Macamide B Pretreatment Attenuates Neonatal Hypoxic-Ischemic Brain Damage of Mice Induced Apoptosis by 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 has antioxidant, anti-apoptotic, and enhances autophagy functions and can prevent cell death, and protect neurons from ischemic damage. Macamide B, an effective active ingredient of maca, has a neuroprotective role in neonatal hypoxic-ischemic brain damage (HIBD), and the underlying mechanism of its neuroprotective effect is not yet known. The purpose of this study is to explore the impact of macamide B on HIBD-induced autophagy and apoptosis and its potential mechanism for neuroprotection. The modied Rice-Vannucci method was used to induce HIBD on 7-day-old (P7) macamide B and vehicle-pretreated pups. TTC staining was used to evaluate the cerebral infarct volume of pups, brain water content was measured to evaluate the neurological function of pups, neurobehavioral testing was used to assess functional recovery after HIBD, TUNEL and FJC staining was used to detect cell autophagy and apoptosis, and western blot analysis was used to detect the expression levels of the pro-survival signaling pathway phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) and autophagy and the apoptosis-related proteins. The results show that macamide B pretreatment can signicantly decrease brain damage, improve the recovery of neural function after HIBD. At the same time, macamide B pretreatment can induce the activation of PI3K/AKT signaling pathway after HIBD, enhance autophagy, and reduce hypoxic-ischemic (HI)-induced apoptosis. In addition, 3-methyladenine (3-MA), an inhibitor of PI3K/AKT signaling pathway, signicantly inhibits the increase in autophagy levels, aggravates HI-induced apoptosis, and reverses the neuroprotective effect of macamide B on HIBD. Our data indicate that macamide B pretreatment might regulate autophagy through 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 1,000 newborns. In developing countries, up to 26 newborns out of every 1,000 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 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 have become urgent subjects in perinatal medical research.
Lepidium meyenii (Maca) is an annual or biennial herb from South America that is a member of the genus Lepidium L. of the family Cruciferae. It is commonly used to treat degenerative diseases, such as neurodegenerative diseases, cardiovascular diseases, diabetes, cancer, and ageing. Maca can remove common consequence of HIBD, and reducing cell apoptosis is essential to improve the neurological dysfunction caused by HIBD. As we all know, the PI3K/Akt pathway is also a meaningful way to regulate autophagy. PI3K/Akt pathway inhibitors can signi cantly inhibit the expression of autophagy-related proteins induced by HBCDs, suggesting that activation of PI3K/Akt pathway may promote the increase of HBCDs-induced autophagy levels [27]. It has been reported that melatonin reduces neuronal damage caused by I/R by activating the expression of autophagy-related levels [28]. In addition, the induction of mitochondrial autophagy through endoplasmic reticulum stress can prevent transient ischemic brain damage [29]. This indicates that activating the PI3K/Akt pathway to enhance autophagy may be a promising therapeutic strategy for HIBD to induce apoptosis.
PI3K/Akt signaling pathway plays a vital role in the process of cell autophagy and apoptosis [30]. A therapeutic strategy dedicated to activating the PI3K/Akt signaling pathway to enhance autophagy, thereby reducing ischemic penumbra (IP) cell apoptosis seems very promising. However, the ability of macamide B to ameliorate HIBD in neonatal mice, and whether macamide B pretreatment can regulate autophagy through the PI3K/AKT signaling pathway after HIBD, thereby reducing HIBD-induced apoptosis is unclear. Therefore, this study explored the potential of macamide B to regulate autophagy through PI3K/AKT signaling pathway to reduce HIBD in neonatal mice and inhibit cell apoptosis.

Animals
The study selected P7 C57BL/6 pups (male or female), weighing 3 to 5 g, provided by the Guangdong Medical Laboratory Animal Center (Guangzhou, Guandong). Each squirrel cage allows one female mouse to take care of its pups. Mice are reared in an environment with a temperature of 20 to 24°C and a humidity of 40% to 70%. All animal-related experiments done in this study have been approved by the Experimental Laboratory Animal Committee of Guangdong Pharmaceutical University and comply with the Chinese Council on Animal Care guidelines.

HIBD model and macamide B administration
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 [31,32]. Brie y, C57BL/6 pups at 7-9 days of age (P7-9) were given continuous inhalation anaesthesia (iso urane) through a face mask, xed 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 bath controlled hypoxia box, and a mixed gas of oxygen (8%) and nitrogen (92%) was supplied. The pups were exposed to hypoxia for 4 h then returned to the dams to feed, and the model was completed.
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. The protocol is described in Fig. 1c.

