Metformin induced M2 polarization to improve peripheral nerve regeneration by regulating AMPK/PGC-1α/PPAR-γ pathway

doi:10.21203/rs.3.rs-2700955/v1

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Metformin induced M2 polarization to improve peripheral nerve regeneration by regulating AMPK/PGC-1α/PPAR-γ pathway

https://doi.org/10.21203/rs.3.rs-2700955/v1

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Peripheral nerve regeneration is a complex process that involves many signaling pathways, and M2 macrophage polarization was recognized to play a pivotal role in this process. The neuroprotective effects of metformin have attracted wide attention, but few reports focusedon the potentialeffects of metforminin immunomodulatory properties to improve the peripheral nerve regeneration by affecting M2 macrophage polarization. In this study, a rat model of sciatic nerve injury and an inflammatory model of bone marrow-derived macrophage (BMDM) cells were established to examine the potential mechanism of metformin treatment in peripheral nerve repair. Our research demonstrated that metformin treatment was able to accelerate functional recovery, axon regeneration and remyelination, and promote M2 macrophage polarization. In vivo, metformin could transform pro-inflammation macrophages into pro-regeneration M2 macrophages. It was also found that the levels of relative proteins of p-AMPK, PGC-1α, and PPAR-γ were significantly increased after metformin treatment. Moreover, the blockage of AMPK abolished the effects of metformin treatment on M2 polarization. Our data indicated that metformin promoted the macrophage polarization towards M2 phenotype by activating the AMPK/PGC-1α/PPAR-γ signaling axis so as to promote peripheral nerve regeneration. These findings may contribute to a more comprehensive understanding on the molecular mechanism of metformin treatment and its potential in peripheral nerve regeneration.

Metformin

Peripheral nerve regeneration

macrophage polarization

AMPK/PGC-1α/PPAR-γ signaling axis

Peripheral nerve injury (PNI) remains a significant clinical challenge and public health burden worldwide. Despite the peripheral nervous system (PNS) possessing the potential for self-healing, the capacity for regeneration was still limited (Lopes et al., 2022). Moreover, the existing treatments have remained suboptimal, often resulting in poor functional recovery. Recently, accumulating evidences confirmed that macrophages were involved in the peripheral nerve regeneration process as a key component. Macrophages could be classified into the “classically activated” M1 phenotype (possessing the effect of pro-inflammatory) and the “alternatively activated” M2 phenotype (possessing the effect of anti-inflammatory). An increasing number of studies supported that M2 macrophages could secrete a variety of pro-axonal regeneration factors (growth factors, cytokines, chemokines, ECM) to inhibit inflammation, regulate fibroblast regeneration, and promote angiogenesis to synergistically regulate peripheral nerve regeneration. Besides, non-functional or dysfunctional M2 macrophages would inhibit axon regeneration and injure normal axons in the process of nerve regeneration (Kotwal and Chien, 2017). Therefore, orderly and timely M2 polarization was critical for the success of peripheral nerve regeneration.

Metformin, a widely used hypoglycemic agent, is currently one of the commonest medications for type 2 diabetes. Recently, metformin received increasing attention because of its anti-inflammatory properties.(Schuiveling et al., 2018; Ursini et al., 2018) Emerging evidences support the novel hypothesis that metformin has immune-modulatory features.(Jing et al., 2018; Schuiveling et al., 2018) Our previous research also showed that metformin could accelerate M2 macrophage polarization to promote wound healing(Qing et al., 2019). Nonetheless, the immunomodulatory property of metformin to affect M2 macrophage polarization during peripheral nerve regeneration has not been unrevealed.

