IGF2 inhibits hippocampal over-activated microglia and alleviates depression-like behavior in LPS- treated male mice

A B S T R A C T Over-activated microglia and inflammatory mediators are found in patients with depression, while manipulation of the microglia function might represent a potential therapeutic strategy. Insulin-like growth factor 2 (IGF2) has been implicated in bacterial infections and autoimmune disorders


Introduction
Depression, a severe psychiatric condition that affects nearly 15% of people worldwide, significantly increases the risk of suicide (Organization, 2017). Signatures of neuroinflammation, including activated microglia and inflammatory mediators, are commonly found in patients with depression (Salter and Stevens, 2017;Setiawan et al., 2018). In particular, endotoxin stimulation (such as LPS, a well-known immuno-stimulant) can result in microglia-derived pro-inflammatory cytokines release, which is closely related to depression-like behaviors (Guan et al., 2020;Zhao et al., 2019). More importantly, antidepressants alleviate depression-like behaviors and prevent depression-induced microglia-derived pro-inflammatory cytokines production (Li et al., 2021b). It is still in debate whether microglia over-activation-induced neuroinflammation is causal or is an accompanying process associated with depression (Troubat et al., 2021), but manipulation of the microglia might influence its progression.
Under normal conditions, there is a balance of M1/M2 microglia, but the balance is disturbed during the depression (Tang, et al., 2018). Interestingly, correcting this imbalance with antidepressants such as fluoxetine could inhibit M1 activation and improve M2 activation of microglia (Li et al., 2022;Su et al., 2015). Therefore, modulation of microglia polarization represents a potential strategy for treating depression. IGF2 is a pleiotropic polypeptide that is distributed widely in various tissues/organs, including the CNS (Pardo et al., 2019), which is especially facilitating hippocampal neurogenesis. Although IGF2 is expressed in microglia during development and injured CNS (Kaur et al., 2006;Kihira et al., 2007), less information is available on the role of IGF2 in the regulation of microglia in the adult brain. Furthermore, although IGF2 has been implicated in a variety of diseases, including bacterial infections and autoimmune disorders such as multiple sclerosis (Wang et al., 2020), the role of IGF2 on the active phenotype of microglia and neuroinflammation has not been established.
Therefore, IGF2 would be potentially applied to modulate microglia reactive to neuroinflammation in the current study. And we also validated whether IGF2 treatment would benefit the hippocampal neurogenesis in the LPS-treated mice. Since over-expression of IGF2 could alleviate depressive behavior induced by chronic restraint stress (Luo et al., 2015), we further determined whether the IGF2-regulated microglia could also improve the animals' depression-like behavior. And our results showed that IGF2 significantly inhibited LPS-induced microglia over-activation, and neuroinflammatory response as well as preserved hippocampal neurogenesis, and then alleviated the depression-like behavior.

Animals
Since female mice exhibit anxiety-like behavior before estrus, we chose male mice for the experiment to exclude such a disturbance. A total of 120 male ICR mice (25-30 g) were obtained from the Hunan SJA Lab Animal Center. The animals were housed in a temperaturecontrolled room with a 12 h day/night cycle at 24 ℃ and had free access to food and water. All experiments were carried out by the Animal Care and Use Committee of the University of South China and the University of Yangzhou, which conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Behavioral evaluation
All of the behavioral tests were conducted in parallel with all of the animals. The protocol followed the sequence of sucrose preference test (SPT), open field test (OFT), tail suspension test (TST), and forced swimming test (FST) as previously described (Li et al., 2021b). This sequence was chosen to expose the animals to the same experimental conditions. All of the behavioral tests were conducted blindly concerning and drug administration and under conditions of dim light and low noise.

Open field test
Open field test is generally used to evaluate the locomotor activity and anxiety behaviors in a novel environment (Kraeuter et al., 2019). The mice were placed in the quiet experiment room for at least 1 h before the behavior test. The open field apparatus consisted of a square box with the following dimensions: 50 cm × 50 cm × 50 cm (length × width × height), and the bottom of the field was equally divided into 25 squares. Each mouse was placed directly into the center of the open field and allowed to freely explore the area for 5 min. When finished, the animal was returned to its home cage. The behavior was recorded by a video camera connected to an automated tracking system (SuperMaze + , Shanghai Xinruan Information Technology Co., Ltd.), and the total distance traveled during the testing session represented the locomotive activity. Then the apparatus was cleaned with 75% ethanol.