Infarct volume measurement
After HIBD for 24 h, intraperitoneal administration of 10% chloral hydrate to the pups for anaesthesia (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 is 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 anaesthesia (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%. The percentage of brain water content = (wet weight -dry weight) / wet weight × 100%.

Neurobehavioral assessments
Neurological damage caused by HIBD can lead to sensorimotor impairments. Body weight and sensorimotor performance (righting re ex, negative geotaxis, and grip test) were tested 1, 3, and 7 days after the HI procedure in a blinded manner [33].

Righting re ex
The righting re ex is used to evaluate the recovery of the brain of pups. The pups were placed on a at 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 at 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. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining.

Western blot analysis
Because cells are prone to programmed cell death during HI [34]. Therefore, we assessed the apoptotic cells in the brain tissue of the pups by TUNEL Apoptosis Detection Kit ( uorescence) (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 xation. After xation for 24 h, the brain tissue of the pup was rinsed under running water for 16h. After washing with running water, perform gradient dehydration, para n embedding, tissue sectioning and other operations on the brain tissue of pups for subsequent TUNEL detection.
The slices were immersed in xylene and for 15 min each, immersed in 100% , 100% , and 95%, 90%, 80%, 70%, and 50% ethanol solutions for 5 min per solution, and washed with ddH 2 O for 3×5 min. Put that slices into citrate buffer solution (0. 01 mol/L), and place them in a microwave oven at hightemperature 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, followed by PBS washing for 3×5 min. 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 washing with PBS for 3×5 min, counter-stain brain tissue cell nuclei with DAPI for 5 min, then repeat the PBST wash step. After absorbing excess water with lter paper, add 25 μl of anti-uorescence quencher to each brain tissue for mounting, immediately observe with a uorescence microscope (Olympus BX51, Japan) , or store it at -20°C for observation within a week.

Fluoro-Jade C Staining
After HIBD for 24 h, the pups were immediately sacri ced, and brain para n sections were made. Fluoro-Jade C (FJC) staining (Biosensis, USA) was used to detect the degeneration of neurons in the pup's brain tissue.
The slices were immersed in xylene and for 15 min each, immersed in 100% and 100% ethanol solutions for 5 min per solution, immersed in 70% ethanol solutions for 5 min per solution, and washed with ddH 2 O for 2×5 min. Transfer slides to a new Coplin jar containing ddH 2 O for 2 min. Mix 9 parts of ddH 2 O and 1 part of potassium permanganate solution and add to the brain tissue slices and incubate at room temperature for 10 min. Rinse slides for 2 min in ddH 2 O. Mix 9 parts of ddH 2 O, 1 part of FJC solution and 1 part of DAPI solution and add to the brain tissue slices and incubate for 12 min. Wash in ddH 2 O 3×1 min. Put the slices in a 56°C oven and bake for 5 min. The dry slides are then cleared by brief (5 min) immersion in xylene. Mount the slides with an anti-uorescence quencher. Immediately observe with a uorescence microscope, or store it at -20°C for observation within a week.

Tissue immuno uorescence staining
After HIBD for 24 h, the pups were immediately sacri ced, and brain para n sections were made. Tissue immuno uorescence staining was used to detect the expression levels of p53, Bax, Bcl-2, caspase-3, and cleaved caspase-3 in the brain tissue of mice.
Repeat tissue dewaxing and high-temperature repair steps, and incubate with Quick Block TM immunostaining blocking solution for 20 min. Incubate each brain tissue section with the primary antibody at 4°C for 16 h, and add 0.5% Triton X-100 to the corresponding primary antibody for cell rupture. The following primary antibodies were used:Beclin1 (1:200 Proteintech, USA). After 16 h, the membranes were removed from the refrigerator, allowed to return to room temperature for 30 min, and washed again with PBST for 3×10 min. Dylight 488 labelled goat antirabbit uorescent secondary antibody (1:360, Sigma-Aldrich, USA) or Dylight 594 labelled goat anti-rabbit uorescent secondary antibody (1:360, Sigma-Aldrich, USA), incubate for 2 h at room temperature in the dark. After washing with PBS for 3×5 min, counter-stain brain tissue cell nuclei with DAPI for 5 min, then repeat the PBST wash step. Mount the slides with an anti-uorescence quencher. Immediately observe with a uorescence microscope, or store it at -20°C for observation within a week.