Earlier studies demonstrated that the pleiotropic effects of metformin were linked with the activation of AMP-activated protein kinase (AMPK)(Zhao et al., 2017). Besides, numerous studies have affirmed the protective effects of AMPK activation on the nervous system. Peroxisome proliferator-activated receptor-γ co-activator 1α (PGC-1α) is a common downstream protein of AMPK that regulates mitochondrial biogenesis and reactive oxygen species synthesis (Yun et al., 2018; Suntar et al., 2020). Meanwhile, PGC-1α is an important ligand for peroxisome proliferator-activated receptors (PPARs), a class of ligand-activated transcription factors in the nuclear receptor superfamily. Of various PPARs, PPAR-γ (the major isoform in the PPAR family) is prevalent in neural tissues, including neurons, microglia, astrocytes and many other cell types (Corona and Duchen, 2015). The interaction between PGC-1α and PPAR-γ was increasingly valued in neuroprotection and had been demonstrated to have a positive effect on diseases including multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's disease, and Parkinson's disease(Prashantha Kumar et al., 2020). Our previous research also found that metformin was characterized by immunomodulatory properties and exerted anti-inflammation effects by activating the AMPK protein(Qing et al., 2019). Recently, emerging studies confirmed that the AMPK/PGC-1α signaling pathway was involved in anti-inflammatory and macrophage polarization (Wang et al., 2018; Luo et al., 2022). However, the role of AMPK/PGC-1α/PPAR-γ signaling in the immunomodulatory function of metformin needs further clarification. This study aimed to examine the effects of metformin on peripheral nerve regeneration and to elaborate the related mechanism.

Establishment of rat model of PNI and drug treatment

Our experimental protocol was approved by the Animal Care and Use Committee of the Central South University. The sciatic crushing model was established by referring to the procedure in our previous study. In brief, Sprague-Dawley (SD) rats were chosen and separated into two groups: i) the control group; ii) the 50mg/kg metformin group (Wu et al., 2022). The rats were anesthetized with 3% pentobarbital sodium (40 mg/kg) via intraperitoneal (i.p.) injection. Then, a 1.5cm deep incision was made by the sterile hemostatic forceps and tissue scissors at the posterior-thigh muscle to expose sciatic nerve. The exposed nerves were clamped using hemostatic forceps for 10 seconds and then relaxed for 10 seconds, and the procedure was repeated up to three times. Impaired nerves were marked by surgical 10 − 0 nylon monofilament and were harvested at 4 weeks post-operatively to evaluate nerve regeneration. After surgery, metformin (50mg/kg) or equal doses of PBS were administrated to the rats via i.p. injection until the endpoint of observation. The rats were housed with free access to water and food in the Animal Care Center of Xiangya Hospital, Central South of University (Changsha, Hunan, China). All surgical procedures and other manipulations were administrated in compliance with the guidelines of the China Council of Animal Care after obtaining approval from the Central South University Committee on Laboratory Animals.

Assessment of sensory and motor functional recovery

The assessment of sensory and motor functional recovery following PNI has been described in our previous studies (Li et al., 2022a). In brief, after inducing nerve crushing injury, the sensory recovery was assessed by thermo-sensitivity analysis and Von-Frey test. First, the rats were moved onto a metal platform with the temperature controlled between 50°C to 55°C. The time taken to observe a nocifensive behavior, i.e., response latency, was recorded properly, where jumping, stamping, leaning posture and hind paw withdrawal or licking were considered as nocifensive behaviors. Second, Von-Frey test (Scholz et al., 2005; Minett et al., 2011) was conducted with the rats placed on a mesh. Specifically, monofilaments at different forces were applied perpendicularly to the plantar surface of the hind paw for 2–5 s. For observations, a positive response was defined as hind paw withdrawal, licking, or paw shaking. By setting the mechanical withdrawal threshold as the force of the last von Frey monofilament, the force of monofilaments was increased continuously until reaching a response rate of 40% or more.

For assessment of the motor functional recovery of sciatic nerve, the sciatic function index (SFI) and the Basso, Beattie, and Bresnahan (BBB) Locomotor Rating were evaluated at 0, 1, 3, 7, 14, and 28 days post-operatively (Dinh et al., 2009). During the SFI test, the rats, after dipping their hind feet in ink, were allowed to walk through a tunnel which was covered by paper to record footprints. Then, the toe spread [TS] (the distance between the first and fifth toe), intermediary toe spread [ITS] (the distance between the second and fourth toe), and print length [PL] (the distance between the third toe and heel) were evaluated on both the injure (E) side (ETS, EITS and EPL, respectively) and normal (N) side (NTS, NITS and NPL, respectively). Subsequently, the SFI was computed by the following formula: SFI=-38.3 × (EPL − NPL)/NPL + 109.5 × (ETS − NTS)/NTS + 13.3 × (EITS − NITS)/NITS − 8.8 (Bain et al., 1989). An independent observer was engaged in evaluating the BBB Locomotor Rating with the rats exposed to an arena in the open field. Moreover, the function of sciatic nerve was evaluated through ankle kinematics(Li et al., 2022a). For sagittal plane analysis, the ankle angle, represented as the degree at terminal stance, was measured as the intersection of the line extending from the lateral epicondyle to lateral malleolus and from the lateral malleolus to metatarsal head. Further, the muscle weight ratio was evaluated based on the wet weight of bilateral gastrocnemius muscles harvested (muscle weight ratio = the wet weight of the gastrocnemius muscle on the injured side / the wet weight of the gastrocnemius muscle on the contralateral normal side), which was detailed previously (Du et al., 2018b).

