Gastrodin injection relieves hypoxic-ischemic brain injury in newborn rats by regulating the P62/Nrf2/HO-1 pathway

Gastrodin injection relieves hypoxic-ischemic brain injury in newborn rats by regulating the P62/Nrf2/HO-1 pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Gastrodin injection relieves hypoxic-ischemic brain injury in newborn rats by regulating the P62/Nrf2/HO-1 pathway Sha Wu, Zhenkui Ren, Mengting Yang, Ying Xiong, Xianxian Li, Xuxian Wu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6630703/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Nov, 2025 Read the published version in Molecular Neurobiology → Version 1 posted 18 You are reading this latest preprint version Abstract Hypoxic-ischemic encephalopathy (HIE) is the leading cause of death and disability in newborns. Hypothermia treatment (TH) is currently the only method that can improve the survival rate of HIE patients, but it has many limitations. Previous studies have shown that gastrodin injection has a potential neuroprotective effect on brain injury. However, it is unclear whether it has the same effect on HIE and its mechanism of action. This study investigated the neuroprotective effect of gastrodin injection on HIE and its possible mechanism through in vivo and in vitro experiments. For in vivo experiments, a hypoxic-ischemic brain damage (HIBD) model was established in neonatal rats, and immediately after modeling, a transperitoneal injection of gastrodin injection was administered. The model was verified via laser diffuse imaging, and hematoxylin-eosin (H&E), Nissl's staining, and 2,3,5-triphenyltetrazolium chloride (TTC) staining were performed to assess the brain tissue damage and the area of brain infarction, and short- and long-term neurobehavioral assessments were performed. Brain tissue damage, cerebral infarction area, short-term and long-term neurobehavioral assessment, superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH-PX), and catalase (CAT) detection, Western blotting and ELISA were used to analyze the levels of oxidative stress and inflammatory factors in brain tissue. For in vitro experiments, a hypoxia-glucose deprivation/reperfusion (OGD/R) injury model was established in SH-SY5Y cells, and gastrodin injection was injected immediately after model establishment. To determine the mechanism of action of gastrodin injection, XRK3F2, a specific inhibitor of P62, was used, and cell viability, the level of intracellular reactive oxygen species (ROS), the mitochondrial membrane potential, and oxidative stress-related protein levels were measured to further validate the neuroprotective effect of gastrodin injection. The results showed that gastrodin injection could alleviate the area of cerebral infarction, reduce neuronal oxidative stress injury, improve short-term and long-term neurobehavioral deficits, improve mitochondrial function, improve the body's ability to resist oxidative stress, and reduce inflammatory reactions in brain tissues from HIBD rats. In vitro experiments revealed that gastrodin injection increased neuronal cell survival after OGD/R injury and inhibited the occurrence of oxidative stress and apoptosis; however, these neuroprotective effects were attenuated by XRK3F2, a specific inhibitor of P62. The present study suggested that gastrodin injection may attenuate oxidative stress injury and apoptosis and ameliorate brain injury after HBID by activating the P62/Nrf2/HO-1 signaling pathway. HIBD HIE gastrodin injection oxidative stress P62/Nrf2/HO-1 pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1 Introduction Neonatal hypoxic-ischemic encephalopathy (HIE) is a type of neonatal brain injury caused by various forms of perinatal hypoxia and is one of the main causes of neonatal encephalopathy, with an incidence of 1.5‰. Approximately 25% of affected children have neurological sequelae such as epilepsy, cerebral palsy, and cognitive disorders [ 1 ] . In China, the incidence of neonatal HIE approximately 3‰-6‰, and the mortality rate accounts for 15.2% of the mortality rate of children under 5 years of age [ 2 ] . Although subcooling therapy can reduce the incidence of neurological sequelae after HIE, it is limited by the inclusion criteria and time window, which still prevents a larger number of children from undergoing HT [ 3 ] . Therefore, developing effective treatments for HIE is important. The result of multifactorial interactions in HIE injury, including oxidative stress, excitatory amino acid toxicity, inflammatory response, and neuronal apoptosis, etc. After the onset of ischemia and hypoxia, due to the lack of ATP, the inactivation of ATP-dependent ions results in Na + /K+-ATP pump malfunction, resulting in the inward influx of Na + and Ca 2+ into cells, leading to intracellular calcium overload, excessive Ca 2+ uptake triggers a molecular neurotoxic cascade reaction, which further leads to the overproduction of free radicals, mitochondrial dysfunction, a reduction in the mitochondrial membrane potential (ΔΨm), the release of apoptotic substances, cell membrane damage, and DNA breakage; In addition, after hypoxia, oxidative stress injury leads to the production of a large amount of ROS and, at the same time, the activation of microglia, the production of a large number of inflammatory factors (TNF-α, IL-1β, etc.), which further exacerbates tissue injury [ 4 , 5 ] . Therefore, the inhibition of oxidative stress damage may be a novel approach for the treatment of HIE. The Nrf2/HO-1 signaling pathway is a key regulator of important cellular defenses and the antioxidative stress pathway [ 6 ] . Celastrol reduces the area of cerebral infarction, decreases microglial activation, and suppresses the levels of inflammatory factors and oxidative stress in SD rats after HIE by activating the Nrf2/HO-1 signaling pathway [ 7 ] . Gastrodia elata is a traditional Chinese medicine used in China to treat dizziness and limb numbness in adults and infantile spasms, epilepsy, convulsions and tetanus in children [ 8 ] . Studies have shown that Gastrodia elata has a neuroprotective effect and that Gastrodin (GAS), an extract of Gastrodia elata and one of its phenolic compounds, can cross the blood-brain barrier and is used to treat a variety of neurological disorders [ 9 ] . Previous studies have shown that in an animal model of ischemia/reperfusion (I/R) injury, GAS can reduce the expression of inflammatory factors such as IL-1β and TNF-α; increase the expression of antioxidant stress-related proteins such as heme oxygenase-1 (HO-1), SOD and the transcription factor Nrf2; and reduce the incidence of neuronal apoptosis, thereby exerting neuroprotective effects [ 10 ] . Therefore, GAS may be a potential therapeutic drug for HIE. In this study, we established a neonatal rat HIBD model and used SH-SY5Y cells to establish an OGD/R injury model to investigate the neuroprotective role of gastrodin injection in HIBD and whether the P62/Nrf2/HO-1 signaling pathway is involved in this process. 2 Materials and methods 2.1 Reagents Gastrodin injection was purchased from Southwest Pharmaceutical Co., Ltd. (manufacturing batch number: H20064391); primary antibodies: Nrf2 (Abmart, TA0639, 1:1000), HO-1 (Proteintech, 10701-1-AP, 1:2000), Keap1 (affinity, AF5266, 1:2000), P62 (affinity, AF5384, 1:2000), β-actin (Servicebio, GB15003, 1: 2000), Bax (Proteintech, 50599-2-Ig, 1:2000), and Bcl2 (Proteintech, 26593-1-AP, 1:1000); secondary antibodies (Servicebio, GB15003); ELISA kits: TNF-α, TGF-β, and IL-6 (Jianglai Biology, JL1302, JL13643, JL20896); MDA, SOD, GSH-PX and CAT kits (Nanjing Jiancheng, A003-1, A001-3, A005-1, A007-1-1); TTC staining (Solarbio, G3005); JC-1 staining kit (Servicebio, G1515); and reactive oxygen species (ROS) detection kit (Servicebio, G1706); CCK-8 detection kit (C6005M); fetal bovine serum (Gibco, 2565856P); high glucose DMEM (Gibco); glucose-free DMEM (Servicebio, G4528), P62 inhibitor (MCE, HY-112904). 2.2 Neonatal hypoxic-ischemic brain injury model and treatment Sprague-Dawley (SD) rats were obtained from the Animal Center of Guizhou Medical University (SCXK (Gui) 2023-0002), Animal Ethics Batch No. NO240028, maintained at a rearing environment temperature of 20–25℃, humidity of 50–60%, and light exposure of 12 h per day (8: 00–20: 00). Adult SD rats with a body mass of 12–18 g, were freely mated, and neonatal SD rats, male and female, were used and raised by SD females. The samples were randomly divided into five groups: sham, HIBD, L-GAS, M-GAS, and H-GAS groups. Modeling was performed according to the modified Rice- Fannuzzi method [ 11 ] . Neonatal rats were anesthetized via isoflurane inhalation, and the right common carotid artery was isolated within 5 minutes, double-ligated, dissected, and returned to the mother to rest for 1 hour. Neonatal rats were placed in a hypoxic apparatus, a gas mixture (92% N 2 + 8% O 2 ) was delivered at a rate of 1.2 L/min, and the hypoxic apparatus was placed in a 37°C water bath to maintain the chamber temperature at approximately 36 ± 1°C for 2 hours. In the sham-operated group, only the right common carotid artery was isolated without ligation or hypoxia; in the other groups, the rats were generated via the anamnestic method, and the rats were returned to their mothers at the end of the model. The treatment group was injected intraperitoneally with different doses of gastrodin injection (50 mg/kg, 100 mg/kg, 200 mg/kg) immediately after modeling, and the model group was injected intraperitoneally with the same volume of saline [ 12 ] . 2.3 Laser scatter imaging Laser scatter imaging is a noninvasive imaging technique widely used in vascular function studies [ 13 ] . Briefly, the rats were deeply anesthetized with isoflurane after 24 h of modeling, the scalp was removed, the skull was fully exposed, the skull was placed under the camera (approximately 10 cm from the skull), and the original scatter images were acquired via a 785 nm laser beam. Relative blood flow was analyzed by imaging the laser scatter in the region of interest (ROI) via RFLSIZW software (Reward, China). 2.4 2,3,5-Triphenyltetrazolium Chloride (TTC) Staining TTC staining was used to evaluate the volume of cerebral infarction [ 14 ] . Twenty-four hours after HIBD, the brain tissues were removed from the rats after isoflurane anesthesia and execution and sectioned through brain tissue sectioning molds. Coronal sections were generated (thickness of 2 mm), placed in 2% TTC solution, incubated at 37°C for approximately 10–15 min, placed in 4% paraformaldehyde for fixation overnight, and then dried on absorbent paper before being photographed with a digital camera. Images were taken with a digital camera and then processed and analyzed with ImageJ software. The percentage infarct volume was calculated according to the following formula: Infarct volume percentage = infarct side volume/contralateral normal hemisphere volume × 100%. Infarct volume percentage = (total area of normal sections on the contralateral side - sum of normal areas of sections on the infarct side)/sum of sections on the contralateral hemisphere × 100%. 2.6 Pathological staining After HIBD, the rats were deeply anesthetized with isoflurane for 24 hours and 28 days, and the hearts were perfused with 10 ml of PBS and then perfused with an equal volume of 4% paraformaldehyde. The brain tissues were removed, and the brain tissues were removed, immersed in 4% paraformaldehyde for fixation for 24 hours, embedded in paraffin, and cut into coronal sections of 5 µm thickness. Then, the brain sections were deparaffinized, dehydrated, and combined with staining with HE or Nissl staining solution. Finally, the results of histological staining were evaluated and recorded via light microscopy. 2.7 Short-term Behavioral Neurological function was scored via the Longa scale [ 15 ] as follows: no defects and normal activity (0 points); flexion of the left forepaw when the tail is raised (1 point); turning in a circle when crawling (2 points); unsteady standing and falling to the left when walking (3 points); and inability to walk independently and loss of consciousness (4 points). Short-term neurobehavioral testing was conducted 24 hours after drug administration by experienced testers. To avoid subjective bias, the experiments were double-blinded, and the experimenters and data analysts were unaware of the groupings. Negative trend experiment: The rats in each group were placed with their heads and trunks facing down on a rough inclined surface with an angle of inclination of 45°. The time required to rotate the heads and trunks of the newborn rats by 180° was recorded in seconds, and the time of more than 60 s was recorded according to the time of 60 s. The procedure was repeated three times for each rat, with an interval of more than 5 min each. Turning reflex: The rats in each group were placed with their heads and trunks facing down on a rough inclined surface with an angle of inclination of 45°. The time required to rotate the heads and trunks of the newborn rats by 180° was recorded in seconds, and the time of more than 60 s was recorded according to the time of 60 s. The procedure was repeated three times for each rat, with an interval of more than 5 min each. 2.8 Western blot Extracted cerebral cortex tissues or SH-SY5Y cells were lysed in RIPA lysis buffer containing 1 mM PMSF, homogenized with a tissue homogenizer, completely lysed on ice for 30 min, and then centrifuged (4°C, 12,000 rpm, 15 min) to obtain supernatants. The protein concentration was measured, and the proteins were prepared via a BCA kit. Proteins were separated by sodium dodecyl sulfate (SDS)-polyacrylamide separation gel electrophoresis, and the bands were subsequently transferred to a PVDF membrane. After being blocked with 5% nonfat milk solution or 5% BSA solution diluted in TBST for 2–4 hours, the membranes were incubated with primary antibodies: Nrf2 (1:1000), HO-1 (1:1000), P62 (1:1000), Keap1 (1:1000), Bax (1:1000), β-actin (1:5000), and Bcl-2 (1:1000) overnight at 4°C in a refrigerator. The next day, the blots were washed three times with TBST for 10 minutes each and incubated with the appropriate secondary antibody (1:10,000) for 60 minutes. After three washes with TBST, the blots were visualized via an enhanced chemiluminescence (ECL) kit and quantified via an imaging system (Bio-Rad). 