Macamide B Pretreatment Attenuates Neonatal Hypoxic-Ischemic Brain Damage of Mice Induced Apoptosis and Regulates Autophagy via the PI3K/AKT Signaling Pathway

Lepidium meyenii (maca) is an annual or biennial herb from South America that is a member of the genus Lepidium L. in the family Cruciferae. This herb possesses antioxidant and antiapoptotic activities, enhances autophagy functions, prevents cell death, and protects neurons from ischemic damage. Macamide B, an effective active ingredient of maca, exerts a neuroprotective effect on neonatal hypoxic-ischemic brain damage (HIBD), but the mechanism underlying its neuroprotective effect is not yet known. The purpose of this study was to explore the effect of macamide B on HIBD-induced autophagy and apoptosis and its potential neuroprotective mechanism. The modified Rice-Vannucci method was used to induce HIBD in 7-day-old (P7) macamide B- and vehicle-pretreated pups. TTC staining was performed to evaluate the cerebral infarct volume in pups, the brain water content was measured to evaluate the neurological function of pups, neurobehavioural testing was conducted to assess functional recovery after HIBD, TUNEL and FJC staining was performed to detect cellular autophagy and apoptosis, and Western blot analysis was used to detect the levels of proteins in the pro-survival phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) signaling pathway and autophagy and apoptosis-related proteins. Macamide B pretreatment significantly decreases brain damage and improves the recovery of neural function after HIBD. At the same time, macamide B pretreatment activates the PI3K/AKT signaling pathway after HIBD, enhances autophagy, and reduces hypoxic-ischemic (HI)-induced apoptosis. In addition, 3-methyladenine (3-MA), an inhibitor of the PI3K/AKT signaling pathway, significantly inhibits the increase in autophagy levels, aggravates HI-induced apoptosis, and reverses the neuroprotective effect of macamide B on HIBD. Our data indicate that a macamide B pretreatment might regulate autophagy through the PI3K/AKT signaling pathway, thereby reducing HIBD-induced apoptosis and exerting neuroprotective effects on neonatal HIBD. Macamide B may become a new drug for the prevention and treatment of HIBD.


Introduction
Hypoxic-ischemic brain damage (HIBD) is a brain lesion produced by hypoxic-ischemic (HI) of brain tissue due to various causes, which can lead to acute death and chronic nerve damage in infants. In developed countries, HIBD occurs in 1-8 newborns out of every 1000 newborns. In developing countries, up to 26 newborns out of every 1000 suffer from HIBD disease [1,2]. Severe HIBD can lead to neurological sequelae such as mental retardation, cerebral palsy, and cognitive impairment in infants, and even death [3]. It is estimated that HIBD causes more than 1 million deaths every year worldwide [4]. To date, mild hypothermia Xiaoxia Yang and Mengxia Wang contributed equally.
* Guoying Li gzlgying820@163.com * Li Luo josephluoli@hotmail.com therapy is considered the only neuroprotective treatment that can improve the outcome of neonatal HIBD patients, but only 40% of patients survive with normal nervous system development [5]. Therefore, the study of HIBD's pathological mechanism and the search for active and effective drugs for neurofunctional repair has become urgent subjects in perinatal medical research. The phenotypes of autophagy, apoptosis, oxidative stress, inflammation, and necrosis are closely related to the pathological development of HIBD [6,7]. Among these factors, accumulating data indicate that the mechanisms of autophagy and apoptosis play fundamental roles in ischemic brain damage in newborn rodents [8,9]. Currently, many studies have focused on autophagy and apoptosis as therapeutic targets to prevent neonatal HIBD [6,9,10]. Autophagy is a process that exists in wide range of eukaryotic cells and uses the lysosomal pathway to degrade self-damaged organelles, proteins, and macromolecular substances. It maintains cell survival by degrading and restoring overexpressed proteins and organs in cells. It has been considered related to brain aging, as well as various neurodegenerative diseases [11]. Studies of stroke have shown that autophagy-induced damage aggravates the nerve damage induced by cerebral ischemic [12]. In a study of ischemic-reperfusion (I/R) damage, melatonin exerted a neuroprotective effect on I/R damage by activating autophagy [13]. In addition, in focal cerebral ischemic and glucose and oxygen deprivation (OGD) models, autophagy exerts a neuroprotective effect on glial cells [14]. The death and survival of cells usually require the coregulation of apoptosis and autophagy. Reducing neuronal apoptosis is a crucial process for determining neural functional recovery after HIBD. In the I/R model, an electroacupuncture intervention significantly inhibited neuronal apoptosis induced by cerebral ischemic, thereby reducing brain damage after I/R injury [15]. Studies examining acute spinal cord damage have found that inhibiting autophagy significantly aggravates cell apoptosis in the damaged spinal cord area and hinders the recovery of nerve function after spinal cord damage [10]. Therefore, efforts to enhance autophagy and reduce apoptosis may be a promising therapeutic strategy for neonatal mice HIBD.
Lepidium meyenii (maca) is an annual or biennial herb from South America that is a member of the genus Lepidium L. in the family Cruciferae. It is commonly used to treat degenerative diseases, such as neurodegenerative diseases, cardiovascular diseases, diabetes, cancer, and aging. Maca removes free radicals and protects cells from developing oxidative stress [16][17][18]. Studies have shown that maca improves the cognitive function of middle-aged mice by upregulating autophagy-related proteins [19]. Macamide is the product of maca obtained after drying. It is produced by the formation of amide bonds between different amino-containing compounds and various fatty acids. Macamides are part of the unique phytochemical composition of maca, which is rich in nutrients and exerts remarkable medicinal effects. It has functions such as antioxidant, anti-inflammatory, anti-osteoporosis, memory improvement, nerve cell protection, and nervous system regulation [19][20][21][22]. However, macamide B (structure shown in Fig. 1a) is an effective monomer of macamide, and the potential mechanism by which it provides neuroprotection and exerts neuroprotective effects is not yet known. Whether macamide B can prevent neonatal HIBD by regulating autophagy and reducing cell apoptosis is worthy of further investigation.
The phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) pathway is one of the most crucial signaling cascades that regulate the autophagy, apoptosis, proliferation, and growth of mammalian cells (Fig. 1b). It plays a vital role in mediating the pathological process of nervous system injury. In the treatment of neurodegenerative diseases, the PI3K/AKT pathway is considered a target to promote cell survival [23]. Therefore, the neuroprotective effect of the PI3K/AKT pathway on cerebral ischemic has been extensively studied. Geniposide protects against perinatal HI-induced brain damage by activating the PI3K/AKT signaling pathway [24]. Ginkgetin inhibits cell apoptosis induced by I/R by activating the PI3K/AKT pathway [25]. In a rat middle cerebral artery occlusion (MCAO) model, sodium butyrate inhibits neuronal apoptosis through the GPR41/Gbc/PI3K/AKT signaling pathway, thereby reducing brain damage after MCAO and improving functional outcomes [26]. Increased nerve cell apoptosis is a common consequence of HIBD, and reducing cell apoptosis is essential to improve the neurological dysfunction caused by HIBD. The PI3K/AKT pathway is also a meaningful target to regulate autophagy. PI3K/AKT pathway inhibitors significantly inhibit the expression of autophagy-related proteins induced by HBCDs, suggesting that the activation of the PI3K/AKT pathway may promote an increase in HBCD-induced autophagy levels [27]. Melatonin reduces I/R-induced neuronal damage by inducing the expression of autophagy-related proteins [28]. In addition, the induction of mitochondrial autophagy by endoplasmic reticulum stress prevents transient ischemic brain damage [29]. As a potential neuroprotective drug, whether macamide B can regulate autophagy through the PI3K/AKT pathway, reduce HIBD-induced apoptosis, and play a neuroprotective effect on neonatal mice HIBD is worthy of our in-depth study.
In the ischemic penumbra (IP) region of pup brain tissue, there are a large number of brain cells in the dormant or semi-dormant state, which can only maintain their morphological integrity and cannot exercise their original normal functions due to the lack of energy supply. Therefore, therapeutic strategies focused on alleviating neuronal apoptosis in the penumbra seem very promising for the treatment of neonatal HIBD. The PI3K/AKT signaling pathway plays a vital role in the processes of cellular autophagy and apoptosis [30]. A therapeutic strategy designed to activate the PI3K/AKT signaling pathway and subsequently enhance autophagy and reduce cell apoptosis in the IP region seems very promising. However, the ability of macamide B to ameliorate HIBD in neonatal mice and whether macamide B pretreatment regulate autophagy through the PI3K/AKT signaling pathway after HIBD, thereby reducing HIBD-induced apoptosis, are unclear. Therefore, this study explored the potential of macamide B to regulate autophagy through the PI3K/AKT signaling pathway and subsequently reduce HIBD in neonatal mice and inhibit cell apoptosis.