Sucrose preference test
The sucrose preference test (SPT) was conducted as previously described (Zhong et al., 2020). Briefly, the mice were habituated to a 1% sucrose solution for 24 h. For the test, the sucrose and pure water bottles were returned for 24 h with their positions interchanged (every 12 h), and the bottles were weighed before and after the test. Sucrose preference was defined as follows: sucrose preference percentage (%) = sucrose solution consumption (g)/(sucrose solution consumption [g] + water consumption [g]) × 100%.

Tail suspension test
The tail suspension test (TST), which was designed specifically for evaluating depression-like behavior in mice, was performed according to our previous study (Zhong et al., 2020). Briefly, mice were suspended in the middle of a three-walled rectangular compartment with a climb stopper placed around the tail before applying the tape. A 6 min session was videotaped and analyzed for immobility time in the last 4 min by an observer blinded to the experimental grouping. Data were analyzed using the SuperMaze behavioral tracking software system (Xinruan Information Technology Co., Shanghai, China).

Forced swimming test
Mice were put individually in a transparent plexiglass cylinder (15 cm diameter × 25 cm high) filled with water (23-25 ℃) to a depth of 15 cm for 6 min in the forced swimming test. The immobility time of mice, defined by floating without swimming to keep their heads above the water, was measured during the last 4 min. Data were analyzed using the SuperMaze behavioral tracking software system (Xinruan Information Technology Co., Shanghai, China).

Western blot analysis
The proteins were extracted from the hippocampal tissues of Vehi-cle+NS mice, Vehicle+LPS mice, and IGF2 +LPS mice, and centrifugation at 12,000 g for 20 min at 4 ℃. Then the supernatant was collected and quantified by the BCA Assay kit (CWBIO). A quantity of 15-30 μg total protein was then separated by 10%− 15% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) at 120 V for 1.5 h gradient polyacrylamide gel. Membranes were blocked in 5% skim milk solution for 2 h, then incubated with primary antibodies (The primary antibodies used in this study were as listed in Table 1) for 2 h at room temperature, 2 h later they were incubated overnight at 4 ℃. After being rinsed three times in 0.05% Tween-Tris buffered solution, membranes were then incubated in goat anti-rabbit, goat anti-mouse, or rabbit anti-goat conjugated to horseradish peroxidase (HRP) (1:1000, CWBIO) for 2 h followed by development with an enhanced chemiluminescence (ECL) system (CWBIO). β-Tubulin was used as a loading control. The optical density of each band was measured using NIH Image J (NIH, Bethesda, MD, USA) and normalized to β-Tubulin.

Real time-PCR
Total RNA was extracted by the Trizol® reagent (CWBIO), according to the manufacturer's instructions. RNA purity was determined by the A260 nm/A280 nm absorption ratio. Total RNA cDNA synthesis was performed with the RevertAid™ First Strand cDNA synthesis kit (Fermentas) according to the manufacturer's instructions, using 2 μg of total RNA. And the cDNA was stored at − 20 • C. Expression levels of genes were determined by ABI-7500 real-time PCR system employing the TB Green™ Premix Ex Taq™ II (Takara). The GAPDH gene expression was used as an internal control. The Primers were designed with Primer 3 software. A two-step PCR protocol was used according to the manufacturer's instructions. PCR cycling conditions were as follows: 30 s at 95 • C, followed by 40 cycles at 95 • C for 10 s and 60 • C for 30 s. Samples were processed in technical duplicates and a melting analysis was performed for each sample at the end of the PCR. 2 -△△Ct method was used to determine the relative gene expression according to our previous study (Li et al., 2021b).

