubated for 2 h at room temperature with the corresponding fluorescent secondary antibodies as follows: Alexa Fluor® 594 AffiniPure Donkey Anti‑Mouse IgG (H+L) (cat. no. 715‑585‑150; 1:500; Jackson ImmunoResearch  Laboratories, Inc.), Alexa Fluor 488 AffiniPure Donkey  Anti‑Rabbit IgG (H+L) (cat. no. 711‑545‑152; 1:500; Jackson ImmunoResearch Laboratories, Inc.), and Alexa Fluor 488 AffiniPure Donkey Anti‑Chicken IgY (IgG) (H+L)  (cat. no. 703‑545‑155; 1:500; Jackson ImmunoResearch  Laboratories, Inc.). Subsequently, they were stained for cell nuclei using 2 µg/ml DAPI (cat. no. G1012‑100ML; Wuhan Service Technology Co., Ltd.) for 10 min at room temperature. The sections were assessed using an Olympus BX53   fluorescence microscope (Olympus Corporation) and images were captured.

Dihydroethidium (DHE) staining. DHE staining was performed to assess the brain's oxidative stress (28). Frozen brain tissue sections were incubated with 2 µmol/l DHE (Thermo Fisher Scientific, Inc.) at 37˚C for 30 min in the dark. After staining for cell nuclei using 2 µg/ml DAPI for  10 min at room temperature, the sections were assessed using an Olympus BX53 fluorescence microscope. A total of six sections from each brain tissue were randomly selected and six areas in each section were randomly selected for imaging.  The number of DHE‑positive cells was counted using ImageJ  1.4 software (National Institutes of Health) and the mean was calculated and taken as the final percentages for each brain tissue section.

TUNEL staining. TUNEL staining was used to evaluate the percentage of apoptotic neurons (29). Frozen tissue sections were blocked using 5% donkey serum for 2 h at room temperature and incubated overnight at 4˚C with primary antibody anti‑NeuN (rabbit; 1:200; cat. no. 26975‑1‑AP; ProteinTech  Group, Inc.). The tissue sections were then incubated with the fluorescent secondary antibody Alexa Fluor 488 AffiniPure  Donkey Anti‑Rabbit IgG (H+L) (cat. no. 711‑545‑152; 1:500;  Jackson ImmunoResearch Laboratories, Inc.) for 2 h at room temperature. TUNEL staining was performed using the One  Step TUNEL Apoptosis Assay kit (cat. no. C1090; Beyotime  Institute of Biotechnology) according to the manufacturer's protocol. Finally, the tissue sections were stained for cell nuclei using 2 µg/ml DAPI for 10 min at room temperature. Random selection of TUNEL counting locations and calculation of  TUNEL‑positive neurons for each section were performed using the aforementioned method for DHE counting.