Animals and Groups
The study selected 7-day-old (P7) C57BL/6 pups (male and female randomly distributed littermates), weighing 3  A total of 170 pups were used in this study. The mouse pups were randomly divided into Sham, HI + Vehicle (PBS for vehicle), HI + Macamide B (60 mg/kg), HI + Macamide B + Vehicle (0.9% saline for vehicle), and HI + Macamide B + 3-methyladenine (3-MA, 2.48 ug) groups [31], with 34 pups in each group. The protocol is described in Fig. 1c. The pups were pretreated with macamide B administration 20 min before surgery. After the operation, the pups were returned to the dams for feeding and recovery and exposed to hypoxic for consecutive 4 h after 1 h. TTC staining, brain water content measurement and Western blot were conducted on the first day after HI. On the third day after HI, IF, TUNEL, and FJC staining were conducted, and neurobehavioural experiment was conducted on days 1, 3, and 7 after HI, respectively.

HIBD Model
In this experiment, we used a total of 160 P7 C57BL/6 pups. The improved Rice-Vannucci method was used to construct the HIBD model [32,33]. Briefly, C57BL/6 pups at 7-9 days of age (P7-9) were given continuous inhalation anesthesia (isoflurane) through a face mask, fixed on a sterile surgical drape in the supine position, and disinfected at the predetermined surgical incision location on the neck skin. A 1-cm incision was cut along the middle of the neck, the subcutaneous tissue was bluntly separated, and the unilateral common carotid artery (CCA) was separated; a coagulator was used to cut off the CCA, the skin was sutured and disinfected, and the pups were returned to the dams for feeding and recovery. After recovering for 1 h, put the pups in a 37 °C water bathcontrolled hypoxic box, and a mixed gas of oxygen (8%) and nitrogen (92%) was supplied. The pups were exposed to hypoxic for 4 h then returned to the dams to feed, and the model was completed.

Drug Treatments
Macamide B (Shanghai Yuanye Bio-Technology Co., Ltd, China, HPLC ≥98%) was dissolved in PBS solution (1.25 mg/ml). Twenty minutes before the ischemia surgery, the macamide B group mice were intraperitoneally injected with macamide B (60 mg/kg). For pups in the vehicle group, give the same volume of PBS treatment. 3-MA (APExBIO, USA) was dissolved in 0.9% saline (1.24 ug/ul). Twenty minutes before the ischemic surgery, neonatal mice in the HI+Macamide B+3-MA group received 2 ul 3MA (2.48 ug) stereotaxic injection into the brain of the lateral ventricle while receiving intraperitoneal pretreatment with macamide B. Neonatal mice in the HI+Macamide B+Vehicle group received the same volume of normal saline injection into the lateral ventricle.

Infarct Volume Measurement
After HIBD for 24 h, intraperitoneal administration of 10% chloral hydrate to the pups for anesthesia (0.1 ml), and the brains were immediately extracted. We cut the pup's brain into four brain slices in a coronal plane (the intervals between adjacent brain slices are 2 mm). The sections were stained for 20 min in a 2% solution of 2,3,5-triphenyltetrazolium chloride (TTC, Sigma-Aldrich, Germany), turning the brain slices from time to time to make even contact with the staining solution. Then, the images of the brain slice of the pup were captured by a digital camera. The nonischemic necrotic area was red, while the ischemic necrotic tissue was white. Use ImageJ software (version 1.8.0, USA) to analyze the cerebral infarct volume of pups. The percentage of infarct volume = [(contralateral hemisphere − un-infarcted area of ipsilateral hemisphere)/contralateral hemisphere × 2] × 100% [1].

Measurement of Brain Water Content
After HIBD for 24 h, intraperitoneal administration of 10% chloral hydrate to the pups for anesthesia (0.1 ml), and the brains were immediately extracted. The pup's brain was divided into two parts and weighed for wet weight and the cerebral hemispheres were dried in an oven at 106°C for 24 h and weighed for dry weight. Percentage of brain water content = (wet weight-dry weight)/wet weight ×100%.

HE Staining
The liver and kidney was fixed with 4% PFA for 24 h and then embedded in paraffin from which 4-μm-thick sections were obtained. After dewaxing and rehydration, the sections were stained with HE, dehydrated, and were made transparent by placing them in a concentration gradient of ethanol and xylene. Finally, the sections were coverslipped with resin and analyzed using an optical microscope (Zeiss AX10, Germany).

Neurobehavioral Assessments
Neurological damage caused by HIBD can lead to sensorimotor impairments. Body weight and sensorimotor performance (righting reflex, negative geotaxis, and grip test) were tested 1, 3, and 7 days after the HI procedure in a blinded manner [34].

Righting Reflex
The righting reflex is used to evaluate the recovery of the brain of pups. The pups were placed on a flat surface with one hand gently holding the head and the other hand gently holding the hind limbs; the pup was rolled onto its back and released. The time required for the mouse to return to an upright position (all limbs on the ground) was recorded. The maximum testing time was 1 min, and times over 1 min were recorded as 1 min.

