Dual regulation of microglia and neurons by Astragaloside IV‐mediated mTORC1 suppression promotes functional recovery after acute spinal cord injury

Abstract Inflammation and neuronal apoptosis contribute to the progression of secondary injury after spinal cord injury (SCI) and are targets for SCI therapy; autophagy is reported to suppress apoptosis in neuronal cells and M2 polarization may attenuate inflammatory response in microglia, while both are negatively regulated by mTORC1 signalling. We hypothesize that mTORC1 suppression may have dual effects on inflammation and neuronal apoptosis and may be a feasible approach for SCI therapy. In this study, we evaluate a novel inhibitor of mTORC1 signalling, Astragaloside IV (AS‐IV), in vitro and in vivo. Our results showed that AS‐IV may suppress mTORC1 signalling both in neuronal cells and microglial cells in vitro and in vivo. AS‐IV treatment may stimulate autophagy in neuronal cells and protect them against apoptosis through autophagy regulation; it may also promote M2 polarization in microglial cells and attenuate neuroinflammation. In vivo, rats were intraperitoneally injected with AS‐IV (10 mg/kg/d) after SCI, behavioural and histological evaluations showed that AS‐IV may promote functional recovery in rats after SCI. We propose that mTORC1 suppression may attenuate both microglial inflammatory response and neuronal apoptosis and promote functional recovery after SCI, while AS‐IV may become a novel therapeutic medicine for SCI.


| INTRODUC TI ON
Spinal cord injury (SCI) is one of the most disabling central nervous system disorders that can lead to varying degrees of sensory and motor function loss. It has a major impact on the quality of life for patients. 1 However, there is currently no recognized effective therapy to treat SCI. The pathological process of SCI is mainly composed of primary injury and secondary injury. Primary injury is mainly caused by the direct damage to the spinal cord, while it may trigger a series of pathology including inflammation, oxidative stress, ischaemia and local oedema, to induce secondary injury. [2][3][4] Studies have shown that the secondary injury phase is an important time window for SCI therapy. 5 Apoptosis of neurons, especially ventral horn motor neurons (VMN), may lead to the loss of motor function 6,7 and contribute to the secondary injury after SCI. 8,9 Besides, microglia-mediated neuroinflammation also plays a crucial role in the expansion of secondary injury after SCI, and the degree of inflammation has a great impact on the recovery of motor function after SCI. 10 Therefore, we propose that therapeutic strategies for SCI should focus on inhibiting neuronal apoptosis and inflammatory responses.
Autophagy is a conserved cellular degradation process that plays a critical role in cellular survival and homoeostasis. Recent studies revealed that autophagy may protect against apoptosis in neurons 11 ; our group also showed that autophagy activators, such as rapamycin and metformin, may promote functional recovery in rats through apoptosis suppression. 12,13 Meanwhile, microglia polarization is an important target for regulating inflammation; studies have shown that microglia M2 polarization may attenuate inflammation after SCI. 14 Intriguingly, both neuronal autophagy and microglial M2 polarization are regulated by mTORC1 signalling pathway. 15,16 Suppression of mTORC1 signalling pathway may not only promote autophagy in neurons but also induce M2 polarization in microglia; therefore, we hypothesize that mTORC1 suppression may have dual effects on inflammation and neuronal apoptosis and may be a feasible approach for SCI therapy.
Astragaloside IV (AS-IV) is an extract of traditional Chinese medicine Radix Astragali. It has been shown to possess neuroprotective pharmacological activity by suppressing inflammation and oxidative stress in central nervous system diseases. 17 More importantly, a previous study showed that AS-IV could potently inhibit mTORC1 activity with less adverse effects. 18 However, it is still not known whether AS-IV could promote functional recovery after SCI. We propose that AS-IV could reduce the inflammatory response and neuronal apoptosis so as to promote functional recovery after SCI by mTORC1 inhibition.
In this study, we found that AS-IV may suppress mTORC1 signalling both in neuronal cells and microglial cells in vitro and in vivo.
AS-IV treatment may stimulate autophagy in neuronal cells and protect them against apoptosis through autophagy regulation; it may also promote M2 polarization in microglial cells and attenuate neuroinflammation. In vivo study, rats were intraperitoneally injected with AS-IV (10 mg/kg/d) daily after SCI, behavioural and histological evaluations showed that AS-IV may promote functional recovery in rats after SCI. We propose that mTORC1 suppression may attenuate both microglial inflammatory response and neuronal apoptosis and promote functional recovery after SCI, while AS-IV may become a novel therapeutic medicine for SCI.