Genes
Primers

Immunohistochemistry
After behavioral tests, six mice from each group were sacrificed with 10% sodium pentobarbital (100 mg/kg) for immunohistochemistry assay according to our previous study (Li et al., 2021b). Brains were removed and post-fixed in 4% paraformaldehyde (PFA) in phosphate buffer saline (PBS). Next, the brains were suspended in 15% (w/v) sucrose for 1 day and 30% (w/v) sucrose for 1 day. Mouse brain tissues were flash-frozen and 2-3 slices of each brain from -1.50 to -2.5 mm relative to the bregma in stereotaxic coordinates were sliced using a cryostat (LEICA, CM1860, Germany) at 30 µm thickness, and the sections were stored in PBS at 4 • C. Free-floating sections were deactivated via the action of endogenous peroxidase on 3% (v/v) hydrogen peroxide in 0.01 M PBS for 20 min and blocked with 5% (v/v) normal goat serum (Boster) or normal rabbit serum (Boster) in 0.1% (v/v) Triton X-100 for 2 h at room temperature (RT). Sections were next incubated with a primary antibody (rabbit anti-DCX, 1:1000 dilution, CST; rabbit anti-IGF2, 1:1000 dilution, Abcam; goat anti-IBA1, 1:1000 dilution, Abcam) in block solution overnight at 4 • C. After washing, sections were exposed to biotinylated goat anti-rabbit IgG or rabbit anti-goat IgG (Proteintech) for 2 h at RT, washed, and incubated for 2 h at RT with an avidin-avidin-biotin-peroxidase (Vector ABC Kit) prepared according to the manufacturer's instructions. After washing, the peroxidase reaction proceeded using a diaminobenzidine substrate (ZSGB-BIO) prepared according to the manufacturer's instructions. For IGF2 protein, staining localized in the cytoplasm was considered positive. Image-Pro Plus version 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA) was used to assess the area and density of the dyed region, and the integrated optical density (IOD) value of the IHC section. The mean densitometry of the digital image (scale bar = 100/25 µm) was designated as representative IGF2 staining intensity (indicating the relative IGF2 expression level). The signal density of the tissue areas from five randomly selected fields was counted in a blinded manner and subjected to statistical analysis. The total number of IBA1s (540 ×450 µm 2 )/DCX-positive cells in six sections (950 ×800 µm 2 ) from the hippocampus of each mice sample was counted under an optical microscope (Olympus, Japan) with a scale bar = of 50/100 µm by an investigator blinded to the treatment groups.

Sholl analysis
ImageJ software (NIH, USA) was used for all image processing and morphological analyses, and the Sholl analysis was performed according to our previous study. Microglia and neurons were selected and cut with the crop tool to facilitate their analysis when they fulfilled the following criteria: (i) presence of untruncated processes; (ii) consistent and strong ionized calcium-binding adapter molecule1 (IBA1), doublecortin (DCX) staining along the entire arborization field; and (iii) relative isolation from neighboring positive cells to avoid overlap. Afterward, IBA1 and DCX signal was segmented with the threshold tool and converted to binary mask before their skeletonization with the skeletonize tool. The latter tool allowed us to obtain segment length and any possible bifurcation of the skeletonized image analyzed with the Fiji-ImageJ software. Then, the maximum and total branch length of microglia processes, as well as the number of branches were measured with the Analyze Skeleton plugin of Fiji-ImageJ. Further, the plugin Sholl analysis of Fiji-ImageJ was used to place concentric circles around the cell starting from the soma and radiating outward at increasing radial increments of 5-10 µm. For the Sholl analysis, pick 24-36 cells for IBA1/DCX evaluation from six mice. To calculate average values, the averaged data obtained from each animal were used.