Electrophysiology recording

Our previous research has described the protocol for the Electrophysiology recording for peripheral nerve regeneration(Li et al., 2022a). In brief, electrophysiology recordings were conducted at 4 weeks post-operatively using a Tucker-Davis Technologies system (TDT, Alachua, FL) with a preamplifier. The stimulating electrodes were fixed 5mm away from the crushed proximal sciatic nerve of the rats, while the recording electrodes were fixed near the sciatic nerve, and 5 mm away from the crushed distal sciatic nerve. Then, after stimulating the sciatic nerve electrically (0.5mA, stimulation frequency 0.5Hz, and pulse duration 100 µs) through the proximal interfascicular electrode, the recording of compound motor action potential (CMAP) at the distal interfascicular electrode were measured. Peak-to-peak amplitude was analyzed using OriginPro 8 software. Data are then blindly assessed by trained technicians.

Retrograde tracing

The retrograde tracing test was performed following the procedure detailed in our previous study(Li et al., 2022b). In brief, the rats were injected with 10 µl of Dil (1,19-dioctadecyl-3,3,39,39-tetramethylindocarbocyanine perchlorate) into the tibial nerve at 4 weeks post-injury. After allowing retrograde transport for 48 h, the rats were sacrificed by anesthetization and were then perfused with saline plus 4 wt% paraformaldehyde. Further, the L4 and L5 dorsal root ganglia (DRG) were harvested, and were made into serial 20 µm cross-sections by cryosectioning. Lastly, the total number of Dil-labeled cells was counted in every alternate section under a fluorescence microscope.

Immunofluorescence staining

Nerve tissues were excised 4 weeks after injury, then fixed with 4% paraformaldehyde, and further perforated with 0.3% Triton X-100. Subsequently, 5% BSA was administrated for blocking. Thereafter, the tissues were incubated with primary antibodies at 4℃ for 8-12h. The targets of primary antibodies are as follows: Anti-NF200 (neurofilament for axons marker, 1:1000, Abcam), Anti-MBP (myelin marker, 1:1000, Abcam), Anti-CD68 (the macrophage marker, 1:1000, Abcam), Anti-CD206 (the M2 macrophage marker, 1:1000; Abcam). Next, the tissues were treated by the secondary antibody at room temperature for 90 minutes. After TBST washing for three rounds (5min each round), the nucleus of the tissue was stained with DAPI (Invitrogen, Carlsbad, CA) for 15 minutes. A fluorescent Nikon N2-DMi8 inverted microscope (Nikon, Kobe, Japan) was then used to observe the staining slides. For quantitative analysis, 6 different fields were taken randomly from each sample preparation, and the positive areas of NF200, MBP, CD68, CD206 and DAPI were examined using Image J software (Media Cyber- netics).

Extraction and stimulation of bone-marrow-derived macrophages (BMDMs)

As for the isolation of BMDMs, C57BL/6j mice (weighting 22-25g) were selected, and the cell suspension was collected from the femurs and the tibias by the sterile technique as previously described (Ying et al., 2013). The BMDMs were cultured in Iscove's modified Dulbecco's medium (IMEM; Gibco, USA) with 1% penicillin/streptomycin (NCM Biotech, Suzhou), 10% fetal bovine serum (FBS; New Zealand, USA), and 20ng/ml M-CSF (Proteinates, USA). Macrophages were collected on day 7 and seeded in a 6-well plate. On day 8, the cells were treated with 100ng/ml Lipopolysaccharide (LPS, Sigma, USA) for 6h(Qing et al., 2019). Then, BMDMs were divided into three groups: (1) Control group (containing equal amounts of PBS as metformin or compound C in medium); (2) Metformin group (treated with 2mM metformin); (3) Compound C group (treated with 2mM metformin and 10uM compound C) (Qing et al., 2019). After 24h treatment, the cells were collected for subsequent testing purposes.