2.9 Detection of MDA, SOD, GSH-PX and CAT A 9x volume of saline was added at a weight (g): volume (mL) ratio = of 1:9, the tissue, was cut, ground well on ice, and centrifuged at 4000 rpm for 10 min, and after which the supernatant was collected for measurement. The assay was performed according to the respective instructions. 2.10 ELISA detection A 9x volume of PBS was added at a weight (g): volume (mL) ratio = of 1:9, the mixture was mixed well on ice, the homogenate was centrifuged at 5000×g for 10 minutes, and the supernatant was collected for testing. The desired plate strips, were removed, 100 µL of supernatant was added to the wells, the plate was covered with a seal and incubated at 37℃ for 1 hour, the mixture was discarded, a biotinylated antibody was added, the mixture was incubated at 37℃ for 1 hour, the mixture was discarded, the mixture was washed with 1× washing solution for 1 minute each time 3 times, enzyme conjugate working solution was added, the mixture was incubated at 37℃ for 30 minutes, the plate was washed 5 times, 90 µL of substrate was added to each well, the mixture was incubated at 37℃ without light for 15 minutes, and finally, 50 µL of substrate was added to each well. The plate was washed five times, 90 µL of substrate was added to each well, and the mixture was incubated at 37℃ for 15 minutes. Finally, 50 µL of termination solution was added to each well, and the OD value of each well was immediately measured at 450 nm. 2.11 Long-term behavior The Morris water maze (MWM) test is an experiment used to assess the learning and memory ability of animals [ 16 ] . On the 21st day after HIBD injury, we used WMT-100 Morris water maze video analysis to evaluate the learning and memory ability of experimental animals. A black circular pool with a diameter of 120 cm and a height of 50 cm was prepared in a room isolated from noise and light. The depth of the pool was 1 cm greater than that of the moving platform. Colorless and odorless titanium dioxide was used to cloud the edges of the water, and the pool was divided into four equal quadrants. The rats were trained for 5 days and the platform was removed on day 6. Swimming routes, latency times, and the number of platform crossings were recorded. The motor coordination of the rats in each group was tested 21 days after HIBD injury. The training was started 3 days before the formal experiment [ 17 ] , the rats were placed on a rotating rod test and the speed was slowly increased from 4 revolutions per minute (4 rpm) to 40 rpm in 5 min. The rats were recorded 3 times and the average value was taken. 2.12 SH-SY5Y cell culture, grouping and glucose deprivation/reoxygenation (OGD/R) modeling The human neuroblastoma SH-SY5Y cell line was obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. The cells were cultured in medium containing 10% fetal bovine serum and 90% high glucose in an incubator at 37 ℃ and 5% CO 2 .SH-SY5Y cells were classified into the following groups: sham, OGD/R, OGD/R + GAS, and OGD/R + GAS + XRK3F2.To establish the OGD/R injury model [ 18 ] , the cells were washed three times with PBS, supplemented with unsweetened DMEM, rapidly transferred to a three-gas incubator (37 ℃, 94% N 2 , 5% CO 2 , and 1% O 2 ), and treated for 4 h. Sugar-free DMEM was discarded and normal complete medium was added. Different does of gastrodin injection and XRK3F2 were added to the GAS-treated group after modeling, and XRK3F2 was added at a dose of 5 µM [ 19 ] . PBS was added to the OGD/R group, which was subsequently incubated at 37℃in a 5% CO 2 cell culture incubator for further incubation after 24 hours. 2.13 CCK-8 assay for cell viability in each group The cells were inoculated into 96-well plates, cultured and modeled. Cell viability was measured via the CCK-8 assay. SH-SY5Y cells were inoculated into 96-well plates (5000 cells/well) [ 20 ] , and the OGD/R model was established according to the manufacturer’s instructions before the cells were treated with CGA at concentrations of 0, 20, 40, 80, 160, and 320 µmol. Twenty-four hours later, 10 µL of CCK-8 solution was added to each well of the 96-well plate which was subsequently incubated for 1.5 h at 37°C. The absorbance of each well was detected at 460 nm via an enzyme meter. The experiment was repeated three times, with five replicate wells in each group. 2.14 Detection of reactive oxygen species (ROS) The medium in the culture dish was aspirated, DCFH-DA working solution was added according to the instructions, the mixture was mixed well, the mixture was incubated at 37°C in a 5% CO 2 incubator protected from light for 30 minutes, the DCFH-DA working solution was discarded, the mixture was washed three times with PBS to remove excess probe, PBS was added, and the mixture was observed under a fluorescence microscope at an excitation wavelength of 488 nm and a reflection wavelength of 525 nm. 2.15 JC-1 staining After the medium was removed, 1 ml of JC-1 staining buffer was added, the mixture was washed twice, 1 ml of cell culture medium was added, 1 ml of JC-1 staining buffer was added, the mixture was mixed gently, the mixture was incubated in the dark at 37°C in a 5% CO 2 incubator for 20 min, the supernatant was discarded, the mixture was washed twice with JC-1 buffer, and 2 ml of JC-1 staining buffer (1×) was added. The cells were observed under a fluorescence microscope. 2.16 Data analysis Statistical analysis was performed via GraphPad Prism 7 software. All experimental data are expressed as the mean ± standard deviation (x ̅ ± s) . One-way ANOVA was used for multiple group comparisons. Two-way ANOVA was used to asses escape latency in the MWM test. P < 0.05 was considered statistically significant. 3. Results 3.1 Gastrodin injection reduces cerebral infarct size and improves neurological deficits after HIBD Compared with that in the sham group, the blood flow in the right hemisphere of the brain was significantly lower than that in the left hemisphere at 24 hours after modeling, and the difference was statistically significant, indicating that the HIBD model was successful (Fig. 1 A-B). The TTC staining results revealed that the right hemisphere of the brain tissue subjected to HIBD presented large white infarct foci, which also indicated that the model was successful compared with that of the sham group (Fig. 1 B-C). In addition, the area of right hemisphere cerebral infarction was significantly lower in the L-GAS, M-GAS, and H-GAS groups than in the HIBD group, and the differences were statistically significant. The most significant effect was observed at a dose of 100 mg/kg. In addition, the Zea-Longa score, flip-flop reflex and negative ground driving test results revealed that (Fig. 1 E-G), compared with those of the sham group, the neurological deficits of the HIBD group were greater; moreover, the time required for the flop reflex and vestibular and proprioceptive functions were impaired, which could be improved by different doses of gastrodin injection, and the effect was most significant when the dose of gastrodin injection was 100 mg/Kg. These results indicate that gastrodin injection can reduce the area of cerebral infarction after HIBD and improve neurological deficits in rats. 3.2 Gastrodin injection Increases the Antioxidant Stress Capacity of Brain Tissue After HIBD and Reduces Neuronal Apoptosis The protein expression of Nrf2, HO-1, P62, Keap1, Bcl2 and Bax in the right cerebral hemisphere was detected by Wester blotting. Compared with those in the sham group, the expression levels of Nrf2, HO-1, P62, and Keap1 were slightly increased and decreased in the HIBD group, and treatment with different doses of gastrodin injection increased the protein expression levels of Nrf2, HO-1, and P62 and significantly decreased the protein expression levels of Keap1. Moreover, the expression of the proapoptotic protein Bax and antiapoptotic protein Bcl-2 was increased and decreased in the HIBD group compared with the sham group, and treatment with different doses of gastrodin injection increased the ratio of Bcl-2/Bax, indicating that gastrodin injection could reduce the occurrence of apoptosis in neuronal cells after HIBD (Fig. 2 A-F). These results indicated that the antioxidative stress capacity of brain tissue was slightly increased and that increased apoptosis was increased after HIBD, but treatment with gastrodin injection could further increase the antioxidative stress capacity and reduced neuronal apoptosis in organic rats. 3.3 Gastrodin injection Increases Antioxidant Stress Factor Levels and Suppresses Inflammatory Responses After HIBD Here, we investigated the expression levels of the oxidative stress related indices SOD, GSH, CAT and MDA in brain tissues from the injured side. As shown in Fig. 3 A-D, compared with those in the sham group, the expression levels of SOD, GSH, and CAT were decreased, and the MDA level was increased in the right sided brain tissue of the HIBD group; the above indices were increased, and the MDA level was decreased in the right sided brain tissue after treatment with different doses of gastrodin injection, and the differences between the M-GAS and H-GAS groups was statistically significant. In addition, the levels of TNF-α, TGF-β and IL-6 in the brain tissue on the injured side were detected via ELISA, as shown in Fig. 3 E-G. The results revealed that the expression of TNF-α and IL-6 increased and the expression of TFG-βdecreased after HIBD, and these effects was reversed by gastrodin injection treatment. These results indicate that gastrodin injection can increase the body's antioxidative stress and anti-inflammatory ability and reduce the occurrence of lipid peroxidation, thus exerting neuroprotective effects. 3.4 Gastrodin injection improves mitochondrial structure in brain tissue after HIBD When hypoxia occurs, it can affect the function of mitochondria in brain tissue, resulting in impaired energy metabolism [ 21 ] . The results (Fig. 4 ) revealed that the morphology and structure of the neurons in the sham group were more normal, the nuclei were larger and almost ovoid, and the chromatin staining was brighter, with mainly euchromatin and less heterochromatin; the mitochondria were elliptical with clearly visible cristae, plate-like or tubular, and the matrix was uniformly dense with electrons; the nuclei of the neurons in the HIBD group were crumpled in an irregular shape, and the staining was lighter, with mainly euchromatin and less heterochromatin; and many mitochondria were obviously swollen, with the cristae reduced, broken or fractured, resulting in energy metabolism disorders [ 21 ] . In the HIBD group, the nuclei of the neurons were wrinkled and irregularly shaped, the chromatin was lighter in color, with more euchromatin and less heterochromatin; a large number of mitochondria were obviously swollen; the cristae were reduced, broken or has disappeared; the matrix was flocculent or even vacuolated, and the electron density was reduced. The neuronal and mitochondrial structures were improved to different degrees by treatment with gastrodin injection at different doses. The results showed that gastrodin injection improved the structural abnormalities of neurons and mitochondria in brain tissue after HIBD. 3.5 Gastrodin injection reduces brain tissue damage after HIBD The neuroprotective effect of gastrodin injection on HIBD was evaluated by HE staining and Nissl staining. HE staining revealed (Fig. 5 A) that the brain tissue of the sham group was structurally intact and that the nerve cells were structurally intact and regularly arranged. The brain tissue of the HIBD injury group was structurally incomplete, with disorganized nerve cell arrangement and the nuclei of the nerve cells were solidified; however, these injuries were partially restored by gastrodin injection treatment. Nissl’s staining was used to determine the condition of the Nissl cells in the brain tissue (Fig. 5 B). Nissl cells were more numerous, structurally complete and regularly arranged in the sham group, Nissl cells were significantly reduced, and the cell arrangement was disorganized during HIBD injury, which could be improved by gastrodin injection. These results suggest that gastrodin injection can reduce HIBD-induced neuronal damage. 