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 rst 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 were compared using Student's t-test. A p < 0. 05 indicated that the difference between the two groups was statistically signi cant.

Macamide B pretreatment attenuated HIBD in newborn mice
In order to determine the optimal dose of macamide B pretreatment to treat neonatal HIBD, three doses were used for dose exploration: 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, compared with the vehicle group, the medium dose (60 mg/kg) of macamide B signi cantly reduced the percentage of infarct volume (p<0.05).
Compared with the vehicle group, there was no signi cant difference in infarct volume between the lowdose (30 mg/kg) and high-dose (90 mg/kg) groups (p>0.05). It shows that the pretreatment of macamide B at a medium dose (60 mg/kg) can signi cantly reduce the cerebral infarction volume of HIBD pups.
Brain water content was used to assess brain edema damage in pups, as shown in the gure (Fig. 2c). The water content of the ipsilateral cerebral hemisphere in the vehicle group was signi cantly higher than that in the sham group (p<0.0001). Macamide B pretreatment signi cantly reduced the water content of the ipsilateral cerebral hemisphere in the vehicle group (p<0.0001). The above results indicate that macamide B pretreatment signi cantly reduces HI-induced brain damage and has neuroprotective effects on HIBD in newborn mice.

Macamide B pretreatment improved neurobehavioral performance after neonatal HIBD
To explore whether macamide B can promote general health and recovery of neurological function, we conducted the following neurobehavioral tests at 1, 3, and 7 days after surgery: righting re ex, 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 can signi cantly reverse the weight loss of pups after HIBD compared with the vehicle group (Fig. 3a). In the righting re ex (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 indicated that macamide B could improve neurobehavioral performance following HIBD.

Macamide B pretreatment activates PI3K-AKT signaling pathway
Whether the PI3K-AKT signaling pathway is involved in the neuroprotective mechanism of macamide B after HI damage is not yet known. Western blot was used to detect the expression levels of proteins related to the PI3K-AKT signaling pathway. Western blot experiment results showed that compared with sham, the expression levels of p-PI3K and p-AKT in the vehicle group were signi cantly decreased (p <0.0001, Fig. 4b, c). Compared with the vehicle group, macamide B pretreatment signi cantly increased the expression levels of p-PI3K and p-AKT protein (p <0.0001, Fig. 4b, c). Western blot showed that macamide B pretreatment signi cantly activates the PI3K-AKT signaling pathway. Macamide B has a neuroprotective effect on HIBD in neonatal mice, which may be mediated by the PI3K-AKT signaling pathway.

Macamide B pretreatment signi cantly enhances the autophagy level of HIBD neonatal mice
To evaluate the effect of macamide B pretreatment on autophagy. Tissue immuno uorescence staining and western blot were used to detect the expression levels of autophagy-related proteins Beclin1 (Fig. 5a), LC3B (Fig. 5b), and p62 (Fig. 5c). Tissue immuno uorescence staining results showed that after HIBD, the number of Beclin1 and LC3B positive cells was signi cantly reduced, and the number of p62 positive cells was signi cantly increased. Macamide B pretreatment reversed this result. The results of western blot experiments showed (Fig. 6) that, compared with sham, the expression levels of Beclin1 and LC3B in the vehicle group were signi cantly decreased (p<0.0001), and the expression levels of p62 were signi cantly increased (p<0.001). After pretreatment with macamide B, macamide B signi cantly up-regulated the expression levels of Beclin1 and LC3B (p<0.0001; p<0.05), and down-regulated the expression levels of p62 (p<0.01). Tissue immuno uorescence staining and western blot results indicate that macamide B pretreatment promotes autophagy, and macamide B may exert neuroprotective effects on HIBD in neonatal mice by enhancing autophagy.

Macamide B pretreatment signi cantly reduces HIBDinduced apoptosis
In order to detect the effect of macamide B pretreatment on cell apoptosis of HIBD neonatal mice. TUNEL and FJC staining were used to evaluate neuronal apoptosis and degeneration. Tissue immuno uorescence staining and western blot were used to detect the expression levels of apoptosisrelated proteins p53, Bax, Bcl-2, caspase-3, and cleaved caspase-3.