Cell viability assay

Cell viability assay was conducted according to the instructions on the CCK-8 kit (NCM Biotech, #C6030). Specifically, BMDMs were seeded using 96-well plates at the density of 5×10³/well. Once the cells appeared to be adherent, metformin at different concentrations (0.5,1,2,4,8 and 16mmol/L) was administrated to the cells, which were then incubated for 24h or 48 h. Next, a volume of 10ul CCK-8 solution was dropped into each well and the cells were further incubated at 37℃ for an appropriate time. Finally, the absorbance of each well was measured with a microplate reader (Thermo Fisher Scientific) at 450nm. The cell viability could be calculated by the following equation:

$$Cell vitability=\left(\frac{Absorbance value of metformin group}{Absorbance value of control group}\right)*100\%$$

Flow cytometry

BMDMs were trypsinized and resuspended in the pre-chilled PBS solution. Then, the anti-mouse F4/80-BV421 (BD Biosciences, USA) was utilized to identify the total macrophages. The M1 macrophages were identified by anti-mouse CD86-APC (BD Biosciences, USA) and the M2 macrophages were identified by anti-mouse CD206-AF488 (BioLegend, USA). Next, 1–10*10⁵ cells were collected and incubated with the above antibodies on ice for 30 min. Finally, the macrophages were detected on the flow cytometry (Verse, BD Bioscience) and the subpopulations of macrophages were analyzed using Flow Jo (Trestar software, Ashland, OR, USA).

Real-time quantitative PCR

The total RNA from BMDMs was extracted by Trizol Reagent (Invitrogen, USA). Synthesis of cDNA was performed following guidelines in the manufacturer’s protocols (Vazyme, Nanjing, China). The amplification of real-time quantitative polymerase chain reaction (real-time qPCR) was operated with an SYBR-Green RT-PCR kit (Vazyme, Nanjing, China). Taking GAPDH as the reference, the expression of associated RNA was quantified by the (2^−ΔΔCT) technique. The primer sequences were obtained from NCBI and synthesized by Tsing Ke Biotech as follows: Inducible Nitric Oxide Synthase (iNOS), 5’-GTTCTCAGCCCAACAATACAAGA-3’ (forward) and 5’-GTGGACGGGTCGATGTCAC-3’ (reverse); Tumor necrosis factor(TNF-α), 5’-CCCTCACACTCAGATCATCTTCT-3’ (forward) and 5’-GCTACGACGTGGGCTACAG-3’ (reverse); Interleukin-1β (IL-1β), 5’-GCAACTGTTCCTGAACTCAACT-3’ (forward) and 5’-ATCTTTTGGGGTCCGTCAACT-3’ (reverse); Arginase-1 (Arg-1), 5’-CTCCAAGCCAAAGTCCTTAGAG-3’ (forward) and 5’-AGGAGCTGTCATTAGGGACATC-3’ (reverse); Interleukin-10 (IL-10), 5’-CTTACTGACTGGCATGAGGATCA-3’ (forward) and 5’-GCAGCTCTAGGAGCATGTGG-3’ (reverse); Chitinase 3-like 3 (Ym-1), 5’- CAGGTCTGGCAATTCTTCTGAA-3’ (forward) and 5’- GTCTTGCTCATGTGTGTAAGTGA-3’ (reverse).

Western Blot Analysis

Separation of protein extracts from BMDMs (30ug) was performed by SDS-PAGE. The separated extracts were placed onto PVDF membranes, which were then blocked by non-fat milk (dissolved in TBS with 0.1% Tween 20) at room temperature for 2 h. Subsequently, the membranes were exposed to the primary antibodies against AMPK-α-1 (1:2500, Abcam), Phospho-AMPK-α-1 (Thr183) + Phospho-AMPK-α-2 (T172) (1:5000, Abcam), PGC-1α (1:1000, Abcam), PPAR-γ (1:1000, Abcam), and GAPDH (1:1000, Abclonal) at 4°C overnight. Further, after washing with TBST for three rounds (5 minutes each round), the bands were incubated with HRP-conjugated secondary antibodies (Abclonal, USA) for 2 h. The signal of the bands was measured by the ChemiDoc XRS + Imaging system (Bio-Rad). Imaging Lab (Bio–Rad, United States) was utilized to quantify the expression of target proteins.