ELISA assay
Mouse IL-1β, IL-18, IL-6, and monocyte chemoattractant protein-1 (MCP-1) ELISA Kits (Elabscience) were used to determine the serum IL-1β, IL-18, IL-6, and MCP-1 levels, according to the manufacturer's instructions. Briefly, after being anesthetized with 10% sodium pentobarbital (100 mg/kg), mice were sacrificed, and eyeball blood was collected and centrifuged at 1500g for 20 min at room temperature. The supernatants were collected and used to measure the IL-1β, IL-18, IL-6, and MCP-1 levels of each sample. 100 μL of standard or sample was added to each well and incubated at 37 ℃ for 2 h. After washing, 100 μL of antibody was added to each well and incubated for 1 h at 37 ℃. After washing, 100 μL of HRP-conjugate was added to each well and incubated for 40 min at 37 ℃. Wells were then developed using tetramethylbenzidine reagent in the dark, and the absorbance was measured at 450 nm.
The hippocampus IGF2 was determined by ELISA assay (Boster) The dissolved proteins were collected after centrifugation at 12,000 g for 20 min at 4 ℃, and then the supernatant was collected for detection. 100 μL of standard or sample was added to each well and incubated at 37 ℃ for 2 h. After washing, 100 μL of antibody was added to each well and incubated for 1 h at 37 ℃. After washing, 100 μL of HRP-conjugate was added to each well and incubated for 40 min at 37 ℃. Wells were then developed using tetramethylbenzidine reagent in the dark, and the absorbance was measured at 450 nm. (H) Immobility time in the forced swim test (Vehicle+NS=17, Vehicle+LPS=22, IGF2 +LPS=16, and IGF2 +NS=9). P < 0.05 was considered significant and is shown as *P < 0.05, **** P < 0.0001 compared to the Vehicle+NS mice and # P < 0.05, ## P < 0.01, #### P < 0.0001 compared to the IGF2 +LPS mice using one-way ANOVAs followed by Bonferroni's post hoc test. The results are shown as the mean ± SD.

Statistical analysis
Statistical analysis was performed using Prism 7.0 software (GraphPad Software, San Diego, CA, USA). The results are presented as the mean ± standard deviation (SD). We used the Kolmogorov-Smirnov test (KS test) method to test for normal distribution, and the data were considered to satisfy normality when the test result p > 0.05. Significant differences were determined using Student's t-test or one-way ANOVA and two-way ANOVA followed by posthoc Bonferroni test, and statistical significance was established as P < 0.05. All of the data were conducted blindly to the experimental conditions.

Systemic IGF2 treatment prevents depression-like behavior in LPStreated mice
It has been shown that IGF2 alleviates depression in behavioral and cellular models of depression (Luo et al., 2015;Zhong et al., 2020), but the effect of IGF2 on depression associated with inflammation is unknown. The experiment was designed according to the steps in Fig. 1 (Fig. 1B). Bonferroni post-test analysis found that LPS treatment resulted in a significant decrease in the total distance traveled (t = 11.88, P < 0.0001), which was decreased by IGF2 treatment (t = 6.808, P < 0.0001). Two-way ANOVA showed that there was a main effect of LPS [F (1,60) = 9.141, P = 0.0037], IGF2 [F (1,60) = 55.45, P < 0.0001] or the LPS×IGF2 interaction [F (1,6) = 21.26, P < 0.0001] in the time spent in the central area (Fig. 1 C). Bonferroni post-test analysis found that LPS treatment resulted in a significant decrease in the time spent in the central area (t = 8.437, P < 0.0001), which was reversed by IGF2 treatment (t = 12.09, P < 0.0001). Two-way ANOVA showed that there was a main effect of LPS [F (1,70) (Fig. 1D). Bonferroni post-test analysis found that LPS treatment resulted in a significant decrease in the inner area distance traveled (t = 4.221, P = 0.0133), which was reversed by IGF2 treatment (t = 3.948, P = 0.0190).