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 has an inhibitory effect on class III PI3K [35]. To determine whether macamide B exerts 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 HIBD newborn mice, 3-MA signi cantly inhibited p-PI3K, p-AKT protein expression (Fig. 9c, d; p <0.0001), and blocked the activation of PI3K-AKT signaling pathway induced by macamide B pretreatment. At the same time, the results of the TTC experiment showed (Fig. 9a, b) that compared with the macamide B + vehicle 2 group, the 3-MA intervention signi cantly increased the cerebral infarction area of the pups p <0.0001 ; The brain water content measurement results showed (Fig. 2c) that compared with the macamide B + vehicle 2 group, 3-MA intervention signi cantly aggravated the brain edema damage of pups (p <0.001); In the behavioural experiment results, compared with the macamide B + vehicle 2 group, 3-MA intervention signi cantly aggravated the weight loss of HIBD neonatal mice at 1, 3, and 7 days after HIBD (Fig. 9c), 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 3-MA is an inhibitor of the PI3K-AKT signaling pathway, and is also a well-known autophagy inhibitor, which works by inhibiting the formation of autophagosomes [35]. In our study, immuno uorescence (Fig.  10) showed a signi cant increase in Beclin1 and LC3B positive cells and a signi cant decrease in p62 positive cells after 3-MA intervention compared to the macamide B + vehicle group. The results of western blot (Fig. 11) were consistent with the trend of immuno uorescence results. 3-MA intervention signi cantly inhibited the expression levels of Beclin1 and LC3B proteins (p <0.0001), and up-regulated the expression level of p62 protein (p <0.001). In apoptosis-related studies, uorescence experiments ( Fig. 12) showed that 3-MA intervention resulted in a signi cant increase in the number of TUNEL-positive cells, FJC-positive neurons, and p53, Bax, caspase-3, and cleaved caspase-3 cells, and a signi cant decrease in the expression of Bcl-2 positive cells, compared with the macamide B + vehicle group. The results of western blot experiments showed (Fig. 13) that compared with the macamide B + vehicle group, 3-MA intervention signi cantly increased the expression levels of p53, caspase-3 and cleaved caspase-3 (p <0.0001; p <0.001), and reduced Bcl2 / Bax ratio (p <0.0001). The above results indicate that macamide B pretreatment may enhance autophagy by activating the PI3K-AKT signaling pathway, thereby reducing HIBD-induced apoptosis.