3.6 Gastrodin injection improves spatial learning memory and motor coordination in rats after HIBD To investigate the effect of gastrodin injection on the long-term prognosis of the rats in each group after HIBD, we conducted a water maze test and a rotating rod test. As shown in Fig. 6 A -B, as the number of training days increased, the time needed to find the platform in the sham group gradually decreased, whereas the time needed to find the platform in the HIBD group did not change significantly with increasing training time, which could be improved by gastrodin injection treatment. In the test on the 6th day, the spatial memory ability of the rats was determined by the number of times they traversed the platform area after the platform area was withdrawn, as shown in Fig. 6 C -D. In the model group, the number of times that the platform area was traversed after the platform area was withdrawn was determined by the number of times it traversed the platform area after the platform area was withdrawn. In the model group, the number of times the rats crossed the platform was recorded. As shown in Fig. 6 C -D, the number of times the rats in the model group traversed the platform was lower than that in the sham group, was whereas the gastrodin injection increased the number of times the rats traversed the platform after HIBD. In the rotating bar experiment, the rats were trained for the first 3 days and tested on the 4th day. As shown in Fig. 6 E, compared with the sham group, the rats in the HIBD group had a shorter residence time on the rotating rod; and treatment with gastrodin injection increased the residence time of rats. These results indicate that gastrodin injection improved the spatial learning memory ability and motor coordination ability of rats after HIBD and improved long-term neurological prognosis after HIBD. 3.7 Gastrodin injection improves brain damage in rats after HIBD The effects of gastrodin injection on the long-term prognosis of brain tissues were evaluated by HE staining and Nissl staining. Previous experimental results revealed that the most significant effect was observed at a gastrodin injection dose of 100 mg/kg, so this dose was used to treat rats for long-term behavioral evaluation. As shown in Fig. 7 A, HE staining revealed that at the age of 28 days, the brain tissue atrophy on the damaged side and part of the brain tissue, especially in the hippocampal region, were absent in the HBID group, whereas in the gastrodin injection treated group, brain tissue atrophy on the damaged side was restored, and brain deficits, especially in the hippocampal region, were reduced compared with those in the HBID group. Similarly, as shown in Fig. 7 B, Nissl staining revealed that the number of Nissl cells was lower in the HIBD group, especially in the hippocampal region, than in the sham group at the age of 28 days, which was ameliorated by treatment with gastrodin injection. These results indicate that gastrodin injection can ameliorate long-term brain tissue damage after HIBD. 3.8 Gastrodin injection Ameliorates OGD/R-Induced SH-SY5Y Cell Damage A CCK-8 assay was used to determine whether gastrodin injection (0, 20, 40, 80, 160, and 320 µmol) is potentially toxic to SH-SY5Y cells. As shown in Fig. 8 A, the proliferative viability of SH-SY5Y cells was not affected at gastrodin injection concentrations up to 160 µmol/L. Next, SH-SY5Y cells were subjected to OGD/R to determine the effect of gastrodin injection (20, 40, or 80 µmol) on cell viability. As shown in Fig. 8 B, the cell survival rate was significantly lower after OGD/R treatment than in the control, and the cell survival rate was increased by different doses of gastrodin injection. The effect was most significant at a gastrodin injection concentration of 40 µmol, which was used for the following experiments.The oxidative stress response of the cells could be caused by OGD/R, which led to an increase in the production of ROS as shown in Fig. 8 C. As show in Fig. 8 C, green fluorescence increased significantly in the OGD/R group, and treatment with gastrodin injection reduced the green fluorescence and the production of ROS. After OGD/R, the energy supply is impaired, resulting in mitochondrial dysfunction, which leads to a decrease in the mitochondrial membrane potential [ 22 ] . As show in Fig. 8 D, the red fluorescence decreased and green fluorescence increased after OGD/R injury, while the red fluorescence increases and decreases the production of green fluorescence after gastrodin injection treatment These results indicated that gastrodin injection could improve the survival of OGD/R-induced SH-SY5Y cells, reduce the production of ROS, inhibit the occurrence of oxidative stress, increase the mitochondrial membrane potential, and improve mitochondrial function. 3.9 Gastrodin injection Reduces OGD/R-Induced SH-SY5Y Cell Damage by Activating the P62/Nrf2/HO-1 Pathway The results of the in vitro Western blot analysis revealed that the expression levels of the oxidative stress-related proteins Nrf2, HO-1, and P62 tended to increase in the OGD/R cell model, and the expression of these proteins further increased after treatment with gastrodin injection, suggesting that gastrodin injection could inhibit the oxidative stress response of cells after OGD/R. The expression level of the apoptosis protein Bcl-2/Bax decreased after OGD/R, indicating that apoptosis increased, whereas Bcl-2/Bax increased after gastrodin injection treatment, indicating that gastrodin injection reduced apoptosis; however, all these protective effects of gastrodin injection could be attenuated by XRK3F2, a specific inhibitor of P62 (Fig. 9 A-F). and the above results indicated that gastrodin injection reduced SH-SY5Y cell damage injury after OGD/R and that this protective effect may be realized by regulating P62. 4 Discussion According to statistics, approximately 750,000 infants worldwide suffer from moderate or severe HIE each year, resulting in neurodevelopmental disorders in approximately 400,000 infants, and HIE accounts for 2.4% of the total global burden of disease [ 23 ] . Although the incidence of neonatal asphyxia has decreased significantly with the development of asphyxia resuscitation techniques, it is still one of the leading causes of neonatal death. Currently, subcapnia is internationally recognized as an effective treatment for moderate and severe HIE, and only 50–60% of HIE cases eligible for subcapnia treatment are affected by gestational age, birth weight, coagulation and circulatory function [ 24 , 25 ] . Therefore, it is important to find effective treatments for HIE. After the occurrence of HIE, the damage can be divided into three stages: In the first stage, owing to the increase in anaerobic fermentation after hypoxia, the energy supply is rapidly depleted, resulting in a large amount of Ca 2+ and excitatory neurotransmitters flowing into the cells, leading to cellular damage [ 26 ] ; in the second stage, reperfusion injury, resulting in a dramatic increase in reactive oxygen species (ROS), oxygen free radicals, and mitochondrial dysfunction, and secondary energy failure, while oxidative stress injury, excitatory amino acid toxicity, and inflammatory response lead to neuronal damage, apoptosis, and ultimately neuronal death [ 27 , 28 ] ; and in the third stage, sustained brain damage due to chronic inflammation and disruption of neuronal synapses and axon formation [ 27 ] . Therefore, inhibiting oxidative stress damage may be a novel approach for the treatment of HIE. Studies have shown that oxidative stress may mediate the progression of HIE through inflammation. After the onset of ischemia and hypoxia, microglia respond rapidly and produce large amounts of inflammatory factors (TNF-α, IL-1β, etc.), glutamate, nitric oxide (NO), and ROS, etc., and these cytokines induce the progression of HIE, and these cytokines induce neuronal apoptosis and are positively correlated with the severity of HIE [ 29 ] . Second, disruption of the energy supply and accumulation of large amounts of ROS and Ca 2+ influx into the cell lead to mitochondrial damage, which decreases the mitochondrial membrane potential (ΔΨm), increases cytochrome C release, activates apoptosis-associated proteins, induces neuronal apoptosis and ultimately leads to cell death [ 30 ] . Studies have shown that while oxidative stress markers, including superoxide dismutase (SOD), malondialdehyde (MDA), the inflammation-related factors IL-6, TNF-α, and IL-1β, metabolism-related markers lactate dehydrogenase (LDH), etc., in cerebrospinal fluid or serum after HIE, can be used as sentinel markers to determine the prognosis of HIE [ 31 ] . Nrf2 is a key regulator of important cellular defense and antioxidative stress pathways [ 32 ] . Nrf2 consists of 605 amino acid residues including seven homologous structural domains, Neh1-6, of which Neh2 binds to Kelch-like ECH-associated protein (Keap1) in the cytoplasm to maintain its stability [ 33 ] . HO-1 is a molecule downstream of Nrf2 that has antioxidant effects [ 34 ] . Under normal conditions, Neh2 binds to Keap1 in the cytoplasm, and when oxidative stress occurs, Neh2 separates from Keap1 and Nrf2 translocates to the nucleus, where it binds to the antioxidant response element (ARE) and initiates downstream oxidative enzymes (catalase, CAT), superoxide dismutase-1 (SOD-1), HO-1, and other antioxidative stress genes [ 35 ] . When cerebral ischemia occurs, Nrf2 begins to increase within 3 hours and peaks at 24 hours. Studies have shown that P62 is closely related to the crosstalk of the Nrf2 signaling pathway and that P62 can competitively bind Keap1 with Nrf2 to increase the release of Nrf2 and initiate the expression of downstream antioxidant genes. In addition, P62 can promote the degradation of Keap1 via selective autophagy, resulting in an increase in the release of Nrf2, which in turn can regulate the expression of AREs and promote the transcription of downstream antioxidant genes [ 36 ] . Wang et al. reported that knockdown of the P62 gene resulted in a significant decrease in Nrf2 expression and a significant increase in Keap1 expression [ 37 ] , whereas overexpression of the P62 gene increased Nrf2 and decreased Keap1 expression [ 38 ] . Li et al. reported that intraperitoneal injection of echinocystic acid could reduce the area of cerebral infarction and improve neurobehavioral scores by activating the Nrf2/HO-1 signaling pathway, increasing the expression of the antiapoptoic protein Bcl-2 protein, and decreasing the expression of the proapoptotic proteins Bax and cleaved-caspase-3 in HIBD model mice; moreover, observing the long-term prognosis revealed that it could reduce the brain defect area [ 39 ] . In this study, gastrodin injection reduced the cerebral infarct area on the ischemic side of HIBD injury model mice, improved neurological deficits, reduced the production of the inflammatory factors TNFα and IL-6, increased the production of inflammation-suppressing factor TGF-β, and increased the expression of the antioxidative stress-related proteins Nrf2, HO-1, and P62 on the ischemic side of brain tissue, reduced the expression of the apoptosis-related protein Bax in brain tissue, increased the expression of anti-apoptosis-related protein Bcl-2 in brain tissue, and improved mitochondrial function, thus playing a neuroprotective role. Li et al. also reported that the phenolic compound 3,4-dihydroxybenzaldehyde in gastrodin could reduce the release of the inflammatory factors TNF-α, IL-6, and IL-1β, increase the expression of the anti-inflammatory factors TGF-β and TGF-β2; and promote the transition of microglia from the M1- type (proinflammatory) to M2- type (reparative) [ 40 ] . In addition, in an in vitro model of Alzheimer's disease (AD), aspalathin increased the survival rate of β-amyloid-treated neuronal cells, reduced the release of proinflammatory factors and NO, and minimized neuronal cell apoptosis, which protected neuronal cells from neurotoxicity [ 41 ] . These results suggest that gastrodin has neuroprotective effects. In this study, the results of HE staining and Nissl staining revealed that at the early stage of HIBD injury, the site of injury was mainly the cortical region of the brain; however, over time, the main site of injury was the hippocampal region, possibly because after hypoxia and ischemia occur in brain tissues, the blood supply to the brain preferentially supplies the regions of the hippocampus, thalamus, and hippocampus, whereas the terminal supply regions of cerebral arteries, such as the cerebral cortex, are prone to damage, and with the recovery of the blood supply, the brain tissue becomes extremely sensitive to ischemia-reperfusion injury; the damage to the hippocampus region, which affects learning, memory ability and motor coordination, and these neurological sequelae can be ameliorated by gastrodin injection. In addition, the present study further confirmed that SH-SY5YS cell survival was reduced after OGD/R injury and that gastrodin injection increased the expression of antioxidative stress proteins and antiapoptotic proteins, decreased the expression of proapoptotic proteins, reduced the production of ROS, and inhibited the attenuation of the mitochondrial membrane potential after OGD/R injury. However, all of these protective effects could be attenuated by a P62-specific inhibitor, suggesting that these neuroprotective effects may be related to the activation of the P62/Nrf2/HO-1 signaling pathway. The present study also has several limitations: P62 is an important substrate in the process of autophagy [ 42 ] ; however, the role of autophagy in neurological disorders is still controversial, and recent studies have shown that severe acute cerebral hypoxia and ischemia overactivate autophagy, leading to neuronal cell injury and death. However, chronic or mild hypoxia can moderately activate autophagy to remove damaged organelles to exert protective effects [ 43 ] ; however, the relationship between P62 and autophagy was not investigated in depth in this study, which is a future research direction. 