| SCI model and AS-IV treatment
A total of 48 adult female SD rats (220-250 g) were purchased from the SLAC Laboratory Animal Company. All experimental procedures were performed in accordance with Chinese Guidelines of Animal Care and Welfare, and this study was approved by Wenzhou Medical University Animal Care and Use Committee. Rats were divided randomly into three groups (the Sham group, the SCI group and the SCI + AS-IV group). Before surgery, rats were anaesthetized with 1% (w/v) sodium pentobarbital (40 mg/kg, i.p.). Next, we cut-off the muscles and ligaments around the T8 spinous process and performed a laminectomy at T8 vertebral level to expose the spinal cord. SCI was induced by a vascular clip (15 g forces; Oscar) to clamp the spinal cord for 1 min.
The same surgical procedure was performed on the Sham group, but without compression of the spinal cord. After surgery, the bladders of rats were emptied manually twice a day. Besides, AS-IV was injected (10 mg/kg i.p.) in the SCI + AS-IV group (the AS-IV group) immediately and given daily 10 mg/kg doses until they were killed. Simultaneously, the other groups were injected with an equal dose of saline.

| Locomotion recovery evaluation
In our research, locomotor function of rats was assessed using the Basso, Beattie and Bresnahan (BBB) locomotor recovery scale, inclined plane test and footprint test. The BBB scale is a 21-item scale that scored results ranging from 0 to 21 points in the open-field test.
For the inclined plane test, it was performed to assess functional improvement at each time-point, and the maximum angle at which a rat could retain its position on a testing apparatus in two positions without falling for 5 seconds was recorded. The BBB scores and inclined plane test were performed on days 1, 3, 7, 14 and 28 after SCI.
The footprint test was also performed and the footprints collected were scanned and evaluated. Each rat was observed by four evaluators who were blinded to the treatment.

| Histological staining
On day 7 and 28 after SCI, the rats were anaesthetized and perfused with 0.9% NaCl through the heart, followed by fixation with 4% paraformaldehyde (PFA). The T7-T9 spinal cord segment containing the lesion site was collected and fixed in 4% PFA for 48 hours, then embedded in paraffin for paraffin sections. Transverse sections with the thick of 5μm were prepared for histological staining. In order to observe the histological changes of each group, HE staining and Nissl staining were performed to paraffin sections according to the instructions of the manufacturer. All images were acquired using light microscopy (Olympus, Tokyo, Japan).

| Cell culture and treatment
The rat-derived microglial cell line (HAPI cells, kept in our laboratory) and the neuronal cell line (PC12 cells, purchased from the Shanghai Institute of Cell Biology) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum (FBS), 100 units/mL penicillin and 100 μg/mL streptomycin. The cells were cultured at 37°C constant temperature incubator with a humidified atmosphere containing 5% CO 2 .
To promote polarization to the M1 phenotype, HAPI cells were treated with LPS (1 μg/mL) for 24 hrs. In PC12 cells, oxidative stress, a common pathological mechanism of apoptosis, was induced by TBHP (100 μmol/L) for 4hrs. 3-MA (10 mmol/L) was used to block autophagy. The treatment of AS-IV (1 or 10 μmol/L) was used to study the effects of AS-IV.