Statistical analysis

All statistical analyses were conducted using GraphPad Prism 8.0 Software (La Jolla, CA, USA), and all data were presented as mean ± SD. The student’s t-test was utilized for the comparison between two sets of data. The one-way analysis of variance (ANOVA) and Dunnett’s post hoc test were utilized for the comparison among multiple groups. P < 0.05 represented statistical significance.

Metformin improved functional recovery after peripheral nerve injury

To demonstrate the functional recovery of sciatic nerve after metformin treatment, we investigated the recovery of motor and sensory functions of rat models after inducing a sciatic nerve injury as described in our previous study(Li et al., 2022a). Specifically, the SFI, BBB score, ankle angle and wet weight mass ratio of the gastrocnemius muscle were examined to assess the recovery of the motor function. At week 4 post-operatively, the SFI was found to be significantly improved in the metformin treatment group versus the control group (P < 0.01) (Figure.1A). The BBB score also supported an improved motor performance achieved by metformin treatment (Figure.1B). Moreover, the ankle angle and the wet weight mass ratio of the gastrocnemius muscle were both increased in the metformin treatment group with statistical significance (P < 0.01) (Figures.1C&D). Above results suggested that metformin treatment was effectively in improving the sensory function recovery of the sciatic nerve after injury in terms of response to mechanical force stimulation and heat (Figure.1E&F). This was indicated by a lower threshold under the condition of mechanical stimulation and a shorter latency period under the condition of heat stimulation (P < 0.01) at post-operatively week 4.

Previous studies demonstrated that improved nerve regeneration was accompanied by an electrophysiological improvement (i.e., increased CMAP).(Du et al., 2018a; Du et al., 2018b) Therefore, to examine the effect of metformin on the PNI, the electrophysiology (CMAP) at 2, 4 weeks was detected. The metformin treatment group presented a significant elevation in the peak amplitude of CMAP versus the control group (Figure.1G, H; P < 0.01) at 4 weeks, but no significant difference was observed at 2 weeks. The data suggested that the metformin treatment group achieved significant electrophysiological recovery after the PNI. Taken together, metformin treatment is able to improve the functional recovery of hind limb following a peripheral nerve injury.

Metformin promoted axonal regeneration and SC remyelination after PNI

We performed immunofluorescence staining to detect NF200 (axon marker) and MBP (remyelination maker) in order to further find out whether metformin treatment could promote axon regeneration and nerve remyelination at 4 weeks post-operatively. From Figures.2A-C, it could be seen that the metformin treatment group had higher levels of MBP and NF200 as compared to the control group (P < 0.05). Then, we further performed retrograde labeling with Dil to examine the peripheral nerve regeneration in both groups, and the results indicated a significantly increased number of Dil positive cells in the treatment group versus control (P < 0.01), as shown in Figure.1I, J. Altogether, these findings support that metformin treatment is able to promote axon regeneration and remyelination of PNI in crushing rat models.

Metformin promoted regeneration of peripheral nerve by inducing M2-type macrophage polarization

According to our previous research, the regeneration of peripheral nerve was associated with M2 macrophage polarization(Li et al., 2022a). Metformin had been demonstrated to exert anti-inflammation effects and possess immunomodulatory properties. Therefore, we hypothesized that metformin could potentially accelerate M2 macrophage polarization. To elaborate whether metformin could facilitate M2 macrophage polarization after PNI, we performed immunofluorescence staining on the impaired nerve at 7 days post-operatively, where the macrophage subpopulation in the nerve injury site was identified based on CD68 (marker for macrophages) and CD206 (marker for M2 macrophages) (Figure.2D). The staining results suggested that the number of CD68⁺CD206⁺ cells was increased in the metformin treatment group versus the control at 1 week post-operatively (Figure.2E; P < 0.01). The combined evidence illustrated that metformin promoted the peripheral nerve regeneration by inducing M2 macrophage polarization.

