Baicalin Ameliorates L-Glutamate-induced Hippocampal Oxidative Stress Injury and Apoptosis in Mice by Regulating Nrf2/HO-1 Signaling Pathway

Objective: To explore the effect and mechanism of baicalin on L-Glutamate-induced oxidative stress injury in the hippocampus of mice. Methods: Forty mice were randomly assigned to five groups: Sham, Model, N-Acetyl-L-Cysteine (NAC), and baicalin (BA-7.5mg/kg and BA-15mg/kg). A model of excitatory amino acid toxicity with oxidative stress injury was induced by injecting L-Glutamate into the lateral ventricle. The drugs were then injected intraperitoneally. Six hours later, behavioral tests were performed. The lesions of brain were observed using HE staining, while the apoptosis of neurons was assessed through TUNEL staining. The levels of superoxide dismutase (SOD) and malondialdehyde (MDA) were determined using biochemical methods. The expression of Cytochrome C (CytC) was assessed by immunohistochemistry. Fluorescent staining was employed to detect the expression of reactive oxygen species (ROS). The levels of Nrf2, HO-1, SOD2 and Catalase (Cat) were detected by qPCR and WB. Results: The behavioral tests showed that the motion distance and pain threshold were reduced. MDA, ROS, and CytC were increased, SOD and Cat were decreased. The CA3 region of the hippocampus exhibited pathological changes, and the rate of TUNEL-positive increased. Baicalin could reverse these changes, especially BA-7.5mg/kg. Conclusion: Baicalin can reduce the hippocampal injury induced by L-Glutamate. This may be related to the activation of the Nrf2/HO-1 signaling pathway. Special Collection:“Pharmacological Studies of Traditional Chinese Medicines”. Baicalin Ameliorates L-Glutamate-induced Hippocampal Oxidative Stress Injury and Apoptosis in Mice by Regulating Nrf2/HO-1 Signaling Pathway Feng LI 1,2, Zishan HUANG 1,2,3, Huanyu GOU 1,2, Jiarui ZHENG 1,2,4, Mingjiang YAO 1,2* 1 Institute of Basic Medical Sciences, Xiyuan Hospital of China Academy of Chinese Medical Sciences, No.1 Xiyuan Caochang, Haidian District, Beijing, 100091, PR China 2 Key Laboratory of Pharmacology of Chinese Materia Medica, Beijing, 100091, PR China 3 Research Institute of Traditional Chinese Medicine (TCM) of Guangdong Pharmaceutical University, Guangzhou,Guangdong, 510006, PR China 4 Academy of Traditional Chinese Medicine (TCM) of Heilongjiang, Haerbing, Heilongjiang, 150036, PR China First author: Feng LI. E-mail: [email protected]. Research field: Cerebrovascular pharmacology and neuropharmacology; * Corresponding author: Mingjiang YAO (Ph.D. Prof.). E-mail: [email protected]. Institute of Basic Medical Sciences, Xiyuan Hospital of China Academy of Chinese Medical Sciences, No.1 Xiyuan Caochang, Haidian District, Beijing, 100091, PR China. E-mail addresses: [email protected] (Feng LI), [email protected] (Zishan HUANG), [email protected] (Huanyu GOU), [email protected] (Jiarui ZHENG), [email protected] (Mingjiang YAO).

Baicalin; L-Glutamate; Brain damage; Oxidative Stress; Nrf2/HO-1 signaling pathway

Objective: To explore the effect and mechanism of baicalin on L-Glutamate-induced oxidative stress injury in the hippocampus of mice. Methods: Forty mice were randomly assigned to five groups: Sham, Model, N-Acetyl-L-Cysteine (NAC), and baicalin (BA-7.5mg/kg and BA-15mg/kg). A model of excitatory amino acid toxicity with oxidative stress injury was induced by injecting L-Glutamate into the lateral ventricle. The drugs were then injected intraperitoneally. Six hours later, behavioral tests were performed. The lesions of brain were observed using HE staining, while the apoptosis of neurons was assessed through TUNEL staining. The levels of superoxide dismutase (SOD) and malondialdehyde (MDA) were determined using biochemical methods. The expression of Cytochrome C (CytC) was assessed by immunohistochemistry. Fluorescent staining was employed to detect the expression of reactive oxygen species (ROS). The levels of Nrf2, HO-1, SOD2 and Catalase (Cat) were detected by qPCR and WB. Results: The behavioral tests showed that the motion distance and pain threshold were reduced. MDA, ROS, and CytC were increased, SOD and Cat were decreased. The CA3 region of the hippocampus exhibited pathological changes, and the rate of TUNEL-positive increased. Baicalin could reverse these changes, especially BA-7.5mg/kg. Conclusion: Baicalin can reduce the hippocampal injury induced by L-Glutamate. This may be related to the activation of the Nrf2/HO-1 signaling pathway.

