Gramine promotes functional recovery after spinal cord injury via ameliorating microglia activation

Abstract In recent years, a large number of studies have reported that neuroinflammation aggravates the occurrence of secondary injury after spinal cord injury. Gramine (GM), a natural indole alkaloid, possesses various pharmacological properties; however, the anti‐inflammation property remains unclear. In our study, Gramine was investigated in vitro and in vivo to explore the neuroprotection effects. In vitro experiment, our results suggest that Gramine treatment can inhibit release of pro‐inflammatory mediators. Moreover, Gramine prevented apoptosis of PC12 cells which was caused by activated HAPI microglia, and the inflammatory secretion ability of microglia was inhibited by Gramine through NF‐κB pathway. The in vivo experiment is that 80 mg/kg Gramine was injected orthotopically to rats after spinal cord injury (SCI). Behavioural and histological analyses demonstrated that Gramine treatment may alleviate microglia activation and then boost recovery of motor function after SCI. Overall, our research has demonstrated that Gramine exerts suppressed microglia activation and promotes motor functional recovery after SCI through NF‐κB pathway, which may put forward the prospect of clinical treatment of inflammation‐related central nervous diseases.

Chemically reactive substances extract from dietary plants performed multiple bioactivates, such as anti-angiogenesis, antiinflammation, anti-proliferation, anti-apoptosis and chemoprevention effects. 9,10 Mass of research has demonstrated that the green barley extracts could exhibit excellent pharmacological effects in nervous system and cardiovascular system. 11,12 The main ingredients from young barley (Hordeum vulgare L.) are Gramine, which is indole alkaloid and performed remarkable pharmacological activities including anti-oxidant, anti-proliferation and anti-inflammation reaction. [13][14][15] However, the anti-inflammation of Gramine during spinal cord injury and its underlying mechanism are not yet elucidated.
Recent studies have showed that NF-κB pathway involved in the neuroinflammatory responses process of microglia activation, which is the vital influence factor of secondary injury after SCI. 16,17 After necrosis or damage of cells, the NF-κB signalling pathway is passively released, which activates microglia to secrete a large number of inflammatory cytokines and gradually amplifies the inflammatory response. 10,16,17 Generally, NF-κB exists in an inactive form in the cytoplasm through binding with IκBα, 18 which could be activated by inflammatory factors secreted by microglia with phosphorylation and degradation.
Subsequently, the inactive form of NF-κB transformed to active form and translocated to the cellular nucleus, which regulates the expression of inflammation-related chemokines and cytokines. 19,20 A large number of researches have testified that NF-κB signalling pathway participates in many diseases, such as neuroinflammation, 21 experimental colitis 22 and sepsis. 23 Hence, regulate NF-κB signalling pathway could ameliorate the pathology process of these disease. In our study, we explored whether Gramine could regulate NF-κB signalling pathway involved in promoting motor functional recovery after SCI.
The underlying mechanism of Gramine-treated microglia may need to be further evaluated, and inhibition of activated microglia by reducing the secretion of inflammatory factors is considered a promising treatment strategy for spinal cord injury. Therefore, our research team tested the possible effect of Gramine on the inflammatory response after SCI in rats and studied the underlying molecular mechanism through LPS-stimulated microglia.

| Cell culture and treatment
The microglial cell line (HAPI, CRL-2815) was obtained from the American Type Culture Collection (ATCC, Manassas, USA). The PC12 cell line was purchased from Cell Biology (Shanghai, China). HAPI and PC12 cells were cultured with high glucose DMEM (Invitrogen, Carlsbad, USA) added 10% foetal bovine serum (FBS, Gibco BRL Co., Ltd., USA), 100 units/ml penicillin and 100 μg/ml streptomycin and incubated at 37℃ in a humidified atmosphere containing 5% CO2 and passaged twice a week. Gramine (Catalog No.S2304, purity ≥98%, Selleckchem, USA) was prepared a stock solution first, which was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich) to form a 10 mM final concentration. The stock solution of Gramine was diluted appropriately with cell culture medium (final DMSO concentration ≤1‰) for in vitro study. Lipopolysaccharide (LPS, Escherichia coli 055:B5, Sigma-Aldrich) was dissolved in saline and prepared as a stock solution with the final concentration of 5 mg/mL. HAPI cells were pre-treated with varying dose of Gramine (10, 20, 40, or 80 μM) with or without 1 μg/mL LPS stimulation for 24 hours Gramine was dissolved directly in normal saline before in vivo experiments.