Discussion
Neonatal HIBD is one of the common causes of neonatal death and disability. This condition usually causes sequelae such as cerebral palsy, visual impairment, and mental retardation [36,37]. Mild hypothermia therapy is considered to be an effective method to reduce the mortality of HIBD. However, the disability and mortality rate of HIBD is still high [38]. At the same time, due to limitations in equipment, human and nancial resources, and other objective reasons, mild hypothermia therapy is not yet entirely popularized in developing countries. Therefore, nding new safe and effective therapeutic drugs is the primary task of HIBD research. As a precious herb, maca has been proven to have neuroprotective effects in previous studies [9,10]. However, the potential therapeutic impact of maca's effective monomer, macamide B, in providing neuroprotection in HIBD has remained unclear, and the key pathways and potential mechanisms of its neuroprotective effects are not yet known.
In this study, we con rmed that Macamide B has a neuroprotective effect on HIBD in newborn mice. Speci cally, we showed that (1) macamide B pretreatment signi cantly reduces HI-induced brain damage, (2) pretreatment with macamide B can signi cantly improve neurobehavioral results after HIBD, (3) macamide B pretreatment signi cantly activates the PI3K/Akt signaling pathway, enhances autophagy, and inhibits cell apoptosis, and (4) the neuroprotective effect of macamide B may be through the PI3K/Akt signaling pathway to regulate autophagy, thereby reducing HIBD-induced apoptosis. In this study, we clari ed that macamide B pretreatment protects neonatal mouse brains from HIBD and improves general conditions and neurobehavior.
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 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 in ammation and promote the recovery of cell death and neuronal damage in focal ischemic stroke and many neurodegenerative diseases [39,40]. 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" [41][42][43]. 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 is signi cantly reduced in mice treated with maca, and antiapoptosis, antioxidation, prevention of cell death, and protection of neurons from ischemia damage are its primary mechanisms of action [9]. In vitro studies showed that the viability of cray sh neurons was signi cantly enhanced by treatment with maca extract, which also showed the neuroprotective effects of maca [9]. 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 [44]. Autophagy disorders are thought to be related to brain ageing and a variety of neurodegenerative diseases. Maca can promote the up-regulation 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 rst discovered the unique active substance macamide in maca [44]. Macamide, a type of benzylated or 3-methoxybenzylated alkanamide alkaloid, is a unique secondary metabolite in maca [44]. Puri ed macamide and synthetic analogues of macamide can exert neuroprotective effects by acting on the endocannabinoid system [45]. Studies have used a zebra sh model of dopaminergic neuron loss to evaluate the neuroprotective effect of macamide. It was found that macamide extract exerts a signi cant neuroprotective effect on zebra sh neurons [46]. Whether macamide B, a unique monomer of macamide, has a neuroprotective effect in neonatal HIBD was investigated in this study, which also included an indepth study of the potential mechanism of 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 rst pass through the blood-brain barrier (BBB) from the blood and enter the extracellular uid of the CNS to be effective. [47]. Therefore, it is necessary to con rm 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 [52,33]. The results of TTC staining showed that there was a clear cerebral infarction area on the ligation side of the brain, which veri ed that the brain tissue damage caused by surgery and hypoxia was, as proposed by Towfghi J et al., mainly con ned 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 signi cantly reduced, indicating that macamide B has a signi cant neuroprotective effect on HIBD in newborn mice. In this experiment, P7 pups were selected as the model mice, mainly because the brains of P7 mouse pups are similar in histological structure to the 32-34 week fetus or newborn [54].
Increasing evidence has shown that apoptosis is a crucial pathological trigger involved in neurological de cits 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]. Studies have found that 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 pro-apoptotic protein Bax [58]. In our study, macamide B pretreatment signi cantly reduces the expression level of p53, and has a regulatory effect on the apoptotic pathway. Apoptosis is a kind of programmed cell death that occurs through regulating genes and their products in cells. Reducing apoptosis is an essential component in the recovery of neurological function in mammals with brain damage. Caspase-3 is a critical protein in the apoptosis signaling pathway, which can induce cell apoptosis in animal models of ischemic stroke [59]. In I/R research, caspase-3 is a key protein . LC3B is currently the most widely used autophagy marker protein, re ecting the number of autophagosomes. When autophagy occurs, LC3B is converted to LC3B , accompanied by the formation of autophagosomes. Beclin1 is the rst autophagy-promoting protein found 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, the remote limbic postconditioning (RIPoC) mitigates I/R damage by activating autophagy [67]. Similarly, Astragaloside IV can play a neuroprotective effect on brain damage caused by ischemic stroke by promoting autophagy [68]. When HIBD occurs, autophagy and apoptosis are often inseparable. The signal network between autophagy and apoptosis is staggered, coherent, and complex, and they affect each other. Studies have shown that mild hypothermia reduces microglia activation after traumatic brain damage by inhibiting autophagy and promoting apoptosis, indicating that apoptosis of autophagy is interrelated [69]. It has been reported that activated autophagy can alleviate I/R damage by inhibiting the apoptosis cascade of ischemic stroke [70]. All these studies show that enhancing autophagy and inhibiting apoptosis has a neuroprotective effect on brain damage. However, it has also been reported that autophagy is harmful to I/R damage, and inhibition of autophagy activation may reduce I/R damage [71]. This controversy may be caused by different ischemia time, animal model, animal strain, administration time, and injection time of inhibitor agonist. Xiaowei Sun et al. found that Eugenol played a neuroprotective role in I/R damage by promoting the increase of Beclin1 level and LC3II/I ratio induced by MCAO or OGD/R, and the decrease of p62 level [72]. Consistent with the research of Xiaowei Sun et al., in this study, we proved that enhancing autophagy is bene cial to HIBD.