Intraperitoneal injection of LPS-induced decreased hippocampal IGF2 expression
To assess whether hippocampal IGF2 was altered after LPS stimulation, the expression of IGF2 in the hippocampus was checked by several methods. RT-PCR experiment verified that IGF2 decreased in the hippocampus of LPS group mice (t = 3.13, P=0.0049) compared with NS mice (Fig. 2 A), which was further verified by ELISA assay (t = 2.377, P=0.0361) (Fig. 2 B). Then, we also examined the expression level of IGF2 in the entire hippocampus by immunochemistry. As shown in Fig. 2C, the IGF2 density in the hippocampal CA3 of the LPS mice was significantly increased. Despite a tendency of decreasing IGF2 in hippocampal CA1 and DG of the LPS mice, however, the signal is relatively weak compared with CA3. The high magnification view of IGF2 density in the hippocampal CA3 is shown in Fig. 2C. Due to the lack of specificity of IGF2 antibody for the western blot assay, we did not further validate the result by western blot. Taken together, these results suggest that intraperitoneal injection with LPS can reduce the expression level of IGF2 in the hippocampus.

Systemic IGF2 treatment prevents the over-activation of microglia in the hippocampus of LPS-treated mice
Peripheral LPS challenge resulted in activated microglia, so we evaluated the effect of IGF2 on LPS-induced microglial activation. We first examined changes in the number of IBA1 positive cells among the three groups by immunochemistry staining, shown in Fig. 3 A, B. We counted the microglia morphology in the molecular layer of the dentate gyrus (DG) region of the hippocampus. One-way ANOVA revealed that there were significant differences among the groups in the number of IBA1 positive cells [F (2,35) = 0.1688, P<0.0001]. Bonferroni post-test analysis revealed that the number of IBA1 positive cells of Vehi-cle+LPS mice was significantly increased in the hippocampal DG region compared to Vehicle+NS mice (t = 8.429, P < 0.0001), which was significantly decreased by IGF2 treatment (t = 3.864, P=0.0259). This result implies that abnormal activation of microglia in the DG region is associated with neuronal damage, consistent with previous studies (Brown and Neher, 2014;Rooney et al., 2020).
Moreover, we used sholl analysis to determine the microglia morphological changes among the three groups. There was a main effect of the LPS×IGF2 interaction [F (16,189) = 3.481, P<0.0001] in the number of intersections between branches and sholl rings. And the total number of intersections across all radii between 15 and 30 µm from the cell of microglia soma reduced remarkably in the hippocampus of Vehicle+LPS mice relative to the Vehicle+NS group, which was significantly increased by IGF2 treatment (15 µm, P=0.0023; 20 µm, P<0.0001; 25 µm, P<0.0001; 30 µm, P<0.0001), the data are shown as Fig. 3 C, D. To further confirm the effect of IGF2 treatment on the activation of microglia, we examined the alterations in the protein levels of microglial marker IBA1 by western blot. One-way ANOVA revealed that there were significant differences among the groups in the hippocampal protein of IBA1 [F (2,15) = 11.16, P<0.0001] (Fig. 3E, F). Bonferroni post-test analysis revealed that the expression of IBA1 in Vehicle+LPS mice was significantly enhanced compared to Vehicle+NS mice (IBA1:t = 6.787, P<0.0001), which was significantly decreased by IGF2 treatment (t = 2.959, P=0.0211).

Systemic IGF2 treatment prevents the polarization of M1 microglia in the hippocampus of LPS-treated mice
Since activated microglia usually existed in the form of M1 and M2 phenotypes (Ponomarev et al., 2005), we further evaluated whether IGF2 treatment might affect the polarization of M1/M2 phenotypes in the hippocampus of LPS-treated mice. As the RT-PCR results described in Fig. 5 A-D, one-way ANOVA revealed that there were significant differences among the groups in the mRNA levels of M1 phenotypes makers in the hippocampus (CD86 mRNA [F (2,14)

Fig. 2.
Effect of LPS administration on the expression of IGF2 in the hippocampus. (A) The expression of IGF2 mRNA in the hippocampus, NS= 9, and LPS= 9. (B) The densitometric analysis of IGF2 in the hippocampus, NS= 8, and LPS= 7. (C) Representative of IGF2 immunohistochemistry images in the hippocampus (Bar=100/25 µm), NS= 3, and LPS= 3. P < 0.05 was considered significant and is shown as *P < 0.05, ** P < 0.01 compared to the NS mice using the Student's t-test. The results are shown as the mean ± SD.