Negative Geotaxis
The geotaxis test was used to diagnose vestibular or proprioceptive functions. Place the pup on a flat surface inclined at 45 degrees, with the head of the pup facing the bottom of the plane, and record the time it takes for the head and body of the pup to make a 180° turn. The maximum testing time was 1 min, and times over 1 min were recorded as 1 min.

Grip Test
The pup grasped a metal wire with a diameter of 1.5 mm with both front feet. The metal wire was stretched horizontally in a test box with a width of 50 cm. The distance between the metal wire and the bottom of the box was 15 cm. The bottom was covered with cork chips. Record the time it takes for the pup to grasp the wire to release it. The minimum testing time was 20 s, and times below 20 s were recorded as 20 s.

Western Blot Analysis
Protein samples (15 μl/well) were separated on SDS-PAGE gel (80 V, 110 min) and transferred to 0.22 μm polyvinylidene difluoride (PVDF) membrane (290 mA, 100 min). Five percent nonfat dry milk in TBST buffer was used to block nonspecific sites for 1. were measured by an automatic chemiluminescence image analysis system (Tanon 5200, Shanghai, China). ImageJ software was used to carry out Western blot analysis, and SPSS 21.0 was applied to process the data.

Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling Staining
Because cells are prone to programmed cell death during HI [35], therefore, we assessed the apoptotic cells in the brain tissue of the pups by TUNEL Apoptosis Detection Kit (fluorescence) (Wanleibio, China).
On day three after HI damage, the pups were perfused into the heart, and fresh brain tissue was quickly collected in 4% paraformaldehyde (PFA) for fixation. After fixation for 24 h, the brain tissue of the pup was rinsed under running water for 16 h. After washing with running water, perform gradient dehydration, paraffin embedding, tissue sectioning, and other operations on the brain tissue of pups for subsequent TUNEL detection.
The slices were immersed in xylene I and II for 15 min each; immersed in 100% I, 100% II, and 95%, 90%, 80%, 70%, and 50% ethanol solutions for 5 min per solution; and washed with ddH 2 O 3 × for 5 min each. The slices were placed in a citrate buffer solution (0. 01 mol/l), and place them in a microwave oven at high-temperature repair for 20 min. Add 25 μl of 3% H 2 O 2 buffer to each brain tissue and let stand for 12 min in the dark. Afterwards, three washes with PBS for 5 min each. Then, add 25 μl TUNEL reaction buffer to each brain tissue and incubated in a 37 °C incubator in the dark for 90 min. After three washes with PBS for 5 min each, cell nuclei in brain tissues were counterstained with DAPI for 5 min, and then the PBST wash step was repeated. After absorbing excess water with filter paper, sections were mounted with an anti-fluorescence quencher. The cells were immediately observe with a fluorescence microscope (Olympus BX51, Japan) or stored at − 20 °C for observation within a week.

Fluoro-Jade C Staining
Twenty-four hours after HIBD, the pups were immediately sacrificed, and paraffin sections of the brains were generated. Fluoro-Jade C (FJC) staining (Biosensis, USA) was performed to detect neuronal degeneration in the pup brain tissue.
The slices were immersed in xylene I and II for 15 min each, immersed in 100% I and 100% II ethanol solutions for 5 min per solution, immersed in 70% ethanol solutions for 5 min per solution, and washed with ddH 2 O for 2 × for 5 min each. Sections were transferred to a new Coplin jar containing ddH 2 O and incubated for 2 min. Nine parts of ddH 2 O and 1 part of potassium permanganate solution were mixed, added to the brain tissue slices, and incubated at room temperature for 10 min. Sections were rinsed for 2 min with ddH 2 O. Nine parts of ddH 2 O, 1 part of FJC solution, and 1 part of DAPI solution were mixed, added to the brain tissue slices, and incubated for 12 min. Sections were washed with ddH 2 O 3 × for 1 min each. The slices were placed in a 56 °C oven and baked for 5 min. The dry sections were then cleared by a brief (5 min) immersion in xylene. Sections were mounted with an anti-fluorescence quencher. The cells were immediately observed with a fluorescence microscope or stored at − 20 °C for observation within a week.

Tissue Immunofluorescence Staining
Twenty-four hours after HIBD, the pups were immediately sacrificed, and paraffin sections of the brain were prepared. Immunofluorescence staining was performed to detect the expression levels of p53, Bax, Bcl-2, caspase-3, and cleaved caspase-3 in the mouse brain tissue.
Tissue dewaxing and high-temperature repair steps were repeated and sections were incubated with Quick Block™ immunostaining blocking solution for 20 min. Each brain tissue section was incubated with the primary antibody at 4 °C for 16 h, and 0.5% Triton X-100 was added to the corresponding primary antibody to disrupt the cell membrane. The following primary antibodies were used: Beclin1 Biosciences, USA). After 16 h, the sections were removed from the refrigerator and incubated at room temperature for 30 min before being washed again with PBST 3 × for 10 min each. DyLight 488-labeled goat anti-rabbit fluorescent secondary antibody (1:360, Sigma-Aldrich, USA) or DyLight 594-labeled goat anti-rabbit fluorescent secondary antibody (1:360, Sigma-Aldrich, USA) or Dylight 405-labeled goat anti-rabbit fluorescent secondary antibody (1:425, Sigma-Aldrich, USA) was incubated with sections for 2 h at room temperature in the dark. After three washes with PBS for 5 min each, cell nuclei in brain tissues were counterstained with DAPI for 5 min, and then the PBST wash step was repeated. Sections were mounted with an anti-fluorescence quencher. The cells were immediately observed with a fluorescence microscope or stored at − 20 °C for observation within a week. When performing multiple immunofluorescence labeling, repeat the experimental steps after hightemperature antigen retrieval (except for the immunofluorescence staining blocking step).

Statistical Analysis
All of the experiments were repeated at least three times. The data are presented as the mean ± SEM. Statistical analyses were carried out by SPSS.21.0 and GraphPad Prism (version 8.0, USA). Differences between individual groups were first compared using analysis of variance (one-way ANOVA), and then post hoc testing was analyzed with Tukey or Student-Newman-Keuls multiple comparisons. Difference between the two groups was compared using Student's t-test. A p < 0.05 indicated that the difference between the two groups was statistically significant.