Western blotting (WB). WB was used to semi‑quantify protein expression levels. After perfusion of 4˚C saline from the heart to remove blood from the brain tissue, intact brain tissue was obtained. Previous studies have reported that the SAH model,   induced via left‑sided endovascular puncture, results in a degree of bias towards bleeding events and can be relatively severe in terms of damage to left‑sided brain tissue (30). Therefore,   the left‑sided brain tissue was used to assess the pathological damage following SAH modeling. Brain tissue proteins were extracted using RIPA lysis solution (cat. no. P0013B; Beyotime  Institute of Biotechnology) according to the manufacturer's protocol. The protein concentration of the samples was determined by the BCA assay (cat. no. P0012; Beyotime Institute of Biotechnology) and subsequently adjusted to 5 µg/µl by adding different amounts of double‑distilled water. The 5 µl of 5 µg/µl protein samples were added to each lane and these protein samples were separated on 10% gels by SDS‑PAGE   electrophoresis and transferred to PVDF membranes. The membranes were blocked for 2 h at room temperature using  5% non‑fat powdered milk (cat. no. P0216‑300g; Beyotime  Institute of Biotechnology). The membranes were incu‑  bated overnight at 4˚C with primary antibodies as follows:  Anti‑p‑ASK1 (Ser‑83; 1:1,000; cat. no. MA5‑28020; Thermo  Fisher Scientific, Inc.), anti‑ASK1 (1:1,000; cat. no. 8662;  Cell Signaling Technology, Inc.), anti‑p‑p38 MAPK  (Thr180/Tyr182) (1:1,000; cat. no. 9216; Cell Signaling  Technology, Inc.), anti‑p38 MAPK (1:1,000; cat. no. 8690;  Cell Signaling Technology, Inc.), anti‑phospho‑stress‑acti‑  vated protein kinase/Jun‑amino‑terminal kinase  (phospho‑SAPK/JNK) (Thr183/Tyr185; 1,000; cat. no. 9255;  Cell Signaling Technology, Inc.), anti‑SAPK/JNK (JNK;  1:1,000; cat. no. 9252; Cell Signaling Technology, Inc.), anti‑4   hydroxynonenal (4‑HNE; 1:1,000; cat. no. ab46545; Abcam),   anti‑heme oxygenase 1 (HO‑1; 1:1,000; cat. no. 82206;  Cell Signaling Technology, Inc.), anti‑Bcl‑2 (1:1,000;   cat. no. 26593‑1‑AP; ProteinTech Group, Inc.), anti‑Bax  (1,1000; cat. no. 60267‑1‑Ig; ProteinTech Group, Inc.) and   anti‑β‑actin (1:2,000; cat. no. 4970; Cell Signaling Technology,  Inc.). This was followed by incubation with the appropriate horseradish peroxidase‑conjugated secondary antibodies for 2 h at room temperature: Goat anti‑mouse IgG‑HRP  (cat. no. sc‑2005; 1:5,000; Santa Cruz Biotechnology, Inc.)   or goat anti‑rabbit IgG‑HRP (cat. no. sc‑2004; 1:5,000; Santa  Cruz Biotechnology, Inc.), and the specific bands were visualized using an ECL kit (cat. no. P0018AS; Beyotime Institute of Biotechnology) and imaged using a UVP Che Studio PLUS   system (Analytik Jena GmbH). Relative densitometric analysis of WB bands was performed using ImageJ 1.4 software  (NIH) and the protein expression levels were normalized against β‑actin.

Experimental design

Expression changes and cellular localization of ASK1   following SAH. A total of 36 rats were randomly assigned to six groups (n=6/group) as follows: i) Sham‑operated (sham), ii) 3 h post‑SAH, iii) 6 h post‑SAH, iv) 12 h post‑SAH, v) 24 h post‑SAH and vi) 72 h post‑SAH. WB was used to assess changes in the protein expression levels of endogenous ASK1 and p‑ASK1. A total of 4 additional rats were divided into sham (n=2) and SAH 24 h (n=2) groups and IF staining was used to evaluate the co‑localization of ASK1.

Therapeutic effects of ASK1 inhibitor NQDI‑1 on short‑ and long‑term neurological function following SAH.  A total of 30 rats were divided into 5 groups (n=6/group) as follows: i) Sham, ii) SAH + vehicle, iii) SAH + 1.0 µg/kg  NQDI‑1, iv) SAH + 3.0 µg/kg NQDI‑1 and v) SAH + 10.0 µg/kg  NQDI‑1. The modified Garcia and beam balance scores were used to assess short‑term neurological function 24 h following  SAH. Based on the extent of the short‑term neurological improvement, 3.0 µg/kg NQDI‑1 was assessed to be the most appropriate dose group and this dose of NQDI‑1 was used in subsequent experiments. A total of 30 rats were divided into  3 groups (n=10) as follows: i) Sham, ii) SAH + vehicle, and iii) SAH + NQDI‑1. A rotarod experiment was used to assess long‑term neurological function on days 7, 14, and 21 after  SAH, and the Morris water maze test was performed at week 4   after SAH to assess long‑term neurological function.

Therapeutic effects of ASK1 inhibitor NQDI‑1 on oxidative stress and apoptosis. A total of 12 rats were divided into  3 groups (n=4/group) as follows: i) Sham, ii) SAH + vehicle, and iii) SAH + NQDI‑1. (THE) staining was performed to assess oxidative stress and TUNEL staining was used to assess neuronal apoptosis 24 h after SAH.