5 Conclusion In conclusion, gastrodin injection can increase antioxidant stress resistance, reduce inflammation and apoptosis in the brain tissue of rats after HIBD through the P62/Nrf2/HO-1 signaling pathway, and improve their long-term neurological prognosis. These findings provide new ideals for the treatment of HIE. Declarations Data availability statement The original contributions presented in the study are included in the article, and further inquiries can be directed to the corresponding author. Ethics statement The animal experiments were reviewed and approved by the Animal Experiment Review and Approval and Animal Research Ethics Committee of Guizhou Medical University (No.2400028). Author contributions Sha Wu, Jianwei Xu and Maoqiong Chen designed the research study. Sha Wu and Zhenkui Ren performed the majority of the experiments and data collection. Sha Wu and Mengting Yang analyzed the data to generate graphs. Mengting Yang, Xianxian Li, Xuxian Wu and Ying Xiong provided technical support. Sha Wu and Zhenkui Ren wrote the manuscript. Jianwei Xu and Maoqiong Chen revised the manuscript. Funding This study was funded by the National Natural Science Foundation of China (82160126), the Special Project for Scientific and Technological Research on Traditional Chinese Medicine and Ethnic Medicine of Guizhou Province Administration of Traditional Chinese Medicine (QZYY-2024-012), and the Science and Technology Department of Guizhou Province (ZK [2022] General 415) and Affiliated Hospital of Guizhou Medical University (2021-GMHCT-001). 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6630703","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":473077803,"identity":"8ff33e2d-e379-46a2-a38c-a96d60ec9f3b","order_by":0,"name":"Sha Wu","email":"","orcid":"","institution":"Guizhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Sha","middleName":"","lastName":"Wu","suffix":""},{"id":473077804,"identity":"c97c61e5-d374-438d-88c0-c69b6037db6e","order_by":1,"name":"Zhenkui Ren","email":"","orcid":"","institution":"Second People's Hospital of Guizhou Province","correspondingAuthor":false,"prefix":"","firstName":"Zhenkui","middleName":"","lastName":"Ren","suffix":""},{"id":473077805,"identity":"40b8e925-335d-4593-a752-69ec28ceeeb8","order_by":2,"name":"Mengting Yang","email":"","orcid":"","institution":"Affiliated Hospital of Guizhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Mengting","middleName":"","lastName":"Yang","suffix":""},{"id":473077807,"identity":"f26e9c70-ad9f-4893-9237-77d61520d0b7","order_by":3,"name":"Ying Xiong","email":"","orcid":"","institution":"Guizhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Xiong","suffix":""},{"id":473077808,"identity":"f1a65d15-b6e3-4fb4-b3d5-1e41693f5674","order_by":4,"name":"Xianxian Li","email":"","orcid":"","institution":"Guizhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xianxian","middleName":"","lastName":"Li","suffix":""},{"id":473077809,"identity":"c9974ae2-b3af-47e7-bb0f-6805ea9bb687","order_by":5,"name":"Xuxian Wu","email":"","orcid":"","institution":"Guizhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xuxian","middleName":"","lastName":"Wu","suffix":""},{"id":473077810,"identity":"514f0815-0ddf-4c36-a5d0-387b6ac9dbec","order_by":6,"name":"Jianwei Xu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxklEQVRIiWNgGAWjYBACefb2gw8SKv7V7z/eQKQWw54zyQYPzhxgbDhzgFhrbjiYST5sA2q5kUCkDsYZDGkSCWx3mBlnPt54g6HGJpqgFnbpxsMWCTzP2Jil04otGI6l5TYQtGXOgcQbCRLMPGzSOWYSjA2HCWthuJFgIJFgwCzBI3mGeC1GEgkJhw0kJHiI1AIO5IQDaQkGPEC/JBDjF1BUPvz5zybBgP3wxhsfamyIcBgSAHqKFOUQLaTqGAWjYBSMgpEBAC7vQ5/Jy3rrAAAAAElFTkSuQmCC","orcid":"","institution":"Guizhou Medical University","correspondingAuthor":true,"prefix":"","firstName":"Jianwei","middleName":"","lastName":"Xu","suffix":""},{"id":473077811,"identity":"8ec17c00-fd72-421a-b384-c7a1d0e69708","order_by":7,"name":"Maoqiong Chen","email":"","orcid":"","institution":"Affiliated Hospital of Guizhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Maoqiong","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2025-05-09 17:38:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6630703/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6630703/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12035-025-05522-z","type":"published","date":"2025-11-24T15:57:05+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":85171560,"identity":"7390b6dd-45dd-4231-8254-329ec633b9cb","added_by":"auto","created_at":"2025-06-23 05:44:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":202247,"visible":true,"origin":"","legend":"\u003cp\u003eGastrodin injection attenuates hypoxic-ischemic brain damage in neonatal rats. (A) Laser scatter contrast imaging for dynamic monitoring of cortical surface blood flow after HIBD and cerebral blood flow (CBF) assessment via an ROI. (B) Quantification of CBF in the ROI of the right cerebral hemisphere in both groups; compared with the sham group,\u003csup\u003e\u003cem\u003e ###\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0. 001;\u003cem\u003e n \u003c/em\u003e= 4. (C) TTC-stained cerebral coronal sections at 24 hr after HIBD. (D) TTC-stained (D) Quantification of the cerebral infarct area; compared with the sham group, \u003csup\u003e\u003cem\u003e##\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; compared with the HIBD group, \u003csup\u003e\u003cem\u003e*\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.05,\u003csup\u003e\u003cem\u003e **\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.01, \u003cem\u003en \u003c/em\u003e= 8. (E) Neurological deficit scores of the rats in each group; (F) Negative grounding test, the time required to rotate 180° (s); (G) Turning over reflex test, the time required to turn over from the supine position to the prone position (s); compared with the sham group, \u003csup\u003e\u003cem\u003e##\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01; compared with the HIBD group, \u003csup\u003e\u003cem\u003e##\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; compared with the HIBD group, \u003csup\u003e\u003cem\u003e##\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; compared with the HIBD group,\u003cem\u003e \u003c/em\u003e\u003csup\u003e\u003cem\u003e##\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; \u003cem\u003en\u003c/em\u003e = 4. \u0026lt; 0. 01; compared with HIBD, \u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.01, \u003cem\u003en\u003c/em\u003e = 10.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6630703/v1/1fcea30c922e3414cfc8357a.png"},{"id":85171565,"identity":"791bfeab-6c6e-4fbc-90a9-74cfd935d6d6","added_by":"auto","created_at":"2025-06-23 05:44:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":270162,"visible":true,"origin":"","legend":"\u003cp\u003eGastrodin injection increases the antioxidative stress capacity of brain tissues and reduces neuronal apoptosis after HIBD (A) Protein immunoblotting results for Nrf2, HO-1, P62, Keap1, Bax, and Bcl-2 at 24 hours after HIBD. (B-F) Quantification of protein immunoblotting for Nrf2, HO-1, Keap1, P62, and Bax/Bcl-2; \u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0. 05, \u003csup\u003e\u003cem\u003e##\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0. 01 compared with the sham group;\u003cem\u003e \u003c/em\u003e\u003csup\u003e\u003cem\u003e*\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.05,\u003csup\u003e \u003c/sup\u003e\u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.01 compared with the HIBD group, \u003cem\u003en\u003c/em\u003e = 3.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6630703/v1/a8df2b2a70bdefce1e6b3d7c.png"},{"id":85171568,"identity":"5a8b6f81-3224-4428-b4e8-32613d39692b","added_by":"auto","created_at":"2025-06-23 05:44:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":145488,"visible":true,"origin":"","legend":"\u003cp\u003eGastrodin injection increases the level of antioxidative stress factors and decreases proinflammatory factor formation after HIBD. (A-D) SOD, GSH-PX, CAT and MDA levels in the brain tissues of each group. (E-G) TNF-α, TGF-β and IL-6 levels in the brain tissues of each group. Compared with SHAM, \u003csup\u003e\u003cem\u003e##\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0. 01, \u003csup\u003e\u003cem\u003e####\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0. 0001; compared with HIBD, \u003csup\u003e\u003cem\u003e*\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e P\u003c/em\u003e \u0026lt; 0. 05, \u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e P\u003c/em\u003e \u0026lt; 0. 01, \u003csup\u003e\u003cem\u003e***\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e P\u003c/em\u003e \u0026lt; 0. 001, \u003cem\u003en\u003c/em\u003e = 3.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6630703/v1/27367f6950d08ce9d70294db.png"},{"id":85171579,"identity":"9e6d5113-b9f0-4ba7-b81b-6eee59dbafbe","added_by":"auto","created_at":"2025-06-23 05:44:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":178564,"visible":true,"origin":"","legend":"\u003cp\u003eTransmission electron microscopy of mitochondria in the brain tissue of each group Nuclei (N), mitochondrial swelling (↑); ×20,000.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6630703/v1/4fc009c65b55edb173dc2d34.png"},{"id":85171576,"identity":"a1d6ec42-6bc8-4d1e-b87b-3f753a9d6301","added_by":"auto","created_at":"2025-06-23 05:44:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":304476,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of gastrodin injection on brain tissues 24 hours after HIBD. (A) Representative images of HE stained samples from each group (scale bar: 20 μm); (B) Representative images of Nissl-stained samples from each group (scale bar: 20 μm).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6630703/v1/a96977b03f95485b0c93eb99.png"},{"id":85172316,"identity":"cb84a111-08cf-4dbe-8394-f8ea152404d3","added_by":"auto","created_at":"2025-06-23 05:52:58","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":199186,"visible":true,"origin":"","legend":"\u003cp\u003eGastrodin injection improved long-term neurological prognosis after HIBD. (A-B) The mean latency of the rats in each group increased with increasing training time. (C-D) Number of times the rats in each group crossed the platform on day 6. (E) Duration of stay on the rotating rod in each group. Compared with SHAM,\u003csup\u003e\u003cem\u003e ##\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0. 01; Comparison with HIBD, \u003csup\u003e\u003cem\u003e** \u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0. 01, \u003cem\u003en \u003c/em\u003e= 5.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6630703/v1/66bc5506b25a72573e6214d4.png"},{"id":85171598,"identity":"4fbef12d-091e-4f9a-99a7-f6276e4b1a74","added_by":"auto","created_at":"2025-06-23 05:44:59","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":340875,"visible":true,"origin":"","legend":"\u003cp\u003eStructural effects of gastrodin injection on brain tissue long-term after HIBD. (A) Representative images of HE stained samples from each group (scale bar: 20 μm). (B) Representative images of Nissl-stained samples from in each group (scale bar: 20 μm).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6630703/v1/36e7cd35ba3b365433242e72.png"},{"id":85171593,"identity":"16faca9c-2a0d-4def-b378-f0c17f9a3f87","added_by":"auto","created_at":"2025-06-23 05:44:59","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":501666,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of gastrodin injection on OGD/R-induced damage in SH-SY5Y cells. (A) Viability of SH-SY5Y cells after treatment with 0, 20, 40, 80, 160, or 320 μmol gastrodin injection, compared with control,\u003csup\u003e\u003cem\u003e ##\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0. 01; compared with OGD/R, \u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0. 01, \u003cem\u003en\u003c/em\u003e = 3. (B) Viability of OGD/R-treated (with or without gastrodin injection (20, 40, 80 μmol) treatment) SH-SY5Y cells;\u003cem\u003e n \u003c/em\u003e= 3. (C) Representative fluorescence images of ROS staining, \u003cem\u003en\u003c/em\u003e = 3, scale bar: 50 μm. (D) Representative graphs of JC-1 staining, \u003cem\u003en \u003c/em\u003e= 3, cale bar: 50 μm.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-6630703/v1/f00d57c9caa44ee08ca36d27.png"},{"id":85171592,"identity":"ed71f2e2-a7dd-4cb5-a70f-55ce7dae9244","added_by":"auto","created_at":"2025-06-23 05:44:59","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":265379,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of gastrodin injection on the P62/Nrf2/HO-1 signaling pathway after OGD/R. (A) Results of protein immunoblotting by gastrodin injection on Nrf2, HO-1, P62, Keap1, Bax and Bcl-2 in SH-SY5Y cells after OGD/R. (B-F) Quantification of protein immunoblotting for Nrf2, HO-1, Keap1, P62, and Bax/Bcl-2. Compared with the control, \u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0. 05, \u003csup\u003e\u003cem\u003e##\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0. 01; compared with OGD/R, \u003csup\u003e\u003cem\u003e* \u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0. 05, \u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e P\u003c/em\u003e \u0026lt; 0. 01, \u003cem\u003en\u003c/em\u003e = 3.