Macamide B Pretreatment Attenuated HIBD in Newborn Mice
To determine whether macamide B pretreatment was effective in reducing HIBD in neonatal mice, we used TTC staining to assess the cerebral infarct volume and cerebral water content to detect cerebral edema damage in the neonatal mice. Three doses were used to determine the optimal dose of the macamide B pretreatment for neonatal HIBD: a low dose (30 mg/kg), medium dose (60 mg/kg), and high dose (90 mg/kg). The results of TTC staining (Fig. 2a,b) showed that the medium dose (60 mg/kg) of macamide B significantly reduced the percentage of infarct volume compared with the vehicle group (p < 0.05). Compared with the vehicle group, no significant difference in the infarct volume was observed between the low-dose (30 mg/kg) and high-dose (90 mg/kg) groups (p > 0.05). Pretreatment with macamide B at a medium dose (60 mg/ kg) significantly reduced the cerebral infarct volume in pups with HIBD.
In order to determine the drug safety of 60 mg/kg macamide B, we performed liver and kidney toxicity tests on pups pretreated with 60 mg/kg macamide B. The results of the HE experiment (Fig. 2c) showed that compared with the sham group, the liver and kidney tissues of the 60 mg/kg macamide B group were evenly stained with HE, the hepatocyte cords were arranged neatly, the liver lobule structure was intact, and the nucleus and cytoplasm were normal. The structure of glomeruli and tubules in kidney tissue is clear, no casts and granules are seen in the tubules, and the ratio of balloons is moderate. The liver and kidney tissue morphology and structure of 60 mg/kg macamide B pretreated pups were not significantly different from those in the sham group. The HE staining results of liver and kidney showed that 60 mg/kg macamide B pretreatment has no toxic side effects on the liver and kidney of pups, and it has a certain degree of safety.
The experimental results of brain water content are shown in Fig. 2d. The water content of the ipsilateral cerebral hemisphere in the vehicle group was significantly higher than that in the sham group (p < 0.0001). Macamide B pretreatment significantly reduced the water content of the ipsilateral cerebral hemisphere in the vehicle group (p < 0.0001). Based on these results, macamide B pretreatment significantly reduced cerebral infarct volume and cerebral edema damage in HIBD neonatal mice, and exerts neuroprotective effects on HIBD in neonatal mice.

Macamide B Pretreatment Improved Neurobehavioural Performance After Neonatal HIBD
In order to evaluate the effect of macamide B on the neurological function of HIBD in newborn mice, we explored whether macamide B promoted the general health and recovery of neurological function by conducting the following neurobehavioural tests at 1, 3, and 7 days after surgery: righting reflex, negative geotaxis, and grip tests in the sham, vehicle, and macamide B groups. Body weight is used to evaluate general health. At 1, 3, and 7 days after HIBD, macamide B pretreatment significantly reversed the weight loss of pups with HIBD compared with the vehicle group (Fig. 3a). In the righting reflex (Fig. 3b), negative geotaxis (Fig. 3c), and grip tests (Fig. 3d), mice in the macamide B group exhibited superior performance at 1, 3, and 7 days after surgery than those in the vehicle group. These outcomes indicate that macamide B pretreatment reverses weight loss and improved neurobehavioural performance after neonatal HIBD.

Macamide B Pretreatment Activates the PI3K-AKT Signaling Pathway
To determine whether PI3K-AKT signaling pathway is involved in the neuroprotective mechanism of macamide B after HIBD, the expression level of PI3K-AKT signaling pathway related proteins was detected by Western blot. Western blot results showed that the levels of p-PI3K and p-AKT were significantly decreased in the vehicle group compared with those in the sham group (p < 0.0001, Fig. 4b, c). Compared with the vehicle group, macamide B pretreatment significantly increased the levels of the p-PI3K and p-AKT proteins (p < 0.0001, Fig. 4b, c). Western blot analysis showed that macamide B pretreatment significantly activated the PI3K-AKT signaling pathway. Macamide B exerts a neuroprotective effect on HIBD in neonatal mice, which may be mediated by the PI3K-AKT signaling pathway. a Representative images of coronal brain sections stained with TTC at 24 h after HIBD. b Quantitative analysis of the cerebral infarct volume in pups. Compared with pretreatment with the vehicle, pretreatment with the medium dose (60 mg/kg) of macamide B significantly reduced the infarct volume. c HE staining results of liver and kidney in mice. d Quantitative analysis of the brain water content. The water content of the ipsilateral cerebral hemisphere from the vehicle group was significantly higher than that of the sham group, and the brain water content of the macamide B group was significantly lower than that of the vehicle group. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 compared with the sham group; # p < 0.05, ## p = 0.001, and #### p < 0.0001 compared with the vehicle group; @ p < 0.001 compared with the macamide B group. n = 6 mice per group. Scale bar = 50 μm
Macamide B pretreatment reversed these changes. The results of Western blot experiments showed (Fig. 7) that the expression levels of Beclin1 and LC3B in the vehicle group were significantly decreased (p < 0.0001), and the expression levels of p62 were significantly increased compared with those in the sham group (p < 0.001). Macamide B pretreatment significantly upregulated the expression levels of Beclin1 and LC3 (p < 0.0001; p < 0.05) and downregulated the expression levels of p62 (p < 0.01). Tissue immunofluorescence staining and Western blot results indicate that macamide B pretreatment significantly increases the autophagy level in neonatal mice with HIBD, and macamide B may exert neuroprotective effects on HIBD in neonatal mice by enhancing autophagy. Fig. 3 Macamide B pretreatment improved neurological function and reversed body weight loss in mice after HIBD. a Mice in the macamide B group had significantly higher body weights than mice in the vehicle group at 1, 3, and 7 days postsurgery. The macamide B pretreatment group exhibited noticeably improved neurobehavioural outcomes in the (b) righting reflex, (c) negative geotaxis, and (d) grip tests at 1, 3, and 7 days postsurgery compared to the vehicle group. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 compared with the sham group; ## p < 0.01, ### p < 0.001, and #### p < 0.0001 compared with the vehicle group. n = 8 mice per group

Macamide B Pretreatment Significantly Reduces HIBD-Induced Apoptosis
We next detected the effect of macamide B pretreatment on apoptosis in neonatal mice with HIBD. TUNEL and FJC staining were used to evaluate neuronal apoptosis and degeneration. Tissue immunofluorescence staining and Western blotting were performed to detect the levels of the apoptosis-related proteins p53, Bax, Bcl-2, caspase-3, and cleaved caspase-3.
The number of TUNEL-positive cells (Fig. 8b), FJCpositive neurons (Fig. 8c), and p53- (Fig. 8d), Bax- (Fig. 8e), Bcl-2- (Fig. 8f), caspase-3- (Fig. 8g), and cleaved caspase-3-positive cells (Fig. 8h) in the ischemic penumbra of neonatal mouse brain tissue was observed under a fluorescence microscope. TUNEL and FJC staining results showed that the numbers of TUNEL-positive cells and FJC-positive neurons in the vehicle group were significantly increased compared with those in the sham group. These changes were significantly attenuated by macamide B pretreatment, which significantly reduced the numbers of TUNEL-positive cells and FJC-positive neurons and inhibited apoptosis. The results of tissue immunofluorescence staining showed significantly increased numbers of p53-, Bax-, caspase-3-, and cleaved caspase-3-positive cells, and a significantly decreased Western blot results (Fig. 9) showed significantly higher levels of p53, caspase-3, and cleaved caspase-3 in the vehicle group than those in the sham group (p < 0.0001; p < 0.01; p < 0.001), and the Bcl-2/Bax ratio decreased significantly (p < 0.0001). Macamide B pretreatment significantly inhibited the increase in the levels of p53, caspase-3, and cleaved caspase-3 and increased the Bcl-2/Bax ratio. Based on these results, macamide B pretreatment significantly reduces apoptosis, and macamide B might exert a neuroprotective effect on HIBD in neonatal mice through the antiapoptotic pathway.