| Quantitative Real-Time PCR
Total RNA was extracted from cells using TRIzol reagent (Invitrogen) following the manufacturer's protocol. 1 μg of total RNA from each group was used for reverse transcription. After the total RNA was reverse transcribed, the SYBR Green reagent (Bio-Rad) was used in qPCR for quantification. And, the cDNA was amplified in a 10 μL reaction volume using the CFX96 ® Real-Time PCR System (Bio-Rad).
The PCR cycle began with template denaturation and hot-start Taq activation for 3 minutes at 95°C, followed by 40 cycles of 95°C for 15 seconds and 60°C for 45 seconds. The normalized expression of the target gene was calculated using the comparative CT method, and a fold change was obtained from the equation 2 −ΔΔC t of each gene.

| Western blot analysis
Total protein was extracted from spinal cord tissues and cells.
Then, the extracts were quantified by BCA kit (Beyotime) and were equilibrated to the same quality level. 12% SDS-polyacrylamide gel electrophoresis was used to dissolve protein samples for the next phase that transferring them to polyvinylidene fluoride membranes (Millipore, 0.2 μm). Subsequently, the membranes were blocked for 2 hours using 5% skim milk in TBST (25 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 7.5) and next incubated in the primary antibody solutions overnight at 4°C. Then, membranes were washed three times for 5 min with TBST and incubated with the relative secondary antibodies for 2 hours at room temperature. Finally, the bands were detected by a Chemi DocXRS + Imaging System (Bio-Rad), and quantitative analysis was performed using Image Lab 3.0 software (Bio-Rad). overnight. For secondary antibody incubation, the slides of cells were incubated with appropriate secondary antibodies for 1 hour. Then, the slides of cells were washed with PBS, stained with DAPI for 1 minute and then sealed with a coverslip. Images were captured using a fluorescence microscope (Olympus).

| Statistical analysis
All data are expressed as mean ± standard deviation. All statistical analyses were performed using GraphPad Prism 7.0. Statistical significance was determined by one-way analysis of variance (ANOVA) followed by Tukey's post hoc analysis. Besides, generalized linear mixed models were used to analyse between-group differences in the BBB scores and inclined plane test results. The results were considered to be statistically significant at *P < .05 or **P < .01. Compared with the control group, the apoptosis-related protein markers of Bax and cleaved caspase 3 were markedly increased while Bcl-2 was reduced in TBHP group. However, the expression of Bax, cleaved caspase 3 and Bcl-2 was restored in AS-IV treated PC12 cells ( Figure 1C). Moreover, TUNEL assay also confirmed that AS-IV inhibited TBHP-induced apoptosis in PC12 cells ( Figure S1).
To clarify the effect of AS-IV on autophagy, we detected the expression of autophagy-related protein, p62 and LC3, in PC12 cells.
We found that the expression of p62 and LC3 was both increased in the TBHP-treated group compared with the control group, while p62 expression level was decreased and LC3 was increased in the AS-IV treated group compared with the TBHP-treated group; the expression of p62 has also been confirmed by fluorescence staining ( Figure 2B). These results suggested that although autophagy was activated, autophagic flux was inhibited by TBHP, while the autophagic flux was rescued by the treatment of AS-IV (Figure 2A).
Similarly, another Western blot assay showed that apoptosis-related proteins were increased in the TBHP-treated group compared with the control group, and they were decreased in the AS-IV-treated group ( Figure 2C).
To further define the relationship between autophagy and neuronal apoptosis, we used 3-MA, an autophagy inhibitor, to suppress autophagy. As predicted, autophagy activated by AS-IV and anti-apoptotic effects were both blocked by 3-MA (Figure 2A,C).
Together, these results suggest that AS-IV treatment may suppress mTORC1 signalling and inhibit neuronal apoptosis through autophagy in vitro.