Role of ASK1/p38 and JNK signaling pathway in the therapeutic effects of NQDI‑1. A total of 48 rats were divided   into 8 groups (n=6/group) as follows: i) Sham, ii) SAH,   iii) SAH + vehicle, iv) SAH + NQDI‑1, v) SAH + scrambled  (Scr) short interfering (si)RNA, vi) SAH + ASK1 siRNA,   vii) SAH + BMS‑582949 and viii) SAH + SP600125 groups.  The ASK1 siRNA, the p38 inhibitor BMS‑582949, and the JNK   inhibitor SP600125 were injected to assess whether ASK1,   p38, and JNK were involved in the neuroprotective effects of  NQDI‑1.

Statistical analysis. The data were presented as the mean ± standard deviation and the experimental data of WB   were obtained in 6 independent replicates, while DHE staining and TUNEL staining were performed as 4 independent replicates. Data were analyzed using SPSS version 17 (SPSS,  Inc.). Data from the Morris water maze test were assessed using a mixed ANOVA/two‑way repeated measures ANOVA, followed by the LSD post hoc test. Data from the remaining experiments were tested for normal distribution using the  Shapiro‑Wilk normal distribution test followed by one‑way  ANOVA followed by Tukey's post hoc multiple comparison test. Graphs were plotted using GraphPad Prism 7 (GraphPad  Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

Results

Mortality and SAH severity. A total of 191 SD rats were used in the present study, of which 8 rats were excluded due to only mild SAH (SAH grading score ≤8). Of the SD rats used in the experiments, 34 rats were in the sham group and 149 rats underwent SAH modeling. No rats in the sham group died; however, 23 rats died following SAH modeling (mortality rate 15.4%) (Tables SI‑V). Compared with the sham group, clots in the subarachnoid space were primarily located near the Willis ring and on both sides of the basilar artery following SAH modeling (Fig. 1A). The SAH grading score 24 h post‑SAH modeling demonstrated no significant difference in the severity of hemorrhage between all non‑sham groups and a significant difference between sham and all non‑sham groups (Fig. 1B).

Protein expression changes and cellular localization of ASK1 following SAH. The protein expression levels of ASK1 increased and reached a peak at 24 h in the brain following SAH (Fig. 1C and D). However, the ratio of the protein expression of p‑ASK1/ASK1 demonstrated no significant difference (Fig. 1C and E). Cellular localization of ASK1 with NeuN‑positive neurons, Iba‑1‑positive microglia, and  GFAP‑positive astrocytes was demonstrated in both the sham and SAH 24 h group (Fig. 1F‑H).

ASK1 inhibitor NQDI‑1 improves short‑term neurological functions 24 h after SAH. NQDI‑1 (1, 3, and 10 µg/kg) was injected intracerebroventricularly 1 h after SAH. Rats undergoing SAH modeling had significantly lower modified Garcia and beam balance scores 24 h after SAH modeling; however, all three doses of NQDI‑1 led to a significant partial improvement in short‑term neurological function (Fig. 2A and B). The modified  Garcia scores were significantly higher in the SAH + NQDI‑1  (3.0 µg/kg) group compared with the SAH + NQDI‑1 (1.0 µg/kg)   group; however, no statistically significant differences were demonstrated compared with the SAH + NQDI‑1 (10.0 µg/kg)   group (Fig. 2A and B), which suggested that further increasing the concentration of NQDI‑1 did not elicit further improvement in short‑term neurological function following SAH. Therefore, NQDI‑1 (3.0 µg/kg) was considered to be the effective optimal concentration and was used in subsequent experiments.