3-MA Inhibits the PI3K-AKT Signaling Pathway and Reverses the Neuroprotective Effect of Macamide B on HIBD in Neonatal Mice
3-MA is a selective inhibitor of PI3K and exerts an inhibitory effect on class III PI3K [36]. We determined whether macamide B exerted a neuroprotective effect on HIBD in neonatal mice through the PI3K-AKT signaling pathway. After treatment with 3-MA, an inhibitor of the PI3K-AKT signaling pathway in newborn mice with HIBD, 3-MA significantly reduced p-PI3K and p-AKT protein levels (Fig. 10e, f; p < 0.0001) and blocked the activation of the PI3K-AKT signaling pathway induced by macamide B pretreatment. At the same time, the results of the TTC experiment showed (Fig. 10a, b) that compared with the The upper left brain slice shows the location of immunofluorescence staining (small black box). ****p < 0.0001 and **p < 0.01 compared with the sham group; #### p < 0.0001 and ## p < 0.01 compared with the vehicle group. n = 8 animals per group. Scale = 100 μm macamide B + vehicle 2 group, the 3-MA intervention significantly increased the cerebral infarct area in the pups (p < 0.0001). The brain water content measurement results showed (Fig. 10c) that the 3-MA intervention significantly aggravated the brain oedema and damage in pups compared with the macamide B + vehicle 2 group (p < 0.05). In the behavioral experiment, the 3-MA intervention significantly aggravated the weight loss of neonatal mice at 1, 3, and 7 days after HIBD compared with the macamide B + vehicle 2 group (Fig. 10g). These outcomes indicate that 3-MA inhibits the PI3K-AKT signaling pathway and reverses the neuroprotective effect of macamide B on HIBD in neonatal mice (Fig. 10h, i, g). Macamide exerts a neuroprotective effect on HIBD in newborn mice through the PI3K-AKT signaling pathway.

3-MA Treatment Inhibits Autophagy and Reverses the Protective Effects of Macamide B Pretreatment on Apoptosis
In order to evaluate that the inhibition of apoptosis by macamide B depends on the mediation of autophagy, we used immunofluorescence and Western blot to detect the levels of autophagy and apoptosis, respectively. 3-MA is an inhibitor of the PI3K-AKT signaling pathway and is also a well-known autophagy inhibitor that functions by inhibiting the formation of autophagosomes [36]. In our study, immunofluorescence staining (Fig. 11, 12) showed significant decrease in the numbers of Beclin1-, LC3B-(p < 0.0001), LAMP1-(p < 0.05), and p62, LC3 and LAMP1 co-localization marker positive cells (p < 0.05) and a significant increases in the number of p62-positive cells (p < 0.0001) after the 3-MA intervention compared to the macamide B + vehicle group. The Western blot results (Fig. 13) were consistent with the trend of the immunofluorescence results. The 3-MA intervention significantly reduced the expression levels of the Beclin1 and LC3 proteins (p < 0.0001) and increased the expression level of the p62 protein (p < 0.001).
In apoptosis-related studies, fluorescence experiments ( Fig. 14) showed that the 3-MA intervention resulted in significant increases in the numbers of TUNEL-positive cells, FJC-positive neurons, and p53-, Bax-, caspase-3-, and cleaved caspase-3-positive cells and a significant decrease in the number of Bcl-2-positive cells compared with the macamide B + vehicle group. The results of Western blot experiments showed (Fig. 15) that compared with the macamide B + vehicle group, the 3-MA intervention significantly increased the levels of p53, caspase-3, and cleaved caspase-3 (p < 0.0001; p < 0.001) and reduced the Bcl2/Bax ratio (p < 0.0001). These outcomes indicate that 3-MA treatment inhibits autophagy and reverses the protective effects of macamide B pretreatment on apoptosis. Therefore, macamide B pretreatment may enhance autophagy by activating the PI3K-AKT signaling pathway, subsequently reducing HIBD-induced apoptosis. Fig. 7 The effect of macamide B pretreatment on autophagy in neonatal mice with HIBD. a Representative Western blots and quantification of (b) Beclin1, (c) LC3II/I, and (d) p62 protein levels in the ipsilateral cerebral hemisphere at 24 h after HIBD. Macamide B pretreatment significantly upregulated the expression levels of Beclin1 and LC3 in HIBD pups and downregulated the expression levels of p62. ****p < 0.0001, **p < 0.01, and ***p < 0.001 compared with the sham group; #### p < 0.0001, ### p < 0.001, and ## p < 0.01 compared with the vehicle group. n = 6 animals per group