Macamide B pretreatment signi cantly promoted the increased of Beclin1 and LC3B expression level and the decrease of p62 level induced by HIBD, reduced brain damage induced by HI, and improved neurological de cit. However, a selective inhibitor of PI3K, also known as the autophagy inhibitor 3-MA, reversed this nding and exacerbated apoptosis. Therefore, macamide B may reduce the apoptosis induced by HIBD by enhancing autophagy, and play a neuroprotective role on HIBD in newborn mice.
PI3K/AKT is a vital pro-survival signaling pathway, involved in many critical cellular processes, such as apoptosis, autophagy, and proliferation, and is considered to be an important regulator of autophagy and apoptosis. Studies have shown that this signaling pathway can protect neurons from damage from different brain diseases [73,74]. p-Akt can be observed in both adults and neonates soon after cerebral ischemia, and IP is particularly signi cant [75]. Studies have shown that activation of PI3K/AKT and its downstream pathways can inhibit neuronal apoptosis [76]. When a brain damage occurs, pAkt can inhibit cell apoptosis, and the increase of pAkt protein has a neuroprotective effect on HIBD [77,78]. Xiaohui Tan et al. found that luteolin can reduce neurotoxicity by inhibiting PI3K/Akt pathway-mediated p53 accumulation and p53-triggered apoptotic pathway, and exert neuroprotective effects on rat brain damage [79]. Consistent with the study by Xiaohui Tan et al., in this study, pretreatment with macamide B inhibited the PI3K/Akt pathway through 3-MA, resulting in a large accumulation of p53, thereby aggravating the apoptosis triggered by p53, leading to caspase-3, cleaved caspase -3 and Bax expression levels increased, and Bcl-2 expression levels decreased. On the contrary, the PI3K/Akt pathway activated by macamide B pretreatment can antagonize the apoptosis induced by HIBD and play a neuroprotective effect. Recent studies have shown that autophagy has a neuroprotective effect, and autophagy can alleviate the traumatic brain injection (TBI) by inhibiting mitochondrial apoptosis pathway or neuroin ammation in the rat brain damage model [80]. In addition, Sevo urane post-conditioning promotes autophagy by activating the PI3K/AKT signaling pathway, thereby attenuating TBI-induced neuronal apoptosis [81]. In the study of spinal cord damage, Melatonin enhances autophagy by regulating the PI3K/AKT signaling pathway and reduces cell apoptosis [17]. In this study, we found that Macamide B pretreatment can activate the PI3K/AKT signaling pathway, enhance the expression level of autophagy, and reduce HIBD-induced apoptosis, After intraperitoneal administration of PI3K inhibitor 3-MA, 3-MA signi cantly inhibited the activation of PI3K/AKT signaling pathway and blocked the formation of autophagosomes, resulting in a signi cant decrease in LC3B and Beclin1 expression levels, and a signi cant increase in p62 expression levels, Thereby aggravating the apoptosis induced by HIBD (Fig. 14). We con rmed that macamide B pretreatment might regulate autophagy through PI3K/AKT signaling pathway to reduce apoptosis induced by HIBD.
In summary, macamide B pretreatment can effectively treat or prevent 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 effective prevention and treatment of neonatal HIBD. Consent to participate Not applicable to this study Consent for Publication All authors approve the manuscript for publication Data and materials availability All data and materials are available on request from authors.  Effect of macamide B pretreatment on HIBD in newborn mice. a Representative pictures of coronal brain sections stained with TTC 24 h after HIBD. b Quantitative analysis results of cerebral infarct volume in pups. Compared with pretreatment with the vehicle, pretreatment with the medium dose (60mg/kg) of macamide B signi cantly reduced the infarct volume. c Quantitative analysis results of brain water content. The water content of the ipsilateral cerebral hemisphere of the vehicle group was signi cantly higher than that of the sham group, and the brain water content of the macamide B group was signi cantly lower than that of the vehicle group. *p < 0.05 vs. sham group; **p < 0.001 vs. sham group; ***p = 0.0003 vs. sham group; ****p < 0.0001 vs. sham group; #p < 0.05 vs. vehicle group; ##p = 0.001 vs. vehicle group; ####p < 0.0001 vs. vehicle group; @p < 0.01 vs. macamide B group. n = 6 for each group.       The effect of 3-MA on PI3K-AKT signaling pathway and HIBD neonatal mice pretreated with macamide B. a Representative pictures of coronal brain sections stained with TTC 24 h after HIBD. b Quantitative analysis results of cerebral infarct volume in pups. Vehicle 1 is the vehicle of macamide B, vehicle 2 is the vehicle of 3-MA. Compared with the vehicle 2 group, the cerebral infarct area of the pups treated with 3-MA increased signi cantly. n = 6 for each group. c Representative western blots and quanti cation data of d p-PI3K and e p-Akt protein in the ipsilateral cerebral hemisphere were detected 24 h after HIBD. Macamide B pretreatment signi cantly inhibited the expression of p-PI3K and p-Akt proteins in the HI brain. n = 6 for each group. On postoperative days 1, 3, and 7, 3-MA reversed macamide B-pretreated HIBD pups' increased f body weights and exacerbated neurobehavioral damage in the g righting re ex, h negative geotaxis, and i grip tests compared to the macamide B + vehicle 2 groups. ****p < 0.0001 vs.