| Microglia/neuron co-culture
To investigate the relationship between microglia and neurons, we utilized a transwell co-culture system (Corning, 0.4 μm pores, USA).
HAPI and PC12 cells were separately seeded into 24-well (1 × 10 5 HAPI cells/insert and 2 × 10 5 PC12 cells/well) transwell plates for the co-culture experiment. The HAPI cells were pre-treated Gramine (10 and 20 μM) with or without 1 μg/mL LPS stimulation for 24 hours After stimulation, the HAPI cells were harvested and seeded into inserts being placed into the PC12 wells. After 24 hours of co-culture, we measured neuronal apoptosis and axonal regeneration by immunofluorescence and Western blot assays.

| Cell viability assay
HAPI cells were seeded into a 96-well plate. After 24 hours for adherence, cells were treated with different dose of Gramine (10, 20, 40 or 80 μM) with or without 1 μg/mL LPS stimulation for 24 hours Then, 10 μL of CCK-8 (Dojindo, Kumamoto, Japan) solution was added into each well of 96-well plate and cultured in cell incubator for 4 hours.
After incubation, a spectrophotometer (Bio-Rad, Hercules, USA) was used to detect the OD value at 450 nm.

| Nitric oxide assay
Griess reaction was used to measure the Nitrite, which was representative NO production. Briefly, HAPI cells were seeded into a 96-well plate. After 24 hours for adherence, cells were treated with different dose of Gramine (10 and 20 μM) with or without 1 μg/mL LPS stimulation for 24 hours After incubation, the culture supernatant (100 μL) of each group was added into a 96-well plate, which was mixed with 100 μL Griess reagent (Beyotime, Shanghai, China) for reacting 10 minutes at room temperature in the dark. After incubation, a spectrophotometer was used to detect the OD value at 540 nm.

| ELISA assay
HAPI cells were seeded into a 12-well plate. After 24 hours for adherence, cells were treated with different dose of Gramine (10 and 20 μM) with or without 1 μg/mL LPS stimulation for 24 hours To detect the anti-inflammatory effect of Gramine, the cell-free supernatant of each well was harvested. The concentrations of IL-1β (MLB00C, R&D Systems, USA), IL-6 (M6000B, R&D Systems, USA) and TNFα (MTA00B, R&D Systems, USA) were detected by the ELISA kits according to the manufacturer's protocols, and OD values were measured by a spectrophotometer at 450 nm.

| Real-time PCR
HAPI cells were re-plated in 6-well plates. After 24 hours for adherence, cells were treated with different dose of Gramine (10 and 20 μM) with or without 1 μg/mL LPS stimulation for 24 hours After  Table 1. The membranes were visualized with an electrochemiluminescence plus reagent (Millipore, USA), and images of protein bands were captured on a ChemiDoc XRS + Imaging System (Bio-Rad, Hercules, USA). All experiments were repeated three times. The intensity of bands was normalized to those of GAPDH using Image Lab 3.0 software.

| Molecular modelling
P65 (PDB ID: 2ARM) was chosen for docking studies. Each protein was prepared for docking after being downloaded from PDB (https://www.rcsb.org/). After being minimized using PyMoL (version 1.7.6), the lowest energy conformations for docking were determined via default parameters. The protein-ligand docking analysis was conducted using AutoDockTools (version 1.5.6), which can provide ligand binding flexibility with the binding pocket residues. The images were finally generated using UCSF PyMoL. housed under controlled environmental conditions. Before the surgery, animals were deeply anaesthetized by 1% (w/v) pentobarbital sodium (4 mL/kg, i.p.), and a laminectomy was performed at the T9 vertebrae after the vertebral column was exposed. The spinal cord was fully exposed, and a moderate crushing injury was performed using a vascular clip for 1 minutes (30 g forces, Oscar, China). The rats received a single orthotopic injection of Gramine (80 mg/kg, dissolved in saline) or equal volume of saline 30 minutes after the SCI surgery. Postoperative treatments included injection of penicillin solution (4 × 10 6 units per animal, i.p.) in the first three days and manual bladder emptying twice a day. Subsequently, all rats were deeply re-anaesthetized and perfused with 0.9% NaCl, followed by