Discussion
In this study, we confirmed that macamide B exerts a neuroprotective effect on HIBD in newborn mice. Specifically, we showed that (1) macamide B pretreatment significantly reduces HI-induced brain damage; (2) pretreatment with macamide B significantly improves neurobehavioural outcomes after HIBD; (3) macamide B pretreatment significantly activates the PI3K/AKT signaling pathway, enhances autophagy, and inhibits cell apoptosis; and (4) 3-methyladenine (3-MA), an inhibitor of the PI3K/AKT signaling pathway, significantly inhibits the increase in autophagy levels, aggravates HI-induced apoptosis, and reverses the neuroprotective effect of macamide B on HIBD. In this study, we clarified that macamide B pretreatment protects neonatal mouse brains from HIBD and improves general conditions and neurobehaviours. The neuroprotective effect of macamide B may be mediated by the PI3K/AKT signaling pathway to regulate autophagy, thereby reducing HIBDinduced apoptosis.
The treatment and prevention of neonatal HIBD have always attracted much attention, but due to the complexity of the nervous system, treating this disease poses severe challenges. Medicinal plants can be advantageous in the drug discovery process because they have undergone indirect clinical trials during their long-term use, and their  The effect of macamide B pretreatment on cellular apoptosis in neonatal mice with HIBD. a Representative Western blots and quantification of (b) p53, (c) Bcl-2/Bax, (d) caspase-3, and (e) cleaved caspase-3 levels in the ipsilateral cerebral hemisphere at 24 h after HIBD. Macamide B pretreatment significantly reduced p53, caspase-3, and cleaved caspase-3 protein levels and increased the Bcl-2/Bax ratio compared with the vehicle group. Western blot results showed that macamide B pretreatment significantly reduced apoptosis in neonatal mice with HIBD. ****p < 0.0001, **p < 0.01, and ***p = 0.0001 compared with the sham group; #### p < 0.0001 and ## p < 0.05 compared with the vehicle group. n = 6 animals per group Fig. 10 The effect of 3-MA on the PI3K-AKT signaling pathway in neonatal mice with HIBD that were pretreated with macamide B. a Representative images of coronal brain sections stained with TTC 24 h after HIBD. b Quantitative analysis of the cerebral infarct volume in pups. Vehicle 1 is the vehicle for macamide B, and vehicle 2 is the vehicle for 3-MA. Compared with the vehicle 2 group, the cerebral infarct area in the pups treated with 3-MA increased significantly. n = 6 mice per group. c Quantitative analysis of the brain water content. d Representative western blots and quantification of (e) p-PI3K and (f) p-AKT protein levels in the ipsilateral cerebral hemisphere at 24 h after HIBD. Macamide B pretreatment signifi-cantly reduced the levels of the p-PI3K and p-AKT proteins in the HI-injured brain. n = 6 mice per group. On postoperative Days 1, 3, and 7, 3-MA reversed the increased (g) body weights and exacerbated poor neurobehavioural performance in the (h) righting reflex, (i) negative geotaxis, and (j) grip tests by macamide B-pretreated pups with HIBD compared to the macamide B + vehicle 2 groups. n = 6 animals per group. ****p < 0.0001 and ***p < 0.001 compared with the sham group; #### p < 0.0001 compared with the vehicle 1 group; @@ p < 0.01 and @@@@ p < 0.0001 compared with the macamide B group; &&&& p < 0.0001, &&& p < 0.001, && p < 0.01, and & p < 0.05 compared with the macamide B + vehicle 2 group side effects and toxicity are usually known. Given the complexity of neurodegenerative diseases, natural drugs may be good candidates for targeting phenotypes such as apoptosis, autophagy, oxidative stress, and inflammation and promote the recovery of cell death and neuronal damage in focal ischemic stroke and many neurodegenerative diseases [37,38]. Maca is a rare medicinal and edible plant that originated in the central part of the Peruvian Andes. It has rich nutritional value and can relieve fatigue, improve sleep, and improve sexual function and exhibits antioxidant activity. Maca is called "Peruvian Ginseng" [39][40][41]. Studies have shown that maca has extensive neuroprotective effects both in vivo and in vitro. The cerebral infarct volume of mice with neurons damaged by H 2 O 2 was treated with maca which is significantly reduced in mice treated with maca, and antiapoptosis, antioxidation, prevention of cell death, and protection of neurons from ischemic damage are its primary mechanisms of action [19]. In vitro studies showed that the viability of crayfish neurons was significantly enhanced by treatment with maca extract, which also showed the neuroprotective effects of maca [19]. Maca can be used as a neuroprotective agent alone or synergistically with other protective agents to prevent neurodegeneration and cell death in stroke and other neurodegenerative diseases [42]. Autophagy disorders are thought to be related to brain aging and a variety of neurodegenerative diseases. Maca can promote the upregulation of autophagy-related proteins by activating the autophagy signal in the mouse cortex and improve the cognitive function of middle-aged mice [23].
In 2000, Zheng BL and others first discovered the unique active substance macamide in maca [41]. Macamide, a type of benzylated or 3-methoxybenzylated alkanamide alkaloid, is a unique secondary metabolite in maca [42]. Studies have shown that macamide can act on the cannabinoid system, inhibit the activity of fatty acid amide hydrolase (FAAH), and exert neuroprotective effects on the central nervous system by regulating the release of neurotransmitters [21]. The reason macamide can bind to FAAH enzymes and protect anandamide from enzyme metabolism may be due to the similarity between the chemical structure of macamide and that of endocannabinoids; for example, anandamide has long chain fatty acid and amide groups. While the macamide structure mimics that of anandamide [43], macamide B, as an effective monomer extracted from macamide, also has long-chain fatty acid and amide groups. And studies have shown that purified macamide or synthetic analogs of macamide can play a neuroprotective role by acting on the endogenous cannabinoid system [44]. Therefore, macamide B may also play a neuroprotective role in neonatal mice HIBD by inhibiting the activity of FAAH and regulating the release of neurotransmitters. In addition, the finding that macamide B reversed righting reflex almost fully may suggest neurosteroid-like action on GABA A receptor function. Studies have shown that taurine can play a neuroprotective role by activating GABA A receptors to reduce focal cerebral ischemic injury in mice [45]. Topiramate (TPM) protects hippocampal cells from oxidative stress and neuroinflammation by activating GABA A receptors in rat brain [45]. Whether the neuroprotective effect of macamide B on neonatal mice HIBD also depends on the activation of GABA A receptors deserves deep thinking. SUR1-TRPM4 was activated and upregulated in astrocytes and vascular endothelial cells in the brain tissue at the early stage of injury, which was closely related to the occurrence of cerebral edema [46]. It indicated that blocking the isomer might prevent or limit the generation of cerebral edema and reduce the progression of secondary brain damage in the early stage of injury. Brain water content experiments showed that the brain water content of neonatal mice was significantly reduced after The upper left brain slice shows the location of immunofluorescence staining (small black box). n = 8 animals in each group. ****p < 0.0001 and *p < 0.05 compared with the macamide B + vehicle control group. n = 8 animals per group. Scale = 100 μm Fig. 13 a Representative Western blots and quantification of (b) Beclin1, (c) LC3, and (d) p62 protein levels in the ipsilateral cerebral hemisphere at 24 h after HIBD. The effect of the 3-MA intervention on autophagy-related proteins in HIBD neonatal mice pretreated with macamide B is shown. Vehicle is the vehicle for 3-MA. Compared with the macamide B + vehicle control group, the Beclin1 and LC3 protein expression levels in the 3-MA intervention group decreased significantly, and the p62 protein expression level increased significantly. ****p < 0.0001 and ***p < 0.001 compared with the macamide B + vehicle control group. n = 6 animals per group  (Fig. 2d). The SUR1-TRPM4 complex may be inhibited during this process. In addition, after treatment with 3-MA, an inhibitor of PI3K/AKT signaling pathway, the cerebral edema damage of the pups was also significantly aggravated. The results of brain water content experiment showed that both macamide B and PI3/AKT signaling pathways might be involved in the blocking of SUR1-TRPM4 complex. The blocking of SUR1-TRPM4 complex may be another key mechanism of neonatal HIBD treatment. As a unique monomer of macamide, it is not yet known whether macamide B has a neuroprotective effect on neonatal HIBD and the key mechanism for its neuroprotective effect. In this study, we will in-depth study the potential mechanism and key pathways for its neuroprotective effect.