Glutamate (Glu) is one of the main excitatory neurotransmitters in the Central Nervous System (CNS) of mammals and is primarily stored in vesicles within the cerebral cortex, striatum, hippocampus or other cerebral regions [1][2][3] . When neurons undergo depolarization, these vesicles release substantial amounts of Glu into the synaptic clefts, where it binds to its receptors on the postsynaptic membrane and then triggers a variety of neural signaling pathways [4] . Glu serves numerous physiological functions, including nourishing neurons, promoting neural growth and development, and participating in various CNS activities like learning and memory [5] . Under normal physiological conditions, the concentration of Glu in the CNS is kept within a normal range through processes such as the glutamate-glutamine cycle and the tricarboxylic acid cycle, without causing neuronal damage [6][7][8] . When stimulated by nociceptive stimulus such as hypoxia, ischemia, or trauma, the balance of Glu in the CNS is disrupted and superfluous amount of Glu is then released, therewith transformed into a neurotoxin, causing a sharp increase in its concentration in the extracellular fluid. This results in severe excitatory toxicity to neurons, and ultimately leading to neuronal injury and death [9][10] . In recent years, numerous studies have demonstrated that neuronal damage resulting from excessive Glu release is a crucial factor influencing the prognosis of various CNS-related diseases, including stroke, Alzheimer’s disease, and Parkinson’s disease [11][12][13] . Consequently, it is a pressing issue to investigate the mechanisms of Glu-induced CNS injury and identify safe and effective drugs to prevent this damage. Neuronal death induced by excess Glu is a result of both receptor-mediated excitotoxicity and non-receptor-mediated oxidative stress damage. The receptors that Glu interacts with are primarily ionic receptors, such as the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and the N-methyl-D-aspartate receptor (NMDAR), etc [14][15][16] . Studies have shown that the activation of these receptors is closely linked to ion channels. Overactivation of the AMPAR receptor can trigger a cascade of pathological changes, including Calcium overload, cellular swelling, protein degradation, and ultimately, cellular injury or death [17][18] . Oxidative stress injury refers to an imbalance between oxidation and antioxidant activity in the body, favoring oxidation [19] . The brain is characterized by high oxygen consumption and relatively low antioxidant capacity [20] . Excessive Glu consumption in brain cells leads to the depletion of antioxidants, resulting in the accumulation of reactive ROS and MDA. These highly reactive molecules can directly oxidize and damage biological macromolecules, such as DNA, proteins, and lipids, thereby triggering cell apoptosis [21][22] . Many studies have demonstrated that the cytotoxicity of oxidized neurons may be associated with the Nuclear transcription factor E2-related factor 2 (Nrf2) and its downstream target protein Heme Oxygenase-1 (HO-1) [23][24][25] . Nrf2 serves as a key regulator of the antioxidant defense system in vivo and is involved in signal transduction pathways related to various intracellular defense mechanisms. Under normal physiological conditions, Nrf2, Kelch-like ECH-associated protein 1 (Keap1), and Cullin3 (Cul3) exist as a complex in the cytoplasm and remain in a state of low activity. When the body suffers oxidative stress, Nrf2 dissociates from the Keap1-Cul3 complex [26] . The activated Nrf2 is then transported into the nucleus and binds to the Antioxidant Response Element (Are) [27] . Ultimately, this leads to the promotion of the expression of antioxidant genes such as SOD, Cat, and HO-1. HO-1 is a primary downstream protein of Nrf2 and is widely distributed throughout body tissues, primarily located in the smooth endoplasmic reticulum. Similarly, under normal physiological conditions, the expression of HO-1 is low. However, in response to stress, the