IGF2 decreases the amount of pro-inflammatory cytokines in the serum of LPS-treated mice
Having shown the effect of IGF2 on hippocampal inflammation, we also explored the alteration of serum pro-inflammatory cytokines by ELISA assay. One-way ANOVA suggested that there were significant differences among the groups in the amount of serum of IL-1β [F (2,21) = 2.895, P = 0.0049], 21)

IGF2 preserves the hippocampal neurogenesis in LPS-treated mice
Microglial over-activation is a key mechanism of neurogenesis inhibition under conditions of inflammation (Sierra et al., 2014). Immunochemistry revealed that there were significant differences in the number of DCX-positive cells in the hippocampus among the three groups [F (2,39) = 1.881, P<0.0001] (Fig. 8 A, B). The number of DCX-positive cells in the hippocampus of Vehicle+LPS mice (t = 11.77, P<0.0001) was significantly decreased compared to Vehicle+NS mice, which was reversed by IGF2 treatment (t = 16.05, P<0.0001). Then, we used sholl analysis to investigate the morphology of DCX-positive cells with vertically oriented apical processes in the hippocampus. Cells were systematically randomly sampled, and their dendritic trees were manually traced and subjected to sholl analysis (Fig. 8 C). The branching index of Vehicle+LPS group DCX positive cells was reduced in the suprapyramidal blade beyond ~60 µm from the cell soma [F (22, 1403) = 166.1, P<0.0001] (Fig. 8 D) and infrapyramidal blade beyond ~80 µm from the cell soma [F (18,1235) = 128.2, P<0.0001] (Fig. 8 E) compared to that of Vehicle+NS group and IGF2 +LPS group, Fig. 3. Effect of IGF2 administration on LPS-induced expression of microglial markers in the hippocampus. (A) Representative of IBA1 immunohistochemistry images in the hippocampus (Bar=50 µm). (B) A number of IBA1 positive cells in the hippocampus, Vehicle+NS= 6, Vehicle+LPS= 6, and IGF2 +LPS= 6. (C) Maximum intensity projections of images were converted to binary images and then skeletonized (Bar=50 µm). (D) Sholl analysis plot of microglia, analyses were of 30 cells from Vehicle+NS= 6 animals, 32 cells from Vehicle+LPS= 6 animals, and 32 cells from IGF2 +LPS= 6 animals. (E) Representative bands of IBA1 (n = 6 in each group), and CD68 (n = 6 in each group) in the hippocampus using β-Tubulin as the loading control. (F) The densitometric analysis of IBA1 in the hippocampus. P < 0.05 was considered significant and is shown as *P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 compared to the Vehicle+NS mice and # P < 0.05, ## P < 0.01, ### P < 0.0001, #### P < 0.0001 compared to IGF2 +LPS mice using one-way ANOVAs followed by Bonferroni's post hoc test. The results are shown as the mean ± SD.
suggesting less complex dendritic branching.