In this study, one of the issues we need to consider is the effectiveness of the route of administration of macamide B in the central nervous system (CNS). The prerequisite for the direct effect of drugs on the CNS is that the drug must first pass through the blood-brain barrier (BBB) from the blood and enter the extracellular fluid of the CNS to be effective. [47]. Therefore, it is necessary to confirm whether macamide B can pass through the BBB. Studies have shown that the lipophilicity of certain components of maca can promote their passage through the BBB and affect CNS function. Related reports have shown that some ingredients in maca are active in the CNS [48][49][50]. For some compounds, the neonatal BBB is more permeable than the adult BBB [51]. At the same time, it has also been reported that when pups are injured by HI, the normal function of the BBB will be destroyed, which will increase the permeability of the BBB. This situation also increases the possibility of macamide B successfully reaching the brain through the BBB [51]. Besides, pups have a large peritoneal surface area and strong exchange capacity, and intraperitoneal administration of macamide B enabled macamide B to reach the brain through different signal transduction pathways and thus exert its neuroprotective effect. We used the Rice-Vannucci method to establish a neonatal mouse HIBD model by ligating one side of the common carotid artery of the pups to cause hemi-brain tissue ischemia and then placing the pups in a closed hypoxic tank for 4 h. This model is currently recognized as an ideal model for the study of neonatal HIBD [34,52]. The results of TTC staining showed that there was a clear cerebral infarction area on the ligation side of the brain, which verified that the brain tissue damage caused by surgery and hypoxic was, as proposed by Towfghi J et al., mainly confined to the carotid artery ligation side [53]; these results also indicate that the HIBD model was successfully constructed. Compared with the vehicle group, the cerebral infarct volume of pups pretreated with macamide B was significantly reduced, indicating that macamide B has a significant neuroprotective effect on HIBD in newborn mice. In this experiment, P7 pups were selected as the model mice, Fig. 15 a Representative Western blots and quantification of (b) p53, (c) Bcl-2/ Bax, (d) caspase-3, and (e) cleaved caspase-3 levels in the ipsilateral cerebral hemisphere at 24 h after HIBD. Compared with the macamide B + vehicle control group, the p53, caspase-3, and cleaved caspase-3 protein levels in the 3-MA intervention group were significantly increased and the Bcl-2/Bax ratio was significantly decreased. ****p < 0.0001 and ***p < 0.0001 compared with the the macamide B + vehicle control group. n = 6 animals per group mainly because the brains of P7 mouse pups are similar in histological structure to the 32-34 week fetus or newborn [54].
Based on accumulating evidence, apoptosis is a crucial pathological trigger of neurological deficits after HIBD [55]. Compared to adult brains, apoptosis is more common in immature newborn brains [56]. Inhibition of neuronal apoptosis is strongly recommended as a therapeutic target for neuronal rescue in the neonatal HIBD paradigm [57]. The tumor suppressor p53 may trigger the apoptosis pathway after DNA damage and activate caspase-3 to induce cell death by upregulating the expression of the proapoptotic protein Bax [58]. In our study, macamide B pretreatment significantly reduced p53 expression and exerted a regulatory effect on the apoptotic pathway. Apoptosis is a form of programmed cell death that occurs through the regulation of genes and their products in cells. Reducing apoptosis is an essential component contributing to the recovery of neurological function in mammals with brain damage. Caspase-3 is a critical protein in the apoptosis signaling pathway that induces cell apoptosis in animal models of ischemic stroke [59]. In I/R research, caspase-3 is a key protein involved in inflammation and apoptosis following I/R damage. Both ischemic and reperfusion lead to an increase in caspase-3 activity [60]. Inhibition of caspase-3 activation effectively prevents neonatal HIBD [61]. Bcl-2 and Bax are crucial proteins in the Bcl-2 gene family, and the Bcl-2 cell apoptosis inhibitor gene Bax antagonizes the protective effect of Bcl-2 and induces cell apoptosis [62]. Studies have shown that the ratio of Bcl-2/Bax activity levels is a crucial determinant of cellular susceptibility to apoptosis, not the levels of individual proteins [63]. In rats with I/R-induced damage, ginkgetin restores the mitochondrial membrane potential by increasing the activity levels of Bcl-2/Bax in rats with brain damage, thereby inhibiting apoptosis induced by the caspase-3 pathway and exerting a neuroprotective effect on rats with I/R-induced damage [25]. In MCAO rats and the SH-SY5Y cell model induced by OGD/R, chrysin reduces the levels of Bax and cleaved caspase-3 by increasing the level of Bcl-2, reducing I/R-induced damage and apoptosis of SH-SY5Y cells induced by OGD/R [64]. In our study, macamide B pretreatment reversed the increases in caspase-3, cleaved caspase-3, and Bax expression levels induced by HI stimulation and increased the expression levels of Bcl-2 and the number of TUNEL-and FJC-positive neurons. Therefore, macamide B may effectively prevent neonatal HIBD by modulating the antiapoptotic pathway.
Cell death and survival in neurodegenerative diseases are usually regulated by autophagy and apoptosis. Autophagy maintains cell survival by degrading and restoring dysfunctional organelles and misfolded proteins [63]. LC3B is currently the most widely used autophagy marker protein, reflecting the number of autophagosomes. When autophagy occurs, LC3B I is converted to LC3B II, accompanied by the formation of autophagosomes. Beclin1 is the first autophagy-promoting protein identified in mammals and regulates autophagosome-lysosome fusion. p62 (SQSTM1) is the primary substrate for degradation during autophagy and plays a vital role in the aggregation and removal of ubiquitinated proteins. The accumulation of p62 indicates that the initiation of autophagy is decreased or the fusion of autophagosomes and lysosomes is disordered, which is negatively correlated with the level of autophagy activity [65,66]. In the I/R model, remote limbic postconditioning (RIPoC) mitigates I/R-induced damage by activating autophagy [67]. Similarly, astragaloside IV may exert a neuroprotective effect on brain damage caused by ischemic stroke by promoting autophagy [68]. When HIBD occurs, autophagy and apoptosis are often inseparable. The signaling network between autophagy and apoptosis is staggered, coherent, and complex, and these processes affect each other. Mild hypothermia reduces microglial activation after traumatic brain damage by inhibiting autophagy and promoting apoptosis, indicating that apoptosis and autophagy are interrelated [69]. Activated autophagy alleviates I/R-induced damage by inhibiting the apoptosis cascade in subjects with ischemic stroke [70]. All these studies show that enhancing autophagy and inhibiting apoptosis exert a neuroprotective effect on brain damage. However, autophagy has also been reported to exacerbate I/R-induced damage, and inhibition of autophagy activation may reduce I/R-induced damage [71]. When