expression of HO-1 upregulated rapidly, exhibiting significant antioxidant effects [28] . The antioxidant effect of HO-1 is primarily related to two aspects: (1) HO-1 prevents free heme from participating in oxidative reactions. (2) HO-1, along with its enzymatic hydrolysis products (CO and bilirubin), exhibits antioxidant, anti-inflammatory properties, enhances tissue microcirculation, and inhibits cell apoptosis [29] . Consequently, modulating the Nrf2/HO-1 Signaling pathway may represent an important therapeutic target for addressing oxidative stress-related disorders. Baicalin (BA) is a flavonoid compound extracted from Scutellaria baicalensis Georgi that possesses a range of pharmacological effects, including anti-inflammatory, anti-apoptotic, and antioxidant properties [30] . Numerous basic experiments and clinical studies have demonstrated that BA exerts neuroprotective effects in various CNS diseases [31][32] . Based on our previous in vitro studies, BA can alleviate the oxidative damage induced by L-Glutamate (L-Glu) on mouse hippocampal neurons HT-22 by activating the Nrf2/HO-1 Signaling pathway [33] . In this study, we established a mouse model of hippocampal injury by intracerebroventricular injection of L-Glutamate and further confirmed in vivo that BA can inhibit hippocampal oxidative damage and apoptosis, and the mechanism is closely linked to the Nrf2/HO-1 Signaling pathway. This further validates the neuroprotective and antioxidant potential of BA on CNS diseases associated with glutamate excitatory injury.

and Materials The study was conducted in accordance with the Basic & Clinical Pharmacology & Toxicology policy for natural products [34] . Furthermore, The study was conducted in accordance with the Basic & Clinical Pharmacology & Toxicology policy for experimental and clinical studies [35] . Animals SPF male C57BL/6J mice (23-25g) were purchased from SPF (Beijing) Biotechnology Co. Ltd. [License number: SCXK (Jing) 2019-0010]. All experimental animals were given adequate water and feed with normal circadian rhythm, and the feeding temperature was maintained at 22-24℃. This study complies with the WMA Statement on animal use in biomedical research and was approved by Experimental Ethics Committee at Xiyuan hospital, China Academy of Chinese Medical Sciences (No. 2024XLC001-1). Reagents and Instruments Pentobarbital sodium was purchased from Beijing Chemical Reagent Company; L-Glutamate was purchased from Beijing Solaibao Technology Co., LTD; N-Acetyl-L-Cysteine was purchased from Sigma; Baicalin was purchased from Shanghai Yuanye Biotechnology Co., LTD; Test kits for SOD and MDA were purchased from Shanghai Biyuntian Biotechnology Co., LTD; ROS test kit purchased from Shanghai Beibo Biotechnology Co., LTD; Primers related to Nrf2, SOD2 and Cat were purchased from Shanghai Shenggong Biological Engineering Co., LTD; Rabbit anti Nrf2 polyclonal antibody and rabbit anti HO-1 polyclonal antibody were purchased from Wuhan Sanying Biotechnology Co., LTD; Rabbit anti CytC monoclonal antibody was purchased from Shenyang Wanlei Biotechnology Co., LTD; The goat anti-rabbit IgG (H+L) was purchased from Beijing Huaxing Biotechnology Co., LTD. Tail flick meter(YLS-12A, Yiyan, China); Upright microscope System (BX53, Olympus, Japan); Microplate Reader (Synergy4, BioTek, United States); Chemiluminescence Imaging System (ChemiDoc XRS +, BIO-RAD, USA); UV Spectrophotometer (SMA1000, Merinton, China); Temperature gradient PCR Instrument (T-GRADIENT, Biometre, Germany). Real-time PCR System (StepOnePlus, Applied Biosystems, USA). Animal Grouping and Drug Treatment Forty mice were divided into five groups using the random number table method: Sham, Model, NAC (21mg/kg), BA-7.5mg/kg and BA-15mg/kg, eight in each group. After the model was established, mouse in each group was immediately injected intraperitoneally at volume of 0.1ml/10g. The Sham and Model group were injected intraperitoneally with an equal volume of normal saline. The NAC was dissolved in normal saline. For the BA solution, BA