Discussion
Given the relevance of IGF2 in the modulation of macrophages and dysregulation of the IGF2 involved in immune diseases (Du et al., 2019), we hypothesized that IGF2 might modulate the function of microglia, and resident macrophages in the brain. LPS challenges resulted in reduced hippocampal IGF2 expression. Notably, exogenous IGF2 prevented the over-activation of microglia and re-balanced the microglia polarization induced by LPS challenges. Furthermore, our results confirmed that rescuing IGF2 improved hippocampal neurogenesis and alleviated the behavioral phenotypes associated with depression in LPS-treated mice.
Although the anti-depressant effect of IGF2 has been observed in behavioral and cellular models of depression (Luo et al., 2015;Zhong et al., 2020), the effect of IGF2 on inflammation-associated depression is still unknown. To verify the antidepressant effect of IGF2 in LPS-treated mice, we used SPT, OFT, TST, and FST to assess depressive-like behaviors. Consistent with previous studies (Li et al., 2021b), our data indicated that IGF2 treatment showed an enhanced percent of sucrose preference while decreasing immobility time in TST and FST. Afterward, we explored the mechanism by which IGF2 inhibits inflammation-associated depression.
IGF2 is involved in neural plasticity (Li et al., 2014), which has also been reported to be localized in rod-like activated microglia in the spinal cord of amyotrophic lateral sclerosis patients (Kihira et al., 2007). However, the causality of brain IGF2 expression and microglia-reatived to neuroinflammation is still unclear. When injected systemically, LPS leads to microglia being over-activated which also results in proinflammatory factors released in the brain (Hoogland et al., 2015;Li et al., 2021a). Although a previous study showed that LPS enhanced the IGF2 in microglial cultures (Kaur et al., 2006), the present study demonstrated that LPS reduced the expression of IGF2 in the hippocampus. The chemical properties of IGF2 allow it to cross the blood-brain barrier and exert action within the CNS (Berg et al., 2021;Duffy et al., 1988;Reinhardt and Bondy, 1994). We first systemic delivered IGF2 into the mice and found that IGF2 dramatically reduced microgliosis in their hippocampus after LPS injection, indicated by a reduced number of IBA1 positive cells and decreased protein level of microglia markers including IBA1, CD68, CD11b, and P2RX7 (Li et al., 2021b). Morphologically, IGF2 also prevented the LPS-induced amoeba-like microglia, shown by reserved enlarged cell bodies and shortened processes. In this regard, we propose that IGF2 can directly modulate the function of microglia.
Past findings indicate that IGF2 is suggested to play a role in regulating the phenotype of macrophages to acquire either a pro-or antiinflammatory (Du et al., 2019;Wang et al., 2020). Microglia are resident macrophages in the CNS, "classically activated" M1-like microglia release pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) to initiate destructive effects, while "alternatively activated" M2-like microglia produce a series of neuroprotective/neurotrophic factors, such as Arg1, CD206, IL-4, and IL-10 (Kim et al., 2021;Plociennikowska et al., 2015;Xu et al., 2015). To confirm and extend our findings, we also analyzed for expression of microglia polarization-related genes by RT-PCR. Our data showed that treatment with IGF2 decreased the expression of pro-inflammatory M1 phenotypic markers CD86, CXCL10, CD16, and iNOS while not affecting the M2 phenotypic markers CD206 and Arg1. Since IGF2 treatment mainly prevented the M1 microglia, we further determined the potential signaling pathway. TLR4, expressed on the surface of microglia, plays an important role in activating NF-κb, ultimately causing the M1 response of microglia (Li et al., 2022). Of particular significance were the findings that IGF2 significantly downregulated the expression of hippocampal TLR4, MyD88 and pNFκb/NFκb in LPS-induced mice (Supplemental data. 1). Thus IGF2 might mediate the crosstalk between M1 microglia via regulating TLR4/MyD88/NFκb expression in the hippocampus. Several studies have demonstrated that TLR4-mediated nuclear factor kappa-b (NF-κb) and mitogen-activated protein kinase (MAPK) signaling pathways, including P38, AKT, ERK and JNK, thus triggering the release of proinflammatory cytokines, including TNF-α, IL-1β, IL-6 expression (Kim et al., 2021;Plociennikowska et al., 2015). Here, we found that IGF2 significantly reduced LPS-stimulated p-ERK1/2 levels in the Fig. 4. Effects of systemic administration of IGF2 on microglial markers in LPS mice in the hippocampus. (A) Representative bands of CD68 (n = 6 in each group), CD11b (n = 6 in each group), and P2RX7 (n = 6 in each group) in the hippocampus using β-Tubulin as the loading control. (B) The densitometric analysis of CD68, CD11b, and P2RX7 in the hippocampus. P < 0.05 was considered significant and is shown as *P < 0.05, **** P < 0.0001 compared to the Vehicle+NS mice and ## P < 0.01, ### P < 0.0001, compared to IGF2 +LPS mice using one-way ANOVAs followed by Bonferroni's post hoc test. The results are shown as the mean ± SD. hippocampus, indicating that IGF2 affected TLR4-associated ERK signaling (Supplemental data. 2). Simultaneously, the levels of TNF-α, IL-1β and IL-18 were substantially decreased in the hippocampus. Since the serum levels of IL-1β, IL-6, IL-18, and MCP-1 were also lessened by the administration of IGF2, we could not exclude the peripheral effect of IGF2 on the systematic immune function, which needs further study. As our experiments examined whole hippocampal tissue, we can not exclude that pro-inflammatory cytokines may also be produced by astrocytes and neurons (Alboni et al., 2010;Gruol et al., 1997;Saha and Pahan, 2006), so we will confirm the regulation of IGF2 on microglia by cellular experiments in the future. Taking these into consideration, it could be concluded that the TLR4/MyD88/NFκb-ERK signaling pathway was an important mechanism by which IGF2 regulated M1 microglial activation.
It has been discovered that systemic or intrahippocampal administration of LPS reduces the formation of newborn neurons in the adult hippocampus (Ekdahl et al., 2003;Monje et al., 2003). The close link between neurogenesis dysfunction and neuroinflammation leads us to ask whether IGF2 could also preserve hippocampal neurogenesis in LPS-treated mice. Our findings showed that IGF2 increased the branching index in the suprapyramidal and infrapyramidal blades of LPS-treated mice. This result suggests that IGF2 effectively preserved the hippocampal neurogenesis in the LPS-treated mice. We have shown that IGF2 inhibits over-activated microglia in the DG region, and studies have found that modulating microglia can affect neurogenesis (Ekdahl et al., 2003;Monje et al., 2003). Therefore, we speculate that the mechanism of IGF2 protection against neurogenesis may be to inhibit over-activated microglia. It has been evidence that hippocampal neurogenesis dysfunction is correlated with neuroinflammation-induced depression, IGF2 could rescue the LPS-induced depressive-like behavior and increase neurogenesis by regulating microglia function.