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-6630703/v1/ce7fe59e3a0ee0b9c4416f47.png"},{"id":97178686,"identity":"c8eae500-8da0-4780-af83-17ff629ba27c","added_by":"auto","created_at":"2025-12-01 16:12:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3483351,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6630703/v1/bb77998e-f6c8-401b-a769-5e2d49214a46.pdf"},{"id":85171574,"identity":"db091724-ee57-4765-82be-2838104d82f1","added_by":"auto","created_at":"2025-06-23 05:44:58","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19492636,"visible":true,"origin":"","legend":"","description":"","filename":"WBimageofacellexperiment1.tif","url":"https://assets-eu.researchsquare.com/files/rs-6630703/v1/c1cc49b1271da05f4b223733.tif"},{"id":85172322,"identity":"96ccbc35-d921-4b5e-8927-c47b62939732","added_by":"auto","created_at":"2025-06-23 05:52:59","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":18936268,"visible":true,"origin":"","legend":"","description":"","filename":"WBimageofacellexperiment2.tif","url":"https://assets-eu.researchsquare.com/files/rs-6630703/v1/3f76bc45d1d71ee17d3d569a.tif"},{"id":85173388,"identity":"a4807dc3-a1ab-4590-883b-0038aa5fa2f3","added_by":"auto","created_at":"2025-06-23 06:00:58","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":26267172,"visible":true,"origin":"","legend":"","description":"","filename":"WBimagesofanimalexperiments1.tif","url":"https://assets-eu.researchsquare.com/files/rs-6630703/v1/a2739858890958c821f9071e.tif"},{"id":85171610,"identity":"faffae3f-3196-4782-bc0d-fbb31dbeff86","added_by":"auto","created_at":"2025-06-23 05:45:00","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":26796392,"visible":true,"origin":"","legend":"","description":"","filename":"WBimagesofanimalexperiments2.tif","url":"https://assets-eu.researchsquare.com/files/rs-6630703/v1/991b3b82188f2b8a6c9ec0a9.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Gastrodin injection relieves hypoxic-ischemic brain injury in newborn rats by regulating the P62/Nrf2/HO-1 pathway","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eNeonatal hypoxic-ischemic encephalopathy (HIE) is a type of neonatal brain injury caused by various forms of perinatal hypoxia and is one of the main causes of neonatal encephalopathy, with an incidence of 1.5\u0026permil;. Approximately 25% of affected children have neurological sequelae such as epilepsy, cerebral palsy, and cognitive disorders \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. In China, the incidence of neonatal HIE approximately 3\u0026permil;-6\u0026permil;, and the mortality rate accounts for 15.2% of the mortality rate of children under 5 years of age \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Although subcooling therapy can reduce the incidence of neurological sequelae after HIE, it is limited by the inclusion criteria and time window, which still prevents a larger number of children from undergoing HT \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Therefore, developing effective treatments for HIE is important.\u003c/p\u003e \u003cp\u003eThe result of multifactorial interactions in HIE injury, including oxidative stress, excitatory amino acid toxicity, inflammatory response, and neuronal apoptosis, etc. After the onset of ischemia and hypoxia, due to the lack of ATP, the inactivation of ATP-dependent ions results in Na\u003csup\u003e+\u003c/sup\u003e/K+-ATP pump malfunction, resulting in the inward influx of Na\u003csup\u003e+\u003c/sup\u003e and Ca\u003csup\u003e2+\u003c/sup\u003e into cells, leading to intracellular calcium overload, excessive Ca\u003csup\u003e2+\u003c/sup\u003e uptake triggers a molecular neurotoxic cascade reaction, which further leads to the overproduction of free radicals, mitochondrial dysfunction, a reduction in the mitochondrial membrane potential (ΔΨm), the release of apoptotic substances, cell membrane damage, and DNA breakage; In addition, after hypoxia, oxidative stress injury leads to the production of a large amount of ROS and, at the same time, the activation of microglia, the production of a large number of inflammatory factors (TNF-α, IL-1β, etc.), which further exacerbates tissue injury \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Therefore, the inhibition of oxidative stress damage may be a novel approach for the treatment of HIE. The Nrf2/HO-1 signaling pathway is a key regulator of important cellular defenses and the antioxidative stress pathway \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Celastrol reduces the area of cerebral infarction, decreases microglial activation, and suppresses the levels of inflammatory factors and oxidative stress in SD rats after HIE by activating the Nrf2/HO-1 signaling pathway \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGastrodia elata is a traditional Chinese medicine used in China to treat dizziness and limb numbness in adults and infantile spasms, epilepsy, convulsions and tetanus in children \u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Studies have shown that Gastrodia elata has a neuroprotective effect and that Gastrodin (GAS), an extract of Gastrodia elata and one of its phenolic compounds, can cross the blood-brain barrier and is used to treat a variety of neurological disorders \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Previous studies have shown that in an animal model of ischemia/reperfusion (I/R) injury, GAS can reduce the expression of inflammatory factors such as IL-1β and TNF-α; increase the expression of antioxidant stress-related proteins such as heme oxygenase-1 (HO-1), SOD and the transcription factor Nrf2; and reduce the incidence of neuronal apoptosis, thereby exerting neuroprotective effects \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Therefore, GAS may be a potential therapeutic drug for HIE.\u003c/p\u003e \u003cp\u003eIn this study, we established a neonatal rat HIBD model and used SH-SY5Y cells to establish an OGD/R injury model to investigate the neuroprotective role of gastrodin injection in HIBD and whether the P62/Nrf2/HO-1 signaling pathway is involved in this process.\u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Reagents\u003c/h2\u003e \u003cp\u003eGastrodin injection was purchased from Southwest Pharmaceutical Co., Ltd. (manufacturing batch number: H20064391); primary antibodies: Nrf2 (Abmart, TA0639, 1:1000), HO-1 (Proteintech, 10701-1-AP, 1:2000), Keap1 (affinity, AF5266, 1:2000), P62 (affinity, AF5384, 1:2000), β-actin (Servicebio, GB15003, 1: 2000), Bax (Proteintech, 50599-2-Ig, 1:2000), and Bcl2 (Proteintech, 26593-1-AP, 1:1000); secondary antibodies (Servicebio, GB15003); ELISA kits: TNF-α, TGF-β, and IL-6 (Jianglai Biology, JL1302, JL13643, JL20896); MDA, SOD, GSH-PX and CAT kits (Nanjing Jiancheng, A003-1, A001-3, A005-1, A007-1-1); TTC staining (Solarbio, G3005); JC-1 staining kit (Servicebio, G1515); and reactive oxygen species (ROS) detection kit (Servicebio, G1706); CCK-8 detection kit (C6005M); fetal bovine serum (Gibco, 2565856P); high glucose DMEM (Gibco); glucose-free DMEM (Servicebio, G4528), P62 inhibitor (MCE, HY-112904).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Neonatal hypoxic-ischemic brain injury model and treatment\u003c/h2\u003e \u003cp\u003e Sprague-Dawley (SD) rats were obtained from the Animal Center of Guizhou Medical University (SCXK (Gui) 2023-0002), Animal Ethics Batch No. NO240028, maintained at a rearing environment temperature of 20\u0026ndash;25℃, humidity of 50\u0026ndash;60%, and light exposure of 12 h per day (8: 00\u0026ndash;20: 00). Adult SD rats with a body mass of 12\u0026ndash;18 g, were freely mated, and neonatal SD rats, male and female, were used and raised by SD females. The samples were randomly divided into five groups: sham, HIBD, L-GAS, M-GAS, and H-GAS groups. Modeling was performed according to the modified Rice- Fannuzzi method \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Neonatal rats were anesthetized via isoflurane inhalation, and the right common carotid artery was isolated within 5 minutes, double-ligated, dissected, and returned to the mother to rest for 1 hour. Neonatal rats were placed in a hypoxic apparatus, a gas mixture (92% N\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;8% O\u003csub\u003e2\u003c/sub\u003e) was delivered at a rate of 1.2 L/min, and the hypoxic apparatus was placed in a 37\u0026deg;C water bath to maintain the chamber temperature at approximately 36\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C for 2 hours. In the sham-operated group, only the right common carotid artery was isolated without ligation or hypoxia; in the other groups, the rats were generated via the anamnestic method, and the rats were returned to their mothers at the end of the model. The treatment group was injected intraperitoneally with different doses of gastrodin injection (50 mg/kg, 100 mg/kg, 200 mg/kg) immediately after modeling, and the model group was injected intraperitoneally with the same volume of saline \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Laser scatter imaging\u003c/h2\u003e \u003cp\u003eLaser scatter imaging is a noninvasive imaging technique widely used in vascular function studies \u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Briefly, the rats were deeply anesthetized with isoflurane after 24 h of modeling, the scalp was removed, the skull was fully exposed, the skull was placed under the camera (approximately 10 cm from the skull), and the original scatter images were acquired via a 785 nm laser beam. Relative blood flow was analyzed by imaging the laser scatter in the region of interest (ROI) via RFLSIZW software (Reward, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 2,3,5-Triphenyltetrazolium Chloride (TTC) Staining\u003c/h2\u003e \u003cp\u003eTTC staining was used to evaluate the volume of cerebral infarction \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Twenty-four hours after HIBD, the brain tissues were removed from the rats after isoflurane anesthesia and execution and sectioned through brain tissue sectioning molds. Coronal sections were generated (thickness of 2 mm), placed in 2% TTC solution, incubated at 37\u0026deg;C for approximately 10\u0026ndash;15 min, placed in 4% paraformaldehyde for fixation overnight, and then dried on absorbent paper before being photographed with a digital camera. Images were taken with a digital camera and then processed and analyzed with ImageJ software. The percentage infarct volume was calculated according to the following formula:\u003c/p\u003e \u003cp\u003eInfarct volume percentage\u0026thinsp;=\u0026thinsp;infarct side volume/contralateral normal hemisphere volume \u0026times; 100%.\u003c/p\u003e \u003cp\u003eInfarct volume percentage = (total area of normal sections on the contralateral side - sum of normal areas of sections on the infarct side)/sum of sections on the contralateral hemisphere \u0026times; 100%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Pathological staining\u003c/h2\u003e \u003cp\u003eAfter HIBD, the rats were deeply anesthetized with isoflurane for 24 hours and 28 days, and the hearts were perfused with 10 ml of PBS and then perfused with an equal volume of 4% paraformaldehyde. The brain tissues were removed, and the brain tissues were removed, immersed in 4% paraformaldehyde for fixation for 24 hours, embedded in paraffin, and cut into coronal sections of 5 \u0026micro;m thickness. Then, the brain sections were deparaffinized, dehydrated, and combined with staining with HE or Nissl staining solution. Finally, the results of histological staining were evaluated and recorded via light microscopy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Short-term Behavioral\u003c/h2\u003e \u003cp\u003eNeurological function was scored via the Longa scale \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e as follows: no defects and normal activity (0 points); flexion of the left forepaw when the tail is raised (1 point); turning in a circle when crawling (2 points); unsteady standing and falling to the left when walking (3 points); and inability to walk independently and loss of consciousness (4 points). Short-term neurobehavioral testing was conducted 24 hours after drug administration by experienced testers. To avoid subjective bias, the experiments were double-blinded, and the experimenters and data analysts were unaware of the groupings.\u003c/p\u003e \u003cp\u003eNegative trend experiment: The rats in each group were placed with their heads and trunks facing down on a rough inclined surface with an angle of inclination of 45\u0026deg;. The time required to rotate the heads and trunks of the newborn rats by 180\u0026deg; was recorded in seconds, and the time of more than 60 s was recorded according to the time of 60 s. The procedure was repeated three times for each rat, with an interval of more than 5 min each.\u003c/p\u003e \u003cp\u003eTurning reflex: The rats in each group were placed with their heads and trunks facing down on a rough inclined surface with an angle of inclination of 45\u0026deg;. The time required to rotate the heads and trunks of the newborn rats by 180\u0026deg; was recorded in seconds, and the time of more than 60 s was recorded according to the time of 60 s. The procedure was repeated three times for each rat, with an interval of more than 5 min each.