was first added to normal saline, and then 1mol/L NaOH was added until the solution turned a clear yellow-green color. Establishment of Hippocampal Injury Model of Mice Induced by Lateral Ventricle Injection of L-Glutamate After the mice were anesthetized via intraperitoneal injection of pentobarbital sodium (40 mg/kg), they were positioned prone on a brain stereotaxic apparatus. The skin on the heads of the mice was disinfected, and subsequently, the skin over the median cranial roof was incised to expose the fontanel. Then a hole was drilled on the skull and a microsyringe (Hamilton, 10ul) was inserted into the right lateral ventricle (ML:-1.0mm, AP:+0.2mm, DV:2.5mm). Then, 4μl of L-Glu (10μg/μl) was injected into the right lateral ventricle, and the injection speed was set at 1μl/min. In the Sham group, 4μl of normal saline was injected into the same position instead. After injection, the needle was left in place for five minutes and the incision was sutured and disinfected. The mice were then placed on a 37℃ thermal blanket until recovered. Six hours after modeling, the behavioral changes of the mice were observed. Open Field Test Mice were placed in the open field test instrument to record the movement trajectory. Each mouse was recorded for five minutes. Before recording next mouse, the inside of the instrument was wiped with alcohol to prevent odour and excrement from affecting the experiment. Finally, the total travel distance of mice in each group was analyzed. Tail Flick Test Firstly, set the temperature and intensity of the infrared light. Subsequently, the tail of the mice was exposed to infrared light and the stopwatch starts. When the mouse starts to bend its tail to avoid heat sources, the timer stops automatically, and the latency was recorded. The test was repeated three times for each mouse. Finally, the latency of tail flick in each group was analyzed. Sample Collection After all the behavioral tests, the mice were anesthetized with pentobarbital sodium through intraperitoneal injection. The specific procedures were as follows: Blood samples were collected, centrifuged at 3000 revolutions per minute (rpm) for 10 minutes at 4℃, and the serum was collected and stored at -80℃ for the determination of oxidative stress indexes. The brains of four mice in each group were randomly sampled, and stored at -80℃ for subsequent Western blotting and RT-qPCR analysis. The entire brains of the remaining 4 mice in each group were fixed in 4% paraformaldehyde, followed by routine dehydration, clearing, embedding, and sectioning. These sections were then used for HE staining, TUNEl staining, and immunohistochemical detection, etc. Oxidative Stress Index Detection In strict accordance with the kit instructions, SOD levels in the brain tissue of mice from each group were detected using the WST-8 method, while MDA levels in the serum of mice were determined by colorimetry. ROS levels in the brain tissue were assessed through DHE labeling. Hematoxylin-eosin Staining The mouse brain tissue was sliced after being fixed with paraformaldehyde, subsequently dehydrated using a gradient of alcohols, cleared with xylene, and embedded in paraffin. The brain slices were then stained with hematoxylin and eosin through routine HE staining procedures. The hippocampus was selected as the primary area of observation for analysis. Immunohistochemistry Staining The Immunohistochemical method was employed for detection. Following paraffin embedding, the brain tissue was sliced, dewaxed, and subjected to antigen retrieval. The slices were washed three times with 0.01mol/L PBS and incubated in deionized water for an additional three times. After washing again with 0.01mol/L PBS, the slices were blocked with 5% goat serum at room temperature for 1 hour. Subsequently, drops of 1:200 dilution of the anti-CytC primary antibody were applied, and the slices were placed in a humidified chamber and incubated at 4℃ overnight. On the second day, the slices were washed three times with 0.01mol/L PBS. The secondary