Metformin converted inflammatory macrophage cells to M2 polarization in vitro

To further demonstrate the effect of metformin on macrophage polarization, we explored the question as to whether metformin was able to accelerate M2 macrophage polarization in vitro. We first extracted and purified the BMDMs of mice according to previous studies (Camell et al., 2017) and treated them with LPS (100ng/ml) for 6h to establish an inflammatory model of macrophages in vitro. With the purpose to identify the optimal concentration of metformin for treating BMDMs, we applied a CCK-8 assay to access cell viability. BMDMs were seeded in 96-well plates and treated with metformin at different concentration gradients (0.5, 1, 2, 4, 8 and 16mmol/L) for 24 and 48h. As shown in Figure.3A&B, treating BMDMs cells with 2 mmol/L of metformin yielded the optimal cell viability (P < 0.01). Therefore, subsequent experiments would follow the 2 mmol/L of metformin to treat macrophages. Then, flow cytometry was performed to display the subpopulation of macrophages. CD86⁺ F4/80⁺ was defined as M1 macrophages and CD206⁺ F4/80⁺was defined as M2 macrophage. The flow cytometry data indicated that the distribution and density of M1 macrophages were markedly decreased (P < 0.01) (Figure.3C,D), while the distribution and density of M2 macrophages were markedly increased (P < 0.01) (Figure.3E,F) in the metformin treatment group versus the control.

We also measured the makers of M1 macrophages (iNOS; TNF-α; IL-1β) and M2 macrophages (Arg-1; IL10; Ym-1) by using RT-qPCR. The data indicated that the relative mRNA expressions of M1 macrophage makers were significantly decreased (iNOS: P < 0.05; TNF-α: P < 0.01; IL-1β: P < 0.01) (Fig. 4A-C). In contrast, the relative mRNA expressions of M2 macrophage makers were significantly increased (Arg-1:P < 0.01; IL10: P < 0.01; Ym-1: P < 0.01) (Fig. 4.D-F). Altogether, these data demonstrated that metformin could alleviate the LPS-induced inflammatory response and skew the inflammatory macrophage cells to M2 macrophages.

Metformin skewed inflammatory macrophage cells to M2 polarization by modulating AMPK/PGC-1α/PPAR-γ signaling pathway

We next determined whether metformin could accelerate M2 macrophage polarization via the AMPK/PGC-1α/PPAR-γ signaling pathway so as to elaborate the molecular mechanism of metformin-induced macrophage polarization. Western blot was employed to measure the expression levels of p-AMPK, AMPK, PGC-α and PPAR-γ (Figure.5). As shown by the results, metformin significantly elevated the p-AMPK/AMPK ratio (P < 0.05) (Figure.5B), suggesting that following the metformin treatment, the expression levels of PGC-α and PPAR-γ were significantly increased versus the control (P < 0.01) (Figure.5C, D). By contrast, when co-treated with metformin and compound C (a selected inhibitor of AMPK) in BMDMs, the p-AMPK/AMPK ratio and the expression levels of PGC-α and PPAR-γ were significantly downregulated as compared to the metformin treatment group (P < 0.01) (Figure.5B-D), implying that metformin activated the PGC-1α/PPAR-γ signaling by activating AMPK. Moreover, it was revealed that inhibiting the AMPK activity by compound C was able to reverse the regulatory function of metformin. As shown by the results, the distribution and density of M1 macrophages were increased (Figure.6A, B; P < 0.01), while the distribution and density of M2 macrophages were decreased (Figure.6C, D; P < 0.01) in the compound C group versus the metformin group. Altogether, it was evidenced that metformin regulated macrophage polarization and facilitated macrophage differentiation to M2 by regulating the AMPK/PGC-1α/PPAR-γ signaling pathway.

Metformin was a synthetic derivative of guanidine, extracted from the extracts of Galega officinalis, and represented a worldwide milestone in the treatment of type 2 diabetes (Serrao et al., 2017; Xia et al., 2018). Recently, emerging evidences demonstrated that metformin inhibited the expression of pro-inflammatory cytokines, protected against oxidative damage and directed macrophage polarization both in vitro and in vivo (Zhao et al., 2017; El-Bahy et al., 2018; Schuiveling et al., 2018). Interestingly, increasing evidences indicated that metformin could inhibit inflammation and promote tissue repair by promoting macrophage differentiation to M2 macrophage (Jung et al., 2021; Xian et al., 2021), and M2 macrophage played a crucial role in peripheral nerve regeneration after injury (Peluffo et al., 2015; Salehi et al., 2019). However, the correlations among metformin, M2 macrophage polarization, and peripheral nerve regeneration remained unknown. Therefore, the objective of this study was to find out whether metformin could switch macrophage into M2 subtype to boost the peripheral nerve regeneration. Throughout our research, we systematically evaluated the specific effects of metformin treatment in the process of peripheral nerve regeneration. The results demonstrated that administration of metformin could induce superior regenerative effects in the peripheral nerve injury model, which was proven by improved recovery of motor and sensory functions, as well as increased axon regeneration and myelination. In addition, we observed that metformin induced M2 macrophage polarization in vivo. As for in vitro, metformin promoted the M2 macrophage polarization involved in the AMPK/PGC-1α/PPAR-γ signaling pathway.