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Western blot\u003c/h2\u003e \u003cp\u003eExtracted cerebral cortex tissues or SH-SY5Y cells were lysed in RIPA lysis buffer containing 1 mM PMSF, homogenized with a tissue homogenizer, completely lysed on ice for 30 min, and then centrifuged (4\u0026deg;C, 12,000 rpm, 15 min) to obtain supernatants. The protein concentration was measured, and the proteins were prepared via a BCA kit. Proteins were separated by sodium dodecyl sulfate (SDS)-polyacrylamide separation gel electrophoresis, and the bands were subsequently transferred to a PVDF membrane. After being blocked with 5% nonfat milk solution or 5% BSA solution diluted in TBST for 2\u0026ndash;4 hours, the membranes were incubated with primary antibodies: Nrf2 (1:1000), HO-1 (1:1000), P62 (1:1000), Keap1 (1:1000), Bax (1:1000), β-actin (1:5000), and Bcl-2 (1:1000) overnight at 4\u0026deg;C in a refrigerator. The next day, the blots were washed three times with TBST for 10 minutes each and incubated with the appropriate secondary antibody (1:10,000) for 60 minutes. After three washes with TBST, the blots were visualized via an enhanced chemiluminescence (ECL) kit and quantified via an imaging system (Bio-Rad).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Detection of MDA, SOD, GSH-PX and CAT\u003c/h2\u003e \u003cp\u003eA 9x volume of saline was added at a weight (g): volume (mL) ratio\u0026thinsp;=\u0026thinsp;of 1:9, the tissue, was cut, ground well on ice, and centrifuged at 4000 rpm for 10 min, and after which the supernatant was collected for measurement. The assay was performed according to the respective instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.10 ELISA detection\u003c/h2\u003e \u003cp\u003eA 9x volume of PBS was added at a weight (g): volume (mL) ratio\u0026thinsp;=\u0026thinsp;of 1:9, the mixture was mixed well on ice, the homogenate was centrifuged at 5000\u0026times;g for 10 minutes, and the supernatant was collected for testing. The desired plate strips, were removed, 100 \u0026micro;L of supernatant was added to the wells, the plate was covered with a seal and incubated at 37℃ for 1 hour, the mixture was discarded, a biotinylated antibody was added, the mixture was incubated at 37℃ for 1 hour, the mixture was discarded, the mixture was washed with 1\u0026times; washing solution for 1 minute each time 3 times, enzyme conjugate working solution was added, the mixture was incubated at 37℃ for 30 minutes, the plate was washed 5 times, 90 \u0026micro;L of substrate was added to each well, the mixture was incubated at 37℃ without light for 15 minutes, and finally, 50 \u0026micro;L of substrate was added to each well. The plate was washed five times, 90 \u0026micro;L of substrate was added to each well, and the mixture was incubated at 37℃ for 15 minutes. Finally, 50 \u0026micro;L of termination solution was added to each well, and the OD value of each well was immediately measured at 450 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Long-term behavior\u003c/h2\u003e \u003cp\u003eThe Morris water maze (MWM) test is an experiment used to assess the learning and memory ability of animals \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. On the 21st day after HIBD injury, we used WMT-100 Morris water maze video analysis to evaluate the learning and memory ability of experimental animals. A black circular pool with a diameter of 120 cm and a height of 50 cm was prepared in a room isolated from noise and light. The depth of the pool was 1 cm greater than that of the moving platform. Colorless and odorless titanium dioxide was used to cloud the edges of the water, and the pool was divided into four equal quadrants. The rats were trained for 5 days and the platform was removed on day 6. Swimming routes, latency times, and the number of platform crossings were recorded. The motor coordination of the rats in each group was tested 21 days after HIBD injury. The training was started 3 days before the formal experiment \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e, the rats were placed on a rotating rod test and the speed was slowly increased from 4 revolutions per minute (4 rpm) to 40 rpm in 5 min. The rats were recorded 3 times and the average value was taken.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.12 SH-SY5Y cell culture, grouping and glucose deprivation/reoxygenation (OGD/R) modeling\u003c/h2\u003e \u003cp\u003eThe human neuroblastoma SH-SY5Y cell line was obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. The cells were cultured in medium containing 10% fetal bovine serum and 90% high glucose in an incubator at 37 ℃ and 5% CO\u003csub\u003e2\u003c/sub\u003e.SH-SY5Y cells were classified into the following groups: sham, OGD/R, OGD/R\u0026thinsp;+\u0026thinsp;GAS, and OGD/R\u0026thinsp;+\u0026thinsp;GAS\u0026thinsp;+\u0026thinsp;XRK3F2.To establish the OGD/R injury model \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e, the cells were washed three times with PBS, supplemented with unsweetened DMEM, rapidly transferred to a three-gas incubator (37 ℃, 94% N\u003csub\u003e2\u003c/sub\u003e, 5% CO\u003csub\u003e2\u003c/sub\u003e, and 1% O\u003csub\u003e2\u003c/sub\u003e), and treated for 4 h. Sugar-free DMEM was discarded and normal complete medium was added. Different does of gastrodin injection and XRK3F2 were added to the GAS-treated group after modeling, and XRK3F2 was added at a dose of 5 \u0026micro;M \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. PBS was added to the OGD/R group, which was subsequently incubated at 37℃in a 5% CO\u003csub\u003e2\u003c/sub\u003e cell culture incubator for further incubation after 24 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.13 CCK-8 assay for cell viability in each group\u003c/h2\u003e \u003cp\u003eThe cells were inoculated into 96-well plates, cultured and modeled. Cell viability was measured via the CCK-8 assay. SH-SY5Y cells were inoculated into 96-well plates (5000 cells/well) \u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e, and the OGD/R model was established according to the manufacturer\u0026rsquo;s instructions before the cells were treated with CGA at concentrations of 0, 20, 40, 80, 160, and 320 \u0026micro;mol. Twenty-four hours later, 10 \u0026micro;L of CCK-8 solution was added to each well of the 96-well plate which was subsequently incubated for 1.5 h at 37\u0026deg;C. The absorbance of each well was detected at 460 nm via an enzyme meter. The experiment was repeated three times, with five replicate wells in each group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.14 Detection of reactive oxygen species (ROS)\u003c/h2\u003e \u003cp\u003eThe medium in the culture dish was aspirated, DCFH-DA working solution was added according to the instructions, the mixture was mixed well, the mixture was incubated at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator protected from light for 30 minutes, the DCFH-DA working solution was discarded, the mixture was washed three times with PBS to remove excess probe, PBS was added, and the mixture was observed under a fluorescence microscope at an excitation wavelength of 488 nm and a reflection wavelength of 525 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.15 JC-1 staining\u003c/h2\u003e \u003cp\u003eAfter the medium was removed, 1 ml of JC-1 staining buffer was added, the mixture was washed twice, 1 ml of cell culture medium was added, 1 ml of JC-1 staining buffer was added, the mixture was mixed gently, the mixture was incubated in the dark at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator for 20 min, the supernatant was discarded, the mixture was washed twice with JC-1 buffer, and 2 ml of JC-1 staining buffer (1\u0026times;) was added. The cells were observed under a fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.16 Data analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed via GraphPad Prism 7 software. All experimental data are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation \u003cem\u003e(x ̅ \u0026plusmn; s)\u003c/em\u003e. One-way ANOVA was used for multiple group comparisons. Two-way ANOVA was used to asses escape latency in the MWM test. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Gastrodin injection reduces cerebral infarct size and improves neurological deficits after HIBD\u003c/h2\u003e \u003cp\u003eCompared with that in the sham group, the blood flow in the right hemisphere of the brain was significantly lower than that in the left hemisphere at 24 hours after modeling, and the difference was statistically significant, indicating that the HIBD model was successful (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B). The TTC staining results revealed that the right hemisphere of the brain tissue subjected to HIBD presented large white infarct foci, which also indicated that the model was successful compared with that of the sham group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-C). In addition, the area of right hemisphere cerebral infarction was significantly lower in the L-GAS, M-GAS, and H-GAS groups than in the HIBD group, and the differences were statistically significant. The most significant effect was observed at a dose of 100 mg/kg. In addition, the Zea-Longa score, flip-flop reflex and negative ground driving test results revealed that (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-G), compared with those of the sham group, the neurological deficits of the HIBD group were greater; moreover, the time required for the flop reflex and vestibular and proprioceptive functions were impaired, which could be improved by different doses of gastrodin injection, and the effect was most significant when the dose of gastrodin injection was 100 mg/Kg. These results indicate that gastrodin injection can reduce the area of cerebral infarction after HIBD and improve neurological deficits in rats.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e3.2 Gastrodin injection Increases the Antioxidant Stress Capacity of Brain Tissue After HIBD and Reduces Neuronal Apoptosis\u003c/p\u003e \u003cp\u003eThe protein expression of Nrf2, HO-1, P62, Keap1, Bcl2 and Bax in the right cerebral hemisphere was detected by Wester blotting. Compared with those in the sham group, the expression levels of Nrf2, HO-1, P62, and Keap1 were slightly increased and decreased in the HIBD group, and treatment with different doses of gastrodin injection increased the protein expression levels of Nrf2, HO-1, and P62 and significantly decreased the protein expression levels of Keap1. Moreover, the expression of the proapoptotic protein Bax and antiapoptotic protein Bcl-2 was increased and decreased in the HIBD group compared with the sham group, and treatment with different doses of gastrodin injection increased the ratio of Bcl-2/Bax, indicating that gastrodin injection could reduce the occurrence of apoptosis in neuronal cells after HIBD (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-F). These results indicated that the antioxidative stress capacity of brain tissue was slightly increased and that increased apoptosis was increased after HIBD, but treatment with gastrodin injection could further increase the antioxidative stress capacity and reduced neuronal apoptosis in organic rats.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Gastrodin injection Increases Antioxidant Stress Factor Levels and Suppresses Inflammatory Responses After HIBD\u003c/h2\u003e \u003cp\u003eHere, we investigated the expression levels of the oxidative stress related indices SOD, GSH, CAT and MDA in brain tissues from the injured side. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-D, compared with those in the sham group, the expression levels of SOD, GSH, and CAT were decreased, and the MDA level was increased in the right sided brain tissue of the HIBD group; the above indices were increased, and the MDA level was decreased in the right sided brain tissue after treatment with different doses of gastrodin injection, and the differences between the M-GAS and H-GAS groups was statistically significant. In addition, the levels of TNF-α, TGF-β and IL-6 in the brain tissue on the injured side were detected via ELISA, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-G. The results revealed that the expression of TNF-α and IL-6 increased and the expression of TFG-βdecreased after HIBD, and these effects was reversed by gastrodin injection treatment. These results indicate that gastrodin injection can increase the body's antioxidative stress and anti-inflammatory ability and reduce the occurrence of lipid peroxidation, thus exerting neuroprotective effects.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Gastrodin injection improves mitochondrial structure in brain tissue after HIBD\u003c/h2\u003e \u003cp\u003eWhen hypoxia occurs, it can affect the function of mitochondria in brain tissue, resulting in impaired energy metabolism \u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. The results (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) revealed that the morphology and structure of the neurons in the sham group were more normal, the nuclei were larger and almost ovoid, and the chromatin staining was brighter, with mainly euchromatin and less heterochromatin; the mitochondria were elliptical with clearly visible cristae, plate-like or tubular, and the matrix was uniformly dense with electrons; the nuclei of the neurons in the HIBD group were crumpled in an irregular shape, and the staining was lighter, with mainly euchromatin and less heterochromatin; and many mitochondria were obviously swollen, with the cristae reduced, broken or fractured, resulting in energy metabolism disorders \u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. In the HIBD group, the nuclei of the neurons were wrinkled and irregularly shaped, the chromatin was lighter in color, with more euchromatin and less heterochromatin; a large number of mitochondria were obviously swollen; the cristae were reduced, broken or has disappeared; the matrix was flocculent or even vacuolated, and the electron density was reduced. The neuronal and mitochondrial structures were improved to different degrees by treatment with gastrodin injection at different doses. The results showed that gastrodin injection improved the structural abnormalities of neurons and mitochondria in brain tissue after HIBD.