antibody, corresponding to the primary antibody, was then added and incubated at room temperature for 30 minutes. After washing the slices three times with 0.01mol/L PBS, freshly prepared DAB color developing solution was added. The slices were then counterstained with hematoxylin at room temperature for 3 minutes and washed three times with 0.01mol/L PBS. They were subsequently dehydrated using a gradient ethanol series until transparent. After drying, the slides were sealed with neutral resin. Images were captured using a microscope, and positive expression appeared as a brownish-yellow color. Statistical analysis was performed using Image J software. TUNEL Staining The paraffin-embedded brain tissue samples were sliced, incubated, stained, and sealed following the instructions provided with the TUNEL kit. Upon completion of the procedure, positive expression was observed under a microscope, appearing as a brownish-yellow color. Finally, statistical analysis was conducted using the Image J software. Real-time qPCR The toal RNA was extracted from brain tissue using Trizol. Subsequently, the mRNA concentration was measured through SMA1000 ultra-fine ultraviolet spectrophotometer and was adjusted using RNase Dnase-free water. RNA was reverse transcribed into cDNA using Primescript RT Master Mix (Reaction conditions: 45℃ for 15 minutes, 95℃ for 5 minutes, followed by maintaining at 4℃). SYBR ® Green Real-time PCR Master Mix was used to determine the mRNA expression levels of the target genes by using the StepOnePlus real-time fluorescence quantitative PCR system [33] . The primers used in the study are listed in Table 1 . Each sample was assayed in triplicate, and the relative expression of the target gene was calculated using the 2 -△△CT method after the reaction. Table 1 Primer sequence | Primers | Sequence（5’-3’） | | Nrf2 | F-5’-CAGCCATGACTGATTTAAGCAG-3’ R-5’-CAGCTGCTTGTTTTCGGTATTA-3’ | | SOD2 | F-5’-AAGGGAGATGTTACAACTCAGG-3’ R-5’-GCTCAGGTTTGTCCAGAAAATG-3’ | | Cat | F-5’-CACCTTCAAGTTGGTTAATGCA-3’ R-5’-CATGACCTGGATGTAAAACGTC-3’ | | β-actin | F-5’-CTACCTCATGAAGATCCTGACC-3’ R-5’-CACAGCTTCTCTTTGATGTCAC-3’ | Western Blotting The tissue was placed in a grinding tube, and a RIPA lysis buffer containing phosphatase inhibitors, protease inhibitors, and PMSF was prepared. An amount of RIPA lysis buffer equal to 10 times the tissue weight was added to each tube. The tissue was homogenized using a frozen grinder, and then centrifuged at 4℃ for 10 minutes at 12,000 rpm. The supernatant was collected, and the protein concentration of the samples was adjusted to a uniform level using Bradford protein assay kit. To each sample, 1/4 volume of 5×loading buffer was added, and the mixture was heated at 100℃ for 5 minutes to completely denature the proteins. The samples were then stored at -80℃ for future use. The samples were separated using SDS-PAGE and subsequently transferred to PVDF membrane and blocked with 5% skim milk for 90 minutes at room temperature. The membrane was then washed three times with 1×PBST for 10 minutes each. Next, the membrane was incubated overnight at 4℃ with primary antibodies at the following concentrations: Nrf2 (1:1500), HO-1 (1:1500), LaminB(1:5000), and β-actin (1:10000). On the second day, the membrane was washed three times with 1×PBST for 10 minutes each. Subsequently, the corresponding secondary antibody (1:20000) was added and the membrane was incubated at room temperature for 1 hour. The membrane was then washed three times with 1×PBST for 10 minutes each. Finally, ECL reagent was added to enhance the chemiluminescent reaction, and the protein bands were visualized using the ChemiDoc XRS + system. Semi-quantitative analysis was performed using Image Lab software. Statistical Analysis The experimental data were presented as Mean±SD. In cases where the data exhibited normal distribution and homogeneous variances, one-way ANOVA was employed. If these conditions were not met, non-parametric test (Mann-Whitney U test) was used. P < 0.05 was considered to be statistically significant.