Earlier studies have extensively addressed the neuroprotective effects of metformin and revealed its positive therapeutic effects in animal models of spinal cord injury (SCI) (Wu et al., 2022), peripheral nerve injury (Liu et al., 2019), and cerebral ischemia (Jiang et al., 2014). Mary et al. found that metformin regulated autophagy by reducing reactive oxygen species (ROS) production, and mitigated peripheral nerve damage in non-obese diabetic mice (MKR) (Haddad et al., 2022). Wang et al. found that metformin stabilized microtubule structure, regulated oxidative stress and mitochondria function by activating the Akt-mediated Nrf2/ARE pathway, and finally promoted axonal regeneration after spinal cord injury (Wang et al., 2020). However, few studies had focused on its immunomodulatory property about the macrophage reprogramming during the peripheral nerve regeneration. Therefore, our findings were novel in that we demonstrated that metformin enhanced the function recovery in PNIs animal models by regulating M2 macrophage polarization.

It is well known that periphery nerve regeneration requires the participation of macrophages, inducing M2 macrophage polarization to accelerate the regeneration of axons and myelin sheaths (Zigmond and Echevarria, 2019). Emerging research had illustrated that M2 macrophage could release the VEGF to induce the vascular regeneration at the location of peripheral nerve damage site, which was critical for nerve regeneration (Cattin et al., 2015). Some studies also highlighted that M2 macrophage could promote the Schwann cell maturity (Stratton et al., 2018). Above findings provided strong evidences for the essential role of macrophages in the process of peripheral nerve regeneration and suggested that targeting macrophages might be a promising strategy to improve the outcome of functional recovery after peripheral nerve injury.

Numbers of studies had also demonstrated that maintaining a certain number of anti-inflammatory M2 macrophages was crucial for nerve repair, which was beneficial for the maturation and migration of Schwann cells and regeneration of axons (Peluffo et al., 2015; Dong et al., 2021). In this study, metformin was shown to facilitate the up-regulation of the number and proportion of M2 macrophages. In in vitro experiments, pre-treatment with LPS could stimulate the polarization of primary macrophage toward M1 phenotype, mimicking the activation of inflammation in the early stages of nerve injury, whereas metformin treatment reversed the pro-inflammatory effect of LPS and up-regulated the expression of the marker for M2 macrophages (CD206), but this effect was reversed with the addition of compound C (an AMPK inhibitor). Above experimental results indicated that metformin promoted the regeneration of axons and myelin sheaths through the polarization of macrophage to M2 type, connected with the activation of AMPK.