| Locomotion recovery assessment
Locomotion recovery analyses, including the Basso-Beattie-Bresnahan (BBB) locomotion scale 24 and the inclined plane test, were performed at 0, 1, 3, 7, 14, 21 and 28 days, and footprint analysis 25 was performed at 28 days. Rats were placed in an open experimental field and allowed to move freely for 5 minutes. Crawling ability was assessed by the BBB scale ranging from 0 (no limb movement or weight support) to 21 (normal locomotion), and the footprint analysis was performed by dipping the animal's posterior limb with red dye and forelimb with blue dye. 26 The inclined plane test was also performed to assess functional improvement in each rat at each of the above time points. Outcome measures were obtained by 3 independent examiners who were blinded to the experimental conditions.

| Haematoxylin and eosin staining
The longitudinal sections were dewaxed and washing with PBS.
Then, sections were fixed with acetone for 2 minutes, washed with water for 1-2 s and stained with haematoxylin for 5 minutes at room temperature. After washing, the slides were immersed in 1% acidic alcohol for 5-10 s and rinsed twice (2 minutes per rinse) with distilled water. Eosin solution was added to re-stain the sections for 1-2 minutes, followed by washing and dehydrating through a gradient ethanol series (80%, 90% and 100%; 30 s per step). Finally, the sections were immersed in xylene I (5 minutes) and xylene II (5 minutes) and sealed with neutral gum. The slides were observed, and images were acquired under a light microscopy (Olympus, Tokyo, Japan).

| Luxol fast blue staining
The longitudinal sections were dewaxed followed by washed with PBS. The sections were immersed in 0.1% LFB dye at room temperature overnight, washed and de-stained with 0.05% lithium carbonate solution for 5 minutes, followed by dehydration with a graded series of ethanol (70% 10 s, 95% 25 s, 100% 25 s). Finally, the slides were hyalinized with xylene and mounted with neutral gum. The images were also acquired under a light microscopy (Olympus, Tokyo, Japan).

| Immunofluorescence
In vitro: HAPI cells were seeded into 6-well plates. After 24 hours for adherence, cells were treated with different dose of Gramine (10 and 20 μM) with or without 1 μg/mL LPS stimulation for 24 hours Co-cultured PC12 cells were seeded into 24-well plates.
After adherence, washed twice by PBS. Then, 1 mL 4% paraformaldehyde was added into each well for 30 minutes. Then, 1 mL 0.5% Triton X-100 was added into each well for permeabilization

| Statistical analysis
The results were presented as mean ± SD Statistical analyses were performed using SPSS statistical software program 18.0, which were from three independent experiments. Difference among groups was assessed by the one-way analysis of variance (ANOVA) followed by Tukey test. *P value <0.05 was considered as statistically significant.

| Gramine suppresses microglia secreting proinflammatory cytokines and expressing inflammatoryrelated genes
The chemical structure of Gramine has been exhibited in Figure 1A. To evaluate cytotoxicity of Gramine, HAPI cells were treated with Gramine different concentrations of Gramine. CCK-8, which was used to measure the cell viability, and revealed that Gramine caused no significant cytotoxicity on HAPI cells at concentrations of ≤20 μM ( Figure 1B).
Moreover, the viability of microglia pre-treated with 0-20 μM Gramine followed by LPS (1 μg/mL) treatment was no statistical significance compared with control group ( Figure 1C). Therefore, 10 and 20 μM Gramine were chose for our further study. We evaluated the effect of Gramine on the pro-inflammatory mediator release in HAPI cells in vitro and detected the pro-inflammatory cytokines, such as IL-1β, IL-6, TNFα and NO secreted from LPS-induced microglia. As shown in Figure 1D-F, IL-1β, IL-6 and TNFα those pro-inflammatory cytokines were significantly increased after LPS stimulation, whereas remarkably decreased in dose-dependent manner by Gramine treatment.
Moreover, we detected the extent of NO, which was synthesized by the critical enzyme, inducible nitric oxide synthase (iNOS) of inflammatory response. Our group detected a remarkable increase of NO release by LPS stimulation, whereas it reversed after pre-treatment with Gramine ( Figure 1G). Furthermore, inflammation-related mRNAs were evaluated by qPCR. Data demonstrated that IL-1β, IL-6, TNFα and iNOS were remarkably increased by LPS-stimulated and reversed after