Baicalin increased the total distance of open field movement in mice The results of open field test showed ( Figure 1A ) that compared with the Sham group, the total distance of the mice in the Model group was significantly reduced (P<0.001). Compared with the Model group, the total distance of mice in drug treatment groups was increased (P<0.001), the BA-15 group was slightly better than the BA-7.5 group. Baicalin increased the pain threshold of mice The latency of the tail flick test indicates the sensitivity of the mouse to pain. Compared with the Sham group, the latency of mice in the Model group was significantly lower (P<0.001) and they were more sensitive to pain. The latency of mice in the drug treatment groups were prolonged, especially in the BA-15 group (P<0.05) ( Figure 1B ). Figure 1 Behavioral index evaluation. (A) Comparison of total distance in open field test. (B) Comparison of the latency of the tail flick test. ### P＜0.001 vs Sham group, *** P＜0.001 and * P＜0.05 vs Model group. (n=8). Baicalin Reduced L-Glutamate-Induced Injury to the CA3 Region of Hippocampus As shown in Figure 2, we selected cortex, the hippocampal CA1 and CA3 region for observation. Surprisingly, only the hippocampal CA3 region of mice in each group had different degrees of pathological changes, and there were no obvious changes in the cortex and hippocampal CA1 region. HE staining revealed that the hippocampal structure of mice in the Sham group was normal, with cells closely arranged, clear nucleoli, and uniform color distribution. No morphological alterations were observed in either neuron or glial cells. In the Model group, the structure of the CA3 region of the hippocampus was markedly abnormal (black arrow). The specific performance was as follows: with reduced cell bodies, shrunk nuclei, red cytoplasm, and irregular shapes, indicating a significant decrease in nerve cells. The pathological changes were notably inhibited in the drug treatment groups, where only mild morphological abnormalities in individual nerve cells were observable. Notably, the pathological changes were significantly inhibited in the BA-7.5 group. Figure 2 HE stained images for. (Scale bar: 200 µm). (A) Cortical tissue. (B) Hippocampus CA1 region. (C) Hippocampus CA3 region. (n=4). Effect of baicalin on SOD and MDA expression In order to evaluate the effect of BA on oxidative stress, the expression level of SOD in brain tissue and MDA in serum were detected. SOD is a crucial antioxidant enzyme that catalyzes the disproportionation of superoxide anion radicals into oxygen and hydrogen peroxide, thereby decreasing their activity and safeguarding cells from oxidative damage. This enzyme serves as a significant indicator for assessing the functionality of the antioxidant system. On the other hand, MDA is a product of membrane lipid peroxidation. Our results show that the level of SOD decreased in the Model group compared with the Sham group and increased after being treated with NAC (P<0.05) and BA-7.5 (P<0.05, Figure 3A ). The MDA level significantly increased after treatment with L-Glu (P<0.05) and BA-7.5 have reversed the trend (P<0.01, Figure 3B ). Figure 3 (A) Quantitative analysis of the level of SOD. (B) Quantitative analysis of the level of MDA. # P＜0.05 vs Sham group, ** P＜0.01 and * P＜0.05 vs Model group.(n=4). Baicalin inhibited ROS expression in hippocampal CA3 region ROS is the primary substance involved in oxidative stress, exhibiting high activity within the body and readily attacking cellular structures, resulting in oxidative damage. In a state of imbalance between oxidation and antioxidant defenses in the body, leading to the production of substantial amounts of ROS. As shown in Figure 4, the level of ROS in hippocampal CA3 region significantly increased in the Model group compared with the Sham group (P < 0.01). However, the level of ROS decreased after being treated with of NAC (P < 0.01), BA-7.5 (P < 0.05), and BA-15. Figure 4 Fluorescent images of ROS in the CA3 region of the hippocampus. (Scale bar: 200 µm). (A), (B), (C), (D), and (E) respectively are Sham, Model, NAC, BA-7.5, and BA-15 group images. (F) Quantitative analysis of the level of ROS. ## P＜0.01 vs Sham group, ** P＜0.01 and * P＜0.05 vs Model group. (n=4). Inhibitory Effect of Baicalin on Apoptosis The TUNEL staining results demonstrated that, in comparison to the Sham group, the TUNEL-positive (brownish-yellow) rate in the Model group was significantly elevated (P < 0.05). Conversely, the positive rate was notably decreased in the drug treatment groups (NAC, BA-7.5, BA-15) (P < 0.05, Figure 5A, C ). Baicalin Inhibits the Expression of Cytochrome C Studies have shown that oxidative stress can activate the mitochondrial apoptosis pathway [36][37] . Most of the CytC in the body