Conclusion
In conclusion, our results demonstrate that the mechanism of IGF2 exerts anti-depressive effects through inhibited over-activated microglia and prevented its transformation to pro-inflammatory phenotype, thereby protecting hippocampal neurogenesis. Since neuroinflammation is a common feature of several human neuropsychiatric disorders and the chemical properties of IGF2 allow it to exert action within the brain after crossing the blood-brain barrier, therefore, the discoveries from the present work may provide therapeutic innovation for these diseases.

Ethics approval
The experimental protocol was approved by the Animal Care and Use Committee of the University of South China and conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Consent for participate
Not applicable.

Consent for publication
is Not applicable.

Code availability
Not applicable.

Conflict of interest statement
We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as Fig. 6. Effect of IGF2 administration on LPSinduced inflammatory cytokines in the hippocampus. (A) The expression of hippocampal TNF-α (n = 6 in each group), IL-1β (n = 6 in each group), and IL-18 (n = 6 in each group). (B) The densitometric analysis of TNF-α, IL-1β, and IL-18 in the hippocampus. P < 0.05 was considered significant and is shown as *** P < 0.001, **** P < 0.0001 compared to the Vehicle+NS mice and # P < 0.05, ### P < 0.001 compared to IGF2 +LPS mice using one-way ANOVAs followed by Bonferroni's post hoc test. The results are shown as the mean ± SD. The densitometric analysis of MCP-1 (Vehicle+NS=4, Vehicle+LPS=7, IGF2 +LPS=5) in the serum. P < 0.05 was considered significant and is shown as *P < 0.05, **** P < 0.0001 compared to the Vehicle+NS mice and ## P < 0.01, ### P < 0.001, #### P < 0.0001 compared to IGF2 +LPS mice using one-way ANOVAs followed by Bonferroni's post hoc test. The results are shown as the mean ± SD.
influencing the position presented in.

Data availability
Data will be made available on request.

Appendix A. Supporting information
Supplementary data associated with this article can be found in the online version at doi:10.1016/j.brainresbull.2023.01.001. , and 21 cells from IGF2 +LPS= 6 animals. P < 0.05 was considered significant and is shown as **** P < 0.0001 compared to the Vehicle+NS mice, #### P < 0.0001 compared to IGF2 +LPS mice using one-way ANOVAs followed by Bonferroni's post hoc test. The results are shown as the mean ± SD.