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Gastrodin injection reduces brain tissue damage after HIBD\u003c/h2\u003e \u003cp\u003eThe neuroprotective effect of gastrodin injection on HIBD was evaluated by HE staining and Nissl staining. HE staining revealed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) that the brain tissue of the sham group was structurally intact and that the nerve cells were structurally intact and regularly arranged. The brain tissue of the HIBD injury group was structurally incomplete, with disorganized nerve cell arrangement and the nuclei of the nerve cells were solidified; however, these injuries were partially restored by gastrodin injection treatment. Nissl\u0026rsquo;s staining was used to determine the condition of the Nissl cells in the brain tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Nissl cells were more numerous, structurally complete and regularly arranged in the sham group, Nissl cells were significantly reduced, and the cell arrangement was disorganized during HIBD injury, which could be improved by gastrodin injection. These results suggest that gastrodin injection can reduce HIBD-induced neuronal damage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Gastrodin injection improves spatial learning memory and motor coordination in rats after HIBD\u003c/h2\u003e \u003cp\u003eTo investigate the effect of gastrodin injection on the long-term prognosis of the rats in each group after HIBD, we conducted a water maze test and a rotating rod test. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA -B, as the number of training days increased, the time needed to find the platform in the sham group gradually decreased, whereas the time needed to find the platform in the HIBD group did not change significantly with increasing training time, which could be improved by gastrodin injection treatment. In the test on the 6th day, the spatial memory ability of the rats was determined by the number of times they traversed the platform area after the platform area was withdrawn, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC -D. In the model group, the number of times that the platform area was traversed after the platform area was withdrawn was determined by the number of times it traversed the platform area after the platform area was withdrawn. In the model group, the number of times the rats crossed the platform was recorded. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC -D, the number of times the rats in the model group traversed the platform was lower than that in the sham group, was whereas the gastrodin injection increased the number of times the rats traversed the platform after HIBD. In the rotating bar experiment, the rats were trained for the first 3 days and tested on the 4th day. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, compared with the sham group, the rats in the HIBD group had a shorter residence time on the rotating rod; and treatment with gastrodin injection increased the residence time of rats. These results indicate that gastrodin injection improved the spatial learning memory ability and motor coordination ability of rats after HIBD and improved long-term neurological prognosis after HIBD.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Gastrodin injection improves brain damage in rats after HIBD\u003c/h2\u003e \u003cp\u003eThe effects of gastrodin injection on the long-term prognosis of brain tissues were evaluated by HE staining and Nissl staining. Previous experimental results revealed that the most significant effect was observed at a gastrodin injection dose of 100 mg/kg, so this dose was used to treat rats for long-term behavioral evaluation. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, HE staining revealed that at the age of 28 days, the brain tissue atrophy on the damaged side and part of the brain tissue, especially in the hippocampal region, were absent in the HBID group, whereas in the gastrodin injection treated group, brain tissue atrophy on the damaged side was restored, and brain deficits, especially in the hippocampal region, were reduced compared with those in the HBID group. Similarly, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, Nissl staining revealed that the number of Nissl cells was lower in the HIBD group, especially in the hippocampal region, than in the sham group at the age of 28 days, which was ameliorated by treatment with gastrodin injection. These results indicate that gastrodin injection can ameliorate long-term brain tissue damage after HIBD.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.8 Gastrodin injection Ameliorates OGD/R-Induced SH-SY5Y Cell Damage\u003c/h2\u003e \u003cp\u003eA CCK-8 assay was used to determine whether gastrodin injection (0, 20, 40, 80, 160, and 320 \u0026micro;mol) is potentially toxic to SH-SY5Y cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, the proliferative viability of SH-SY5Y cells was not affected at gastrodin injection concentrations up to 160 \u0026micro;mol/L. Next, SH-SY5Y cells were subjected to OGD/R to determine the effect of gastrodin injection (20, 40, or 80 \u0026micro;mol) on cell viability. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB, the cell survival rate was significantly lower after OGD/R treatment than in the control, and the cell survival rate was increased by different doses of gastrodin injection. The effect was most significant at a gastrodin injection concentration of 40 \u0026micro;mol, which was used for the following experiments.The oxidative stress response of the cells could be caused by OGD/R, which led to an increase in the production of ROS as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC. As show in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC, green fluorescence increased significantly in the OGD/R group, and treatment with gastrodin injection reduced the green fluorescence and the production of ROS. After OGD/R, the energy supply is impaired, resulting in mitochondrial dysfunction, which leads to a decrease in the mitochondrial membrane potential \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. As show in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD, the red fluorescence decreased and green fluorescence increased after OGD/R injury, while the red fluorescence increases and decreases the production of green fluorescence after gastrodin injection treatment These results indicated that gastrodin injection could improve the survival of OGD/R-induced SH-SY5Y cells, reduce the production of ROS, inhibit the occurrence of oxidative stress, increase the mitochondrial membrane potential, and improve mitochondrial function.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.9 Gastrodin injection Reduces OGD/R-Induced SH-SY5Y Cell Damage by Activating the P62/Nrf2/HO-1 Pathway\u003c/h2\u003e \u003cp\u003eThe results of the in vitro Western blot analysis revealed that the expression levels of the oxidative stress-related proteins Nrf2, HO-1, and P62 tended to increase in the OGD/R cell model, and the expression of these proteins further increased after treatment with gastrodin injection, suggesting that gastrodin injection could inhibit the oxidative stress response of cells after OGD/R. The expression level of the apoptosis protein Bcl-2/Bax decreased after OGD/R, indicating that apoptosis increased, whereas Bcl-2/Bax increased after gastrodin injection treatment, indicating that gastrodin injection reduced apoptosis; however, all these protective effects of gastrodin injection could be attenuated by XRK3F2, a specific inhibitor of P62 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA-F). and the above results indicated that gastrodin injection reduced SH-SY5Y cell damage injury after OGD/R and that this protective effect may be realized by regulating P62.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eAccording to statistics, approximately 750,000 infants worldwide suffer from moderate or severe HIE each year, resulting in neurodevelopmental disorders in approximately 400,000 infants, and HIE accounts for 2.4% of the total global burden of disease \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Although the incidence of neonatal asphyxia has decreased significantly with the development of asphyxia resuscitation techniques, it is still one of the leading causes of neonatal death. Currently, subcapnia is internationally recognized as an effective treatment for moderate and severe HIE, and only 50\u0026ndash;60% of HIE cases eligible for subcapnia treatment are affected by gestational age, birth weight, coagulation and circulatory function \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Therefore, it is important to find effective treatments for HIE.\u003c/p\u003e \u003cp\u003eAfter the occurrence of HIE, the damage can be divided into three stages: In the first stage, owing to the increase in anaerobic fermentation after hypoxia, the energy supply is rapidly depleted, resulting in a large amount of Ca\u003csup\u003e2+\u003c/sup\u003e and excitatory neurotransmitters flowing into the cells, leading to cellular damage \u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e; in the second stage, reperfusion injury, resulting in a dramatic increase in reactive oxygen species (ROS), oxygen free radicals, and mitochondrial dysfunction, and secondary energy failure, while oxidative stress injury, excitatory amino acid toxicity, and inflammatory response lead to neuronal damage, apoptosis, and ultimately neuronal death \u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e; and in the third stage, sustained brain damage due to chronic inflammation and disruption of neuronal synapses and axon formation \u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Therefore, inhibiting oxidative stress damage may be a novel approach for the treatment of HIE. Studies have shown that oxidative stress may mediate the progression of HIE through inflammation. After the onset of ischemia and hypoxia, microglia respond rapidly and produce large amounts of inflammatory factors (TNF-α, IL-1β, etc.), glutamate, nitric oxide (NO), and ROS, etc., and these cytokines induce the progression of HIE, and these cytokines induce neuronal apoptosis and are positively correlated with the severity of HIE \u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Second, disruption of the energy supply and accumulation of large amounts of ROS and Ca\u003csup\u003e2+\u003c/sup\u003e influx into the cell lead to mitochondrial damage, which decreases the mitochondrial membrane potential (ΔΨm), increases cytochrome C release, activates apoptosis-associated proteins, induces neuronal apoptosis and ultimately leads to cell death \u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Studies have shown that while oxidative stress markers, including superoxide dismutase (SOD), malondialdehyde (MDA), the inflammation-related factors IL-6, TNF-α, and IL-1β, metabolism-related markers lactate dehydrogenase (LDH), etc., in cerebrospinal fluid or serum after HIE, can be used as sentinel markers to determine the prognosis of HIE \u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNrf2 is a key regulator of important cellular defense and antioxidative stress pathways \u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Nrf2 consists of 605 amino acid residues including seven homologous structural domains, Neh1-6, of which Neh2 binds to Kelch-like ECH-associated protein (Keap1) in the cytoplasm to maintain its stability \u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. HO-1 is a molecule downstream of Nrf2 that has antioxidant effects \u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. Under normal conditions, Neh2 binds to Keap1 in the cytoplasm, and when oxidative stress occurs, Neh2 separates from Keap1 and Nrf2 translocates to the nucleus, where it binds to the antioxidant response element (ARE) and initiates downstream oxidative enzymes (catalase, CAT), superoxide dismutase-1 (SOD-1), HO-1, and other antioxidative stress genes \u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. When cerebral ischemia occurs, Nrf2 begins to increase within 3 hours and peaks at 24 hours. Studies have shown that P62 is closely related to the crosstalk of the Nrf2 signaling pathway and that P62 can competitively bind Keap1 with Nrf2 to increase the release of Nrf2 and initiate the expression of downstream antioxidant genes. In addition, P62 can promote the degradation of Keap1 via selective autophagy, resulting in an increase in the release of Nrf2, which in turn can regulate the expression of AREs and promote the transcription of downstream antioxidant genes \u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. Wang et al. reported that knockdown of the P62 gene resulted in a significant decrease in Nrf2 expression and a significant increase in Keap1 expression \u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e, whereas overexpression of the P62 gene increased Nrf2 and decreased Keap1 expression \u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. Li et al. reported that intraperitoneal injection of echinocystic acid could reduce the area of cerebral infarction and improve neurobehavioral scores by activating the Nrf2/HO-1 signaling pathway, increasing the expression of the antiapoptoic protein Bcl-2 protein, and decreasing the expression of the proapoptotic proteins Bax and cleaved-caspase-3 in HIBD model mice; moreover, observing the long-term prognosis revealed that it could reduce the brain defect area \u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. In this study, gastrodin injection reduced the cerebral infarct area on the ischemic side of HIBD injury model mice, improved neurological deficits, reduced the production of the inflammatory factors TNFα and IL-6, increased the production of inflammation-suppressing factor TGF-β, and increased the expression of the antioxidative stress-related proteins Nrf2, HO-1, and P62 on the ischemic side of brain tissue, reduced the expression of the apoptosis-related protein Bax in brain tissue, increased the expression of anti-apoptosis-related protein Bcl-2 in brain tissue, and improved mitochondrial function, thus playing a neuroprotective role.