the body is lack of nutrition, autophagy can promote cell survival, but excessive autophagy may lead to the death of autophagic cells. The controversy over the advantages and disadvantages of autophagy for cell survival may be attributed to the use of different ischemic times, animal models, animal strains, administration times, and injection times of inhibitor agonists. Xiaowei Sun et al. found that eugenol played a neuroprotective role in I/R damage induced by MCAO or OGD/R by increasing in Beclin1 levels and the LC3II/I ratio and decreasing p62 levels [72]. Consistent with the research of Xiaowei Sun et al., in this study, we proved that enhancing autophagy is beneficial to ameliorate HIBD. Under normal circumstances, when we pretreated HIBD puppies with macamide B, macamide B pretreatment significantly enhanced autophagy and inhibited apoptosis, and exerted a significant neuroprotective effect on HIBD pups. In this process, autophagy might maintain the endoplasmic reticulum function by digesting protein aggregates and misfolded proteins, limiting the apoptosis induced by the endoplasmic reticulum stress response, and exerting a neuroprotective effect on HIBD in newborn mice; When cells encounter energy crisis, autophagy may also provide energy and nutrition for cells by digesting macromolecules such as organelles and proteins, thereby prolonging cell life. However, after intraperitoneal administration of macamide B pretreatment together with intracerebroventricular administration of autophagy inhibitor 3-MA, the expression levels of autophagy-promoting related proteins Beclin1 and LC3 in HIBD pups were significantly decreased, the expression level of autophagy-inhibiting related protein p62 was significantly increased, the neuroprotective effect of macamide B pretreatment was significantly reversed, and the cerebral infarct volume of the pups was significantly increased. Brain edema damage was significantly increased, and the neurobehavioral function was also severely damaged. Autophagy inhibition significantly increased HIBD-induced apoptosis of nerve cells and aggravated HI-induced brain injury. Macamide B pretreatment significantly increased Beclin1 and LC3 expression levels and decreased p62 levels following HIBD, reduced brain damage induced by HI, and improved neurological deficits. However, a selective inhibitor of PI3K, also known as the autophagy inhibitor 3-MA, reversed these changes and exacerbated apoptosis. Therefore, macamide B may reduce HIBD-induced apoptosis by enhancing autophagy and exert a neuroprotective effect on HIBD in newborn mice.
PI3K/AKT is a vital prosurvival signaling pathway that is involved in many critical cellular processes, such as apoptosis, autophagy, and proliferation, and is considered an important regulator of autophagy and apoptosis. Studies have shown that this signaling pathway protects neurons from damage in subjects with different brain diseases [73,74]. Notably, p-AKT is detected in both adults and neonates soon after cerebral ischemic, and IP is particularly significant [75]. Activation of PI3K/AKT and its downstream pathways inhibits neuronal apoptosis [76]. When brain damage occurs, p-AKT inhibits apoptosis, and an increase in p-AKT protein levels exerts a neuroprotective effect on HIBD [77,78]. Xiaohui Tan et al. found that luteolin reduces neurotoxicity by inhibiting PI3K/AKT pathway-mediated p53 accumulation and the p53-triggered apoptotic pathway to exert neuroprotective effects on brain damage in rats [79]. Consistent with the study by Xiaohui Tan et al., in the present study, pretreatment with macamide B inhibited the PI3K/AKT pathway, as confirmed using 3-MA, resulting in the substantial accumulation of p53, thereby aggravating the apoptosis triggered by p53 and leading to increased caspase-3, cleaved caspase-3, and Bax levels and decreased Bcl-2 levels. In contrast, the activation of the PI3K/AKT pathway by macamide B pretreatment antagonized apoptosis induced by HIBD and exerted a neuroprotective effect. Recent studies have shown that autophagy exerts a neuroprotective effect, and autophagy alleviates traumatic brain injury (TBI) by inhibiting the mitochondrial apoptosis pathway or neuroinflammation in a rat brain damage model [80]. In addition, sevoflurane postconditioning promotes autophagy by activating the PI3K/AKT signaling pathway, thereby attenuating TBI-induced neuronal apoptosis [81]. In a study of spinal cord damage, melatonin enhanced autophagy by regulating the PI3K/AKT signaling pathway and reduced cell apoptosis [10]. In the present study, macamide B pretreatment activated the PI3K/AKT signaling pathway, increased the expression level of autophagy, and reduce HIBD-induced apoptosis. After the intraperitoneal injection of the PI3K inhibitor 3-MA, 3-MA significantly inhibited the activation of the PI3K/AKT signaling pathway and blocked the formation of autophagosomes, resulting in a significant decrease in LC3B and Beclin1 expression levels and a significant increase in p62 expression levels, thereby aggravating the apoptosis induced by HIBD. We confirmed that macamide B pretreatment might regulate autophagy through the PI3K/ AKT signaling pathway to reduce HIBD-induced apoptosis.
Autophagy and apoptosis are two important physiological activities of cell survival and death, which are crucial to the overall fate of cells. Bcl-2 and Bcl-xL, as the key anti-apoptotic proteins of the Bcl-2 family, play an important role in the regulation of cell apoptosis. Its important role in autophagy has also been revealed by more and more studies, which indicates that apoptotic guardians Bcl-2 and Bcl-xL can promote Beclin1 to be separated from Vps4/ class III PI3K complex by interacting with Beclin1, the key initiator of autophagy, and then inhibit autophagy [82]. Notably, when Beclin1 is isolated from the Beclin1-Bcl-2/ Bcl-xL complex, the inhibition of autophagy is significantly reversed, thereby enhancing autophagy [83,84]. Studies have found that after knockout/mutation of BH3 domain in Beclin1 or BH3 receptor domain in Bcl-2/Bcl-xL, the Beclin1-Bcl-2/Bcl-xL complex is destroyed, thus enhancing autophagy and inhibiting apoptosis; AMPK promotes the formation of Beclin1-PI3K complex by destroying Bcl-2-Be-clin1 complex, thereby enhancing myocardial autophagy and protecting diabetic cardiomyocytes from apoptosis injury [85]. All the above studies have shown that the dissociation of Beclin1-Bcl-2/Bcl-xL complex plays an important role in enhancing autophagy and inhibiting apoptosis. Macamide B pretreatment promotes the activation of PI3K/AKT and the increase of Beclin1 and Bcl-2 levels, enhances autophagy, and inhibits apoptosis. The destruction of Beclin1-Bcl-2/ Bcl-xL complex may play a key role in this process, which deserves further exploration. Although Beclin1, as the core protein of autophagy, plays a key role in autophagy, some studies have shown that autophagy can also occur in a manner independent of Beclin1. In Parkinson's disease, the neurotoxin MPP + can induce Beclin1-independent autophagy in SH-SY5Y cells [86]; Resveratrol induces Beclin1-independent autophagy in breast cancer cell MCF-7 [87]; Bcl-xL/ Bcl-2 inhibitors can promote the occurrence of autophagy by inducing Beclin1 [81]. At the same time, Beclin1 also has anti-apoptotic effect. Studies have shown that the deletion of Beclin1 can trigger caspase-dependent cell death and promote the occurrence of apoptosis [88]. In addition, pharmacological inhibitors of Beclin1 can induce apoptosis [89,90]. During the process of macamide B pretreatment activating PI3K/AKT signaling pathway, enhancing autophagy, and reducing HIBD-induced apoptosis, the dissociation of Beclin1-Bcl-2/Bcl-xL complex and the independent autophagy and anti-apoptosis ability of Beclin1 may play an important role, which is worthy of further investigation in the next research of our research group [81].
In summary, macamide B pretreatment effectively treats or prevents HIBD in newborn mice, and its effect may be achieved by regulating autophagy through the PI3K/AKT signaling pathway, thereby reducing HIBD-induced apoptosis. Macamide B may be a potential drug candidate for the effective prevention and treatment of neonatal HIBD.