| AS-IV suppresses mTORC1 and promotes M2 polarization in microglia in vitro
We study the effect of AS-IV on the polarization of microglia in

| AS-IV suppresses mTORC1 signalling in rats in vivo
To identify the effect of AS-IV on mTORC1 signalling in vivo, we established SCI model in rats and examined the ratio of p-mTOR/t-mTOR and p-p70S6K/p70S6K in spinal cord tissues by Western blot.
We found both the phosphorylation levels of mTOR and p70S6K were reduced in the AS-IV treated group compared with the SCI group ( Figure 6A). After that, we performed immunofluorescence double staining on the cross-section of spinal cord tissue slice; p-p70S6K was co-stained with NeuN or Iba1, which are markers of neuronal cells and microglia respectively, to reveal changes in mTORC1 signalling in neurons and microglia. As shown in Figure 6B,

| AS-IV promotes locomotor function recovery after SCI in rats
The above studies showed that AS-IV possesses strongly neuroprotective and anti-inflammatory effects in vitro; however, whether they can be beneficial for locomotor function recovery in vivo is still unknown. Therefore, we used three behavioural tests, the BBB locomotion score, the inclined plane test and footprint test, to evaluate motor function recovery in SCI rats. The results showed that all rats lost their hind legs mobility immediately after SCI compared with the sham operation group and gradually recovered in a time-dependent manner. The results of the BBB score and inclined plane test showed that the AS-IV treated group recovered better than the SCI group ( Figure 7A Figure 7C). Together, these results indicate that AS-IV promotes functional recovery in SCI rats.

| AS-IV attenuates neuronal loss in SCI rats
To affirm the neuroprotective effect of AS-IV in vivo, we observed neuronal retention in spinal cord tissue via HE staining and Nissl staining. As shown in Figure 7D,E, HE staining showed that central grey matter and dorsal white matter of SCI rats were seriously damaged compared with the Sham group, while AS-IV treatment significantly attenuated the damage of spinal cord tissue. Also, we counted the numbers of ventral motor neurons (VMNs) via HE staining and Nissl staining, we found SCI induced massive loss of VMNs, while AS-IV could attenuate its loss ( Figure 7F,G). Next, we detected the expression of apoptosis-related proteins at day 7 after SCI. The Western blot results showed that AS-IV treatment effectively reversed the increase of caspase 3-dependent apoptosis induced by SCI ( Figure 8A). This was further supported by immunofluorescence staining of cleaved caspase 3; as shown in Figure 8B,C, the fluorescence intensity was decreased in the AS-IV group compared with the non-treated SCI group. Taken together, the results above suggested that AS-IV could effectively attenuate caspase 3-dependent neuronal apoptosis and restrain neuron loss in SCI rats in vivo.

| AS-IV promotes M2 polarization and suppresses pro-inflammatory mediators release in SCI rats
To explore the effect of AS-IV on microglia polarization in SCI in vivo, Western blot analysis was performed to detect the expression of Arg-1. The results showed that AS-IV promoted the expression of Arg-1 compared with the SCI rats ( Figure 8D). To further investigate the regulatory effect of AS-IV on inflammatory response, we performed Western blot to observe the expression levels of pro-inflammatory mediators, such as iNOS, COX-2 and TNF-α, at day 7 after SCI. The results showed that the pro-inflammatory mediators above were distinctly increased in the SCI group compared with the Sham group; however, they were reduced in the AS-IV-treated group ( Figure 8E). Collectively, these results indicate that AS-IV promotes M2 polarization and inhibits the inflammatory response in SCI rats.