\u003c/p\u003e \u003cp\u003eLi et al. also reported that the phenolic compound 3,4-dihydroxybenzaldehyde in gastrodin could reduce the release of the inflammatory factors TNF-α, IL-6, and IL-1β, increase the expression of the anti-inflammatory factors TGF-β and TGF-β2; and promote the transition of microglia from the M1- type (proinflammatory) to M2- type (reparative) \u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. In addition, in an in vitro model of Alzheimer's disease (AD), aspalathin increased the survival rate of β-amyloid-treated neuronal cells, reduced the release of proinflammatory factors and NO, and minimized neuronal cell apoptosis, which protected neuronal cells from neurotoxicity \u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. These results suggest that gastrodin has neuroprotective effects. In this study, the results of HE staining and Nissl staining revealed that at the early stage of HIBD injury, the site of injury was mainly the cortical region of the brain; however, over time, the main site of injury was the hippocampal region, possibly because after hypoxia and ischemia occur in brain tissues, the blood supply to the brain preferentially supplies the regions of the hippocampus, thalamus, and hippocampus, whereas the terminal supply regions of cerebral arteries, such as the cerebral cortex, are prone to damage, and with the recovery of the blood supply, the brain tissue becomes extremely sensitive to ischemia-reperfusion injury; the damage to the hippocampus region, which affects learning, memory ability and motor coordination, and these neurological sequelae can be ameliorated by gastrodin injection.\u003c/p\u003e \u003cp\u003eIn addition, the present study further confirmed that SH-SY5YS cell survival was reduced after OGD/R injury and that gastrodin injection increased the expression of antioxidative stress proteins and antiapoptotic proteins, decreased the expression of proapoptotic proteins, reduced the production of ROS, and inhibited the attenuation of the mitochondrial membrane potential after OGD/R injury. However, all of these protective effects could be attenuated by a P62-specific inhibitor, suggesting that these neuroprotective effects may be related to the activation of the P62/Nrf2/HO-1 signaling pathway. The present study also has several limitations: P62 is an important substrate in the process of autophagy \u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e; however, the role of autophagy in neurological disorders is still controversial, and recent studies have shown that severe acute cerebral hypoxia and ischemia overactivate autophagy, leading to neuronal cell injury and death. However, chronic or mild hypoxia can moderately activate autophagy to remove damaged organelles to exert protective effects \u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e; however, the relationship between P62 and autophagy was not investigated in depth in this study, which is a future research direction.\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eIn conclusion, gastrodin injection can increase antioxidant stress resistance, reduce inflammation and apoptosis in the brain tissue of rats after HIBD through the P62/Nrf2/HO-1 signaling pathway, and improve their long-term neurological prognosis. These findings provide new ideals for the treatment of HIE.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eData availability statement\u003c/p\u003e\n\u003cp\u003eThe original contributions presented in the study are included in the article, and further inquiries can be directed to the corresponding author.\u003c/p\u003e\n\n\u003cp\u003eEthics statement\u003c/p\u003e\n\u003cp\u003eThe animal experiments were reviewed and approved by the Animal Experiment Review and Approval and Animal Research Ethics Committee of Guizhou Medical University (No.2400028).\u003c/p\u003e\n\n\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003eSha Wu, Jianwei Xu and Maoqiong Chen designed the research study. Sha Wu and Zhenkui Ren performed the majority of the experiments and data collection. Sha Wu and Mengting Yang analyzed the data to generate graphs. Mengting Yang, Xianxian Li, Xuxian Wu and Ying Xiong provided technical support. Sha Wu and Zhenkui Ren wrote the manuscript. Jianwei Xu and Maoqiong Chen revised the manuscript.\u003c/p\u003e\n\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis study was funded by the National Natural Science Foundation of China (82160126), the Special Project for Scientific and Technological Research on Traditional Chinese Medicine and Ethnic Medicine of Guizhou Province Administration of Traditional Chinese Medicine (QZYY-2024-012), and the Science and Technology Department of Guizhou Province (ZK [2022] General 415) and Affiliated Hospital of Guizhou Medical University (2021-GMHCT-001).\u003c/p\u003e\n\n\u003cp\u003eConflict of interest\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePark YJ, Borlongan CV, Dezawa M. Cell-based treatment for perinatal hypoxic-ischemic encephalopathy. 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Gastrodin Protects Neural Progenitor Cells Against Amyloid \u0026beta; (1-42)-Induced Neurotoxicity and Improves Hippocampal Neurogenesis in Amyloid \u0026beta; (1-42)-Injected Mice. J Mol Neurosci. 2016 Sep;60(1):21-32. doi: 10.1007/s12031-016-0758-z.\u003c/li\u003e\n\u003cli\u003eMorishita H, Mizushima N. Diverse Cellular Roles of Autophagy. Annu Rev Cell Dev Biol. 2019 Oct 6; 35:453-475. doi: 10.1146/annurev-cellbio-100818-125300.\u003c/li\u003e\n\u003cli\u003eHu Y, Luo Y, Zheng Y. Nrf2 Pathway and Autophagy Crosstalk: New Insights into Therapeutic Strategies for Ischemic Cerebral Vascular Diseases. Antioxidants (Basel). 2022 Sep 2;11(9):1747. doi: 10.3390/antiox11091747.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"moln","sideBox":"Learn more about [Molecular Neurobiology](https://www.springer.com/journal/12035)","snPcode":"12035","submissionUrl":"https://submission.nature.com/new-submission/12035/3","title":"Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"HIBD, HIE, gastrodin injection, oxidative stress, P62/Nrf2/HO-1 pathway","lastPublishedDoi":"10.21203/rs.3.rs-6630703/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6630703/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHypoxic-ischemic encephalopathy (HIE) is the leading cause of death and disability in newborns. Hypothermia treatment (TH) is currently the only method that can improve the survival rate of HIE patients, but it has many limitations. Previous studies have shown that gastrodin injection has a potential neuroprotective effect on brain injury. However, it is unclear whether it has the same effect on HIE and its mechanism of action. This study investigated the neuroprotective effect of gastrodin injection on HIE and its possible mechanism through in vivo and in vitro experiments. For in vivo experiments, a hypoxic-ischemic brain damage (HIBD) model was established in neonatal rats, and immediately after modeling, a transperitoneal injection of gastrodin injection was administered. The model was verified via laser diffuse imaging, and hematoxylin-eosin (H\u0026amp;E), Nissl's staining, and 2,3,5-triphenyltetrazolium chloride (TTC) staining were performed to assess the brain tissue damage and the area of brain infarction, and short- and long-term neurobehavioral assessments were performed. Brain tissue damage, cerebral infarction area, short-term and long-term neurobehavioral assessment, superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH-PX), and catalase (CAT) detection, Western blotting and ELISA were used to analyze the levels of oxidative stress and inflammatory factors in brain tissue. For in vitro experiments, a hypoxia-glucose deprivation/reperfusion (OGD/R) injury model was established in SH-SY5Y cells, and gastrodin injection was injected immediately after model establishment. To determine the mechanism of action of gastrodin injection, XRK3F2, a specific inhibitor of P62, was used, and cell viability, the level of intracellular reactive oxygen species (ROS), the mitochondrial membrane potential, and oxidative stress-related protein levels were measured to further validate the neuroprotective effect of gastrodin injection. The results showed that gastrodin injection could alleviate the area of cerebral infarction, reduce neuronal oxidative stress injury, improve short-term and long-term neurobehavioral deficits, improve mitochondrial function, improve the body's ability to resist oxidative stress, and reduce inflammatory reactions in brain tissues from HIBD rats. In vitro experiments revealed that gastrodin injection increased neuronal cell survival after OGD/R injury and inhibited the occurrence of oxidative stress and apoptosis; however, these neuroprotective effects were attenuated by XRK3F2, a specific inhibitor of P62. The present study suggested that gastrodin injection may attenuate oxidative stress injury and apoptosis and ameliorate brain injury after HBID by activating the P62/Nrf2/HO-1 signaling pathway.\u003c/p\u003e","manuscriptTitle":"Gastrodin injection relieves hypoxic-ischemic brain injury in newborn rats by regulating the P62/Nrf2/HO-1 pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-23 05:44:51","doi":"10.21203/rs.3.rs-6630703/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-04T23:20:04+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-01T14:44:32+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-01T01:13:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-29T09:23:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"201614817766346104766664453385761191210","date":"2025-06-27T01:24:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"136971899725733126668925819684428649933","date":"2025-06-24T17:53:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"22838146412472405993780263196390036177","date":"2025-06-23T02:26:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"312255845940852013952374072030339669006","date":"2025-06-20T01:55:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"104670634763833604782227758874332948156","date":"2025-06-20T00:53:17+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-18T11:38:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"156646224111979035267918178314249177025","date":"2025-06-18T11:21:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"147207914331270645961589608877570664925","date":"2025-06-18T02:54:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"173975240013332486607967464012786684104","date":"2025-06-18T00:36:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"148674893896705406888085141975097856686","date":"2025-06-18T00:03:44+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-17T16:57:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-13T01:24:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-13T01:23:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Neurobiology","date":"2025-05-09T17:26:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"moln","sideBox":"Learn more about [Molecular Neurobiology](https://www.springer.com/journal/12035)","snPcode":"12035","submissionUrl":"https://submission.nature.com/new-submission/12035/3","title":"Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1a8f664a-1481-4148-9bcc-a1794bb035de","owner":[],"postedDate":"June 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-01T16:05:42+00:00","versionOfRecord":{"articleIdentity":"rs-6630703","link":"https://doi.org/10.1007/s12035-025-05522-z","journal":{"identity":"molecular-neurobiology","isVorOnly":false,"title":"Molecular Neurobiology"},"publishedOn":"2025-11-24 15:57:05","publishedOnDateReadable":"November 24th, 2025"},"versionCreatedAt":"2025-06-23 05:44:51","video":"","vorDoi":"10.1007/s12035-025-05522-z","vorDoiUrl":"https://doi.org/10.1007/s12035-025-05522-z","workflowStages":[]},"version":"v1","identity":"rs-6630703","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6630703","identity":"rs-6630703","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}