Macrophage polarization has been recognized as a component in the regulation of energy metabolism (Kumari et al., 2018; Li et al., 2020b). AMPK, the common target for Metformin, was essential for the cellular metabolism and growth. In previous studies, activated AMPK had been involved in tissue repair of a multitude of pathological processes, such as ulcerative colitis(Nasr et al., 2022), rheumatoid arthritis(Cai et al., 2022), and diabetic ulcers(Huang et al., 2021; Wang et al., 2021b). Interestingly, the AMPK/PGC-1α/PPAR-γ signaling axis also had been illustrated in the literature to play a key role in maintaining mitochondrial homeostasis and energy metabolism. Recent studies reported that the regulations of energy metabolism and innate immunity were linked through an antagonistic crosstalk between NF-κB and the AMPK/PGC-1α/PPAR-γ signaling pathway(Li et al., 2020a; Zhang et al., 2020; Yang et al., 2022). Ma et al. confirmed that SIRT1 could stimulate oxidative energy production by activating AMPK, PPAR-γ and PGC-1α simultaneously, while these factors were found to suppress NF-kB signaling and inflammation(Ma et al., 2018). Some authors also argued that inhibiting the AMPK/PGC-1α/PPAR-γ signaling pathway could induce chronic inflammation in metabolic diseases(Lee et al., 2021; Wang et al., 2021b; Yang et al., 2022). Thus, we hypothesized that the regulatory function of metformin was associated with the AMPK/PGC-1α/PPAR-γ signaling pathway. In the context, to clarify the molecular mechanism how metformin induced M2 macrophage polarization during the wound healing process, we further investigated the AMPK/PGC-1α/PPAR-γ signaling axis. According to our results, metformin significantly elevated the p-AMPK/AMPK ratio, as well as the expression levels of PGC-α and PPAR-γ. By contrast, the p-AMPK/AMPK ratio, PGC-α and PPAR-γ in cells co-treated with metformin and compound C indicated that metformin activated the PGC-1α/PPAR-γ signaling by activating AMPK. Moreover, we observed that inhibiting the AMPK activity by compound C could reverse the regulatory function of metformin. As shown in our present study, metformin treatment promoted the alternative M2 macrophage polarization by inducing the activation of AMPK. Activating AMPK could upregulate the PGC-1α/PPAR-γ signaling pathway. Therefore, we supposed that the effect of metformin on wound healing might be related to the alteration of macrophage phenotype by regulating the AMPK/PGC-1α/PPAR-γ signaling axis. Notably, our findings revealed a novel molecular mechanism in that we demonstrated that Metformin enhanced the function recovery in PNIs animal models by regulating M2 macrophage polarization. Wang et al. also reported that MAR1 inhibited the inflammatory response in LPS-induced RAW 264.7 macrophages and hPBMCs through the SIRT1/PGC-1α/PPAR-γ pathway(Wang et al., 2021a). Based on previous research findings, we provided further evidence supporting that metformin was a promising therapeutic strategy for inducing M2 macrophage polarization by modulating the AMPK/PGC-1α/PPAR-γ signaling pathway.

In conclusion, this study demonstrated that metformin could induce M2 macrophage polarization to facilitate peripheral nerve regeneration and functional recovery. The therapeutic effect of metformin in this process was achieved by facilitating the switching of macrophage’s phenotype to M2 type. The results also revealed that the immunomodulatory properties of metformin were associated with the AMPK/PGC-1α/PPAR-γ signaling pathway. Our findings may contribute to a more comprehensive understanding on the molecular mechanism of metformin treatment and its potential in peripheral nerve regeneration.

Author Contributions Gaojie Luo: Performed experiments, Writing-review & editing. Zekun zhou: Performed experiments, Writing-original draft, Writing-review & editing, Data curation. Cheng Li, Yaopei Zhang and Wei Chen: Formal analysis, Data curation. Xiaoxiao Li: Writing-review & editing. Juyu Tang: Writing-review & editing, Resources, Project administration. Liming Qing: Writing-review & editing, Resources, Funding acquisition, Project administration.

Acknowledgments We would like to thank Institute of Medical Science, Xiangya Hospital of the Central South University for invaluable assistance in conducting these experiments, and the other members of the research group for useful discussion in preparing this manuscript.

Data Availability The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation

Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Ethics Approval All the operations were performed in accordance with the guidelines of the China Animal Care Committee and the approval of the Laboratory Animal Committee of Central South University.

Funding: This publication was funded in part by the National Natural Science Foundation of China (To Dr tang 81472104 and To Dr Qing, No. Qing, No.81901978).

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No competing interests reported.

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Metformin induced M2 polarization to improve peripheral nerve regeneration by regulating AMPK/PGC-1α/PPAR-γ pathway

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Abstract

Figures

Introduction

Materials And Methods

Establishment of rat model of PNI and drug treatment

Assessment of sensory and motor functional recovery

Electrophysiology recording

Retrograde tracing

Immunofluorescence staining

Extraction and stimulation of bone-marrow-derived macrophages (BMDMs)

Cell viability assay

Flow cytometry

Real-time quantitative PCR

Western Blot Analysis

Statistical analysis

Results

Metformin improved functional recovery after peripheral nerve injury

Metformin promoted axonal regeneration and SC remyelination after PNI

Metformin promoted regeneration of peripheral nerve by inducing M2-type macrophage polarization

Metformin converted inflammatory macrophage cells to M2 polarization in vitro

Metformin skewed inflammatory macrophage cells to M2 polarization by modulating AMPK/PGC-1α/PPAR-γ signaling pathway

Discussion

Conclusion

Declarations

References

Additional Declarations

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