originates from mitochondria, and the release of a substantial amount of CytC is intimately linked to apoptosis, hinting at potential serious pathological alterations within the body. Immunohistochemical results ( Figure 5B, D ) revealed that, in comparison to the Sham group, the number of CytC immunopositive cells in the CA3 region of the hippocampus was significantly elevated in the Model group (P < 0.05), with the cytoplasm appearing brownish-yellow. When compared to the Model group, the number of CytC-positive cells was notably decreased in the drug treatment groups (P < 0.05), with the BA-7.5 group exhibiting better therapeutic effect. These findings align with the results obtained from TUNEL and HE staining. Figure 5 (A) TUNEL staining images. (Scale bar: 200 µm). (B) Effect of BA on CytC protein expression in hippocampus CA3 region. (Scale bar: 200 µm). (C) Quantitative analysis of the level of TUNEL positive cells. (D) Quantitative analysis of the level of CytC. # P＜0.05 vs Sham group, * P＜0.05 vs Model group. (n=4). Effect of Baicalin on the Expression of Genes or Proteins Associated with the Nrf2/HO-1 Signaling Pathway To investigate the mechanism of BA on L-Glu-damaged mice, the effects of BA on the Nrf2/ HO-1 signaling pathway were measured. After treatment with L-Glu, the levels of Nrf2 and HO-1 were increased in the Model group, but there is no statistical significance. In the drug groups, Nrf2 (P < 0.05) and HO-1 (P < 0.01) levels continued to rise, with the increase being more pronounced in the BA-7.5 group ( Figure 6A, B, C ). We also examined the effect of BA on the mRNA expression of Nrf2 ( Figure 6D ). The result was consistent with those obtained through western blotting. The mRNA expression of Nrf2 was increased in the BA-7.5 group (P<0.05). Furthermore, we measured the mRNA expression levels of Cat and SOD2. Compared with the Sham group, the expression of Cat in the Model group (P<0.05) was decreased, while it was elevated in the drug groups, particularly in the BA-7.5 group (P<0.01, Figure 6E ). The levels of SOD2 was decreased in the Model group but there is no statistical significance. The downward trend was reversed in the drug groups, the BA-7.5 group worked best (P<0.05, Figure 6F ). These results indicate that BA can alleviate L-Glu-induced oxidative stress damage to mice through activating the Nrf2/HO-1 signaling pathway. Figure 6 Effect of BA on genes and proteins associated with the Nrf2/HO-1 signaling pathway. (A) Protein bands of Nrf2, HO-1 and internal reference (LaminB and β-actin). (B)(C) Relative Nrf2 and HO-1 protein level. (D)(E)(F) The mRNA relative level of Nrf2, Cat and SOD2. # P＜0.05 vs Sham group, ** P＜0.01 and * P＜0.05 vs Model group. (n=4).

Figure 7 Mechanism of BA regulating oxidative stress by activating the Nrf2/HO-1 signaling pathway. Oxidative stress damage is considered to be an important factor that affect the progression of CNS diseases, such as epilepsy, stroke, or Parkinson’s disease. This damage arises due to the imbalance between the oxidative and antioxidant capacities. The primary manifestation is the generation of a significant number of free radicals, which assault the unsaturated fatty acids in the biofilm, leading to lipid peroxidation. This process results in the production of numerous peroxides, such as MDA, hydroxyl radicals, hydroperoxides and ROS, etc [38] . MDA and ROS are the key products of oxidative stress damage. High levels of MDA and ROS can cause denaturation of proteins and nucleic acids within cells, disrupt cell structure and physiological functions, ultimately leading to apoptosis [39] . However, the body also possesses a self-defense mechanism, comprising both enzymatic and non-enzymatic antioxidant systems. The primary antioxidant enzymes include SOD, Catalase, and GSH, etc. Under normal physiological condition, these enzymes can promptly eliminate the free radicalsautonomously, thereby preventing oxidative stress damage [40][41][42] . Additionally, measuring the levels of MDA and ROS can indirectly indicate the extent of oxidative damage in the body. As mentioned earlier, oxidative stress injury is related to the occurrence and development of various CNS diseases, subsequently, oxidative stress injury involves alterations in a variety of neurotransmitters. Glu is one of the primary excitatory neurotransmitters in the mammalian central nervous system and plays a role in synaptic signaling and neuronal growth processes. In pathological conditions, a large amount of Glu releasing can overactivate the corresponding receptors, leading to excitotoxicity and oxidative neurotoxicity [43][44] . Thus, the trend of developing safe and effective natural plant ingredients as medicines for alleviating excitatory amino acid toxicity has been persisting for a