ASK1 inhibitor NQDI‑1 improves long‑term neurological functions after SAH. Using starting speeds of 5 or 10 rpm for the Rotarod test, fall latency was significantly decreased in the SAH + vehicle group compared with the sham group, whereas the fall latency was significantly prolonged following intracerebroventricular injection of NQDI‑1 compared with the SAH + vehicle group (Fig. 2C and D). Furthermore, during days 1‑5 of the training phase of the Morris water maze test at week 4 post‑SAH, the SAH + vehicle group demonstrated significantly longer escape latency and swimming distance compared with the sham group, whereas NQDI‑1 treatment demonstrated a significant decrease compared with the SAH+ vehicle group (Fig. 2E‑G). In the testing phase with the removal of the underwater circular platform, no significant differences were observed in swimming velocity between the three groups  (Fig. 2H). The probe quadrant duration was significantly shorter in the SAH + vehicle group compared with the sham group and NQDI‑1 treatment significantly prolonged the explo‑  ration time compared with the SAH + vehicle group (Fig. 2I).

ASK1 inhibitor NQDI‑1 decreases oxidative stress and neuronal apoptosis 24 h post‑SAH. The level of oxidative stress was measured using DHE staining and the proportion of apoptotic neurons was assessed using the percentage of TUNEL‑positive neurons 24h after SAH. The percentage of DHE‑positive cells was significantly higher in the SAH + vehicle group compared with the sham group and NQDI‑1 treatment significantly decreased this compared with the SAH + vehicle group (Fig. 3A and). The percentage of TUNEL‑positive neurons was significantly higher in the SAH + vehicle group compared with the sham group. However, the percentage of TUNEL‑positive neurons decreased significantly following treatment with NQDI‑1 compared with the SAH + vehicle group (Fig. 3B and D).

ASK1 siRNA, BMS‑582949, and SP600125 improve short‑term neurological functions 24 h post‑SAH. Compared with the SAH + Scr siRNA group or SAH + vehicle group, administration of ASK1 siRNA, BMS‑582949 or SP600125 demonstrated significant improvement in the modified Garcia and beam balance score (Fig. 4A and B).

NQDI‑1 attenuates oxidative stress and apoptosis after  SAH via decreased phosphorylation of ASK1, p38, and  JNK. The protein expression levels of ASK1, p‑p38, p‑JNK, 4‑HNE, HO‑1, Bax, and Bcl‑2 were significantly higher after SAH compared with the sham group; however, the ratio of   p‑ASK1/ASK1 was not significantly different (Fig. 5A‑M).  The injection of vehicle or Scr siRNA did not induce significant differences in the protein expression levels compared with the SAH group. Compared with the SAH + vehicle group, treatment using NQDI‑1 caused a significant decrease in the protein expression levels of p‑ASK1/ASK1, p‑p38, p‑JNK, 4‑HNE, and Bax, whereas protein expression levels of HO‑1 and Bcl‑2 were significantly increased. Compared with the SAH + Scr siRNA group, protein expression levels of ASK1 significantly decreased following injection of  ASK1 siRNA. Treatment with ASK1 siRNA also resulted in a significant decrease in the protein expression levels of p‑p38, p‑JNK, 4‑HNE, and Bax and a significant increase in  HO‑1 and Bcl‑2 expression compared with the SAH + Scr siRNA group.

p38 inhibitor BMS‑582949 reduces the protein expression levels of p‑p38 but does not affect p‑ASK1, ASK1, or p‑JNK protein expression levels. Treatment with the p38 inhibitor BMS‑582949 demonstrated a significant decrease in protein expression levels of p‑p38, 4‑HNE, and Bax and also significantly increased protein expression of HO‑1 and Bcl‑2 compared with the SAH + vehicle group; however, the protein expression levels of ASK1, p‑ASK1/ASK1 and p‑JNK demonstrated no significant differences (Fig. 6A‑M). JNK inhibitor SP600125 decreases the protein expression level of p‑JNK but does not affect p‑ASK1, ASK1, or p‑p38 protein expression levels. Treatment with JNK inhibitor SP600125   caused a significant decrease in the protein expression levels of p‑JNK, 4‑HNE, and Bax and a significant increase in HO‑1 and  Bcl‑2 protein expression levels compared with the SAH + vehicle group; however, protein expression levels of ASK1, p‑ASK1/ASK1, and p‑p38 were not significantly altered (Fig. 7A‑M).

【For more info: david.deng@wecistanche.com / WhatApp:86 13632399501】