| D ISCUSS I ON
The incidence of SCI has soared particularly among young people, and it brings an enormous burden to the individual and society. 19,20 Thus, it is a priority to find an effective strategy to treat SCI. In recent years, a growing number of traditional Chinese herbal medicine ingredients have been tested in the treatment of SCI. AS-IV is a Chinese herbal extract with low toxicity and minimal side effects.
Many studies have demonstrated that AS-IV possesses neuroprotective effects that can reverse or alleviate various central nervous system diseases, such as Parkinson's disease and cerebral ischaemia. 21,22 In the present study, we provide evidence that AS-IV rendered a beneficial effect on neuroprotection against SCI. We found that (a) AS-IV promotes autophagy and inhibits neuronal apoptosis ical processes for SCI therapy; however, barely any therapies could target both these two pathological processes. In our study, we found that AS-IV possesses 'one stone two birds' potential, which not only inhibits neuronal apoptosis but also suppresses neuroinflammation through inhibiting the mTORC1 signalling pathway (Figure 9). that mTORC1 is a new therapeutic target for SCI. [26][27][28][29] In the process of apoptosis, mTORC1 suppression may promote autophagy, which may further suppress apoptosis, while mTORC1 suppression may also induce M2 polarization that may suppress neuroinflammation. In our study, we found that mTORC1 signalling was decreased in the SCI group when compared with the Sham group, while neuronal apoptosis and neuroinflammation were activated in the SCI group; however, when mTORC1 is an isoform of Rheb that directly regulates the activity of mTORC1.
Akt is another major upstream molecule to regulate mTORC1 signalling. It has been reported that AS-IV may inhibit suppress mTORC1 signalling through Rheb. 18 To answer whether AS-IV may also inhibit mTORC1 signalling by regulating Akt, we assess Akt activity by detecting the phosphorylation of Akt. We found the phosphorylation level of Akt was not changed significantly with the treatment of AS-IV ( Figure 4B), implying that AS-IV does not affect mTORC1 activity Microglial polarization plays a decisive role in the regulation of neuroinflammation after SCI. Generally, microglia can be classically activated into M1 (pro-inflammatory) phenotype or alternatively activated into M2 (anti-inflammatory) phenotype. 39,40 The polarization of both M1 and M2 affects the outcome of the inflammatory response, which may lead to either tissue damage or repair after SCI. Studies have found that a large number of microglia/macrophages polarized into M1 phenotype after SCI and secreted lots of pro-inflammatory mediators, which were not conducive to the repair of SCI. 41 AS-IV has been reported to have protective effects on cerebral ischaemia-reperfusion injury as well as the integrity of the bloodbrain barrier. [45][46][47][48][49][50] With the low toxicity, AS-IV may be expected to be applied to the clinic for the treatment of central nervous system injury in the future. Previous studies have also shown that rapamycin, a classical mTORC1 inhibitor, could exert neuroprotective effects in SCI. 28,[51][52][53] Compared with these mTORC1 inhibitors, such as rapamycin, AS-IV could effectively suppress mTORC1 signalling pathway with mild adverse effects. 18 However, mTORC1 is still a key signalling in the regulation of cellular metabolism, and its suppression may bring a series of adverse reactions. 54 In our study, AS-IV was injected intraperitoneally, although therapeutic effects were shown in the study, the long-term side effects of mTORC1 suppression by AS-IV are still unknown. Nowadays, local delivery of drug with sustained release ability has become a new trend for SCI therapy. [55][56][57][58] Thus, further studies are needed to find a more suitable delivery system for AS-IV.

| CON CLUS ION
AS-IV mediated mTORC1 signalling suppression may inhibit neuronal apoptosis through autophagy and inhibit neuroinflammation through microglial M2 polarization, which leads to locomotor function recovery in SCI rats. Our study indicates that AS-IV may become a potential drug for SCI treatment.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

AUTH O R S' CO NTR I B UTI O N
XYW, XLZ and JLL contributed to conceptualization; CAH contributed to methodology; XQZ contributed to software; MBG and XMC contributed to validation; XXP contributed to formal analysis; F I G U R E 9 Schematic diagram of the effects of AS-IV on SCI. AS-IV treatment suppresses mTORC1 signalling pathway to regulate neuronal autophagy and microglia polarization, which promotes functional recovery of SCI rats ZXS contributed to investigation; XXP and BW contributed to resources; SLH contributed to data curation; JLL contributed to writing-original draft preparation; XLZ and YFZ contributed to writing-review and editing; HL contributed to visualization; YSW, YW, WYG and NFT contributed to supervision; XYW and XLZ contributed to project administration; XYW contributed to funding acquisition.

DATA AVA I L A B I L I T Y S TAT E M E N T
All original data used in this work will be made available upon request.