long time. Many studies have demonstrated that flavonoids exhibit favorable anti-inflammatory, antioxidant, antibacterial, and other pharmacological properties [45][46] . For instance, Luteolin, Baicalin, and Quercetin. In this study, we used the method of injecting L-Glu directly into the lateral ventricles of mice to establish a model of excitatory amino acid toxicity with oxidative stress injury as previously reported [47][48][49] . After injection of specific amount of L-Glu into the lateral ventricle, mice exhibited epileptiform and depression-like manifestations with reduced pain threshold. The behavioral manifestation is that the movement distance of open field and the latency of tail dumping are decreased. Besides, the histomorphological findings further proved that L-Glu can cause hippocampal tissue damage, especially in the CA3 region, where precisely related to learning and memory, emotional regulation, or pain perception [50][51][52] . Meantime, there was a significant increase in the expression of ROS and MDA, accompanied by apoptosis, a classical type of programmed cell death (The positive expression of CytC and TUNEL was increased). This suggests the occurrence of oxidative stress damage. This is consistent with the findings of previous studies. Nevertheless, BA treatment can significantly reverse these changes. This demonstrated the anxiolytic and analgesic effects of BA. The BA treatment groups showed inhibition of the increase in ROS and MDA levels, and promoted the release of SOD and Cat. These results indicate that BA may inhibit L-Glu-induced oxidative neurotoxicity by enhancing the body’s antioxidant system. Most studies have demonstrated strong association between the Nrf2 and oxidative stress damage [53][54], as has been shown in our previous in vitro studies on HT-22 cells [55] . Under normal physiological condition, Nrf2, Keap1, and Cul3 exist in the form of a complex and remain in a low-activity state. Upon stimulation by oxidative stress, Nrf2 dissociates into monomers and translocates to the nucleus. In the nucleus, it binds to the ARE, thereby activating the cell’s antioxidant potential. This promotes the expression of antioxidant genes such as HO-1 and SOD, helping to restore the body’s balance between oxidation and antioxidants. Ultimately, it resists oxidative stress damage and inhibits apoptosis [56][57] . Our study found that BA facilitates this process. The specific mechanism by which BA regulates oxidative stress damage is illustrated in Figure 7 . In this study, Glu stimulation was found to enhance the expression of Nrf2 and HO-1 proteins, indicating that the Nrf2/HO-1 signaling pathway may serves as an endogenous antioxidant mechanism in the body. With BA treatment, the expression levels of Nrf2 and HO-1 proteins were further elevated, which significantly mitigated oxidative stress damage. These findings suggest that the antioxidant effect of BA may be intimately linked to the activation of the Nrf2/HO-1 signaling pathway. Nevertheless, this study has some limitations. Especially, we did not include a group that received co-treatment with Nrf2/HO-1 signaling pathway inhibitors and BA. Secondly, based on the literature and our previous studies related to ischemic stroke [58][59], two dose groups (BA-7.5 and BA-15) was set up, however, low-dose of baicalin (BA-7.5) seemed to show better efficacy in the morphological and following molecular biology tests. We considered that besides the reason for not setting large dose range treatment groups, this phenomenon might attribute to different modeling method, therapeutic time window, or routes of administration. Therefore, the optimal dose and time window, as well as the mechanism of BA should be further screened. These are also our next research plan.

In a word, this study found that BA alleviated the oxidative stress damage caused by excitatory amino acid injury. And it was further proved that the antioxidant effect of BA was closely related to the activation of Nrf2/HO-1 signaling pathway.

This study was supported by Beijing Natural Science Foundation (No.7232317) and CACMS Innovation Fund (No.C12021A01305). Author Contributions Feng LI, Mingjiang YAO designed the study; Feng LI, Zishan HUANG, Huanyu GOU, Jiarui ZHENG, Mingjiang YAO performed the experiments; Feng LI, Mingjiang YAO contributed to manuscript preparation. All authors have read and approved the final manuscript. Ethics statement This study complies with the WMA Statement on animal use in biomedical research and was approved by Experimental Ethics Committee at Xiyuan hospital, China Academy of Chinese Medical Sciences (No. 2024XLC001-1). Conflict of interest statement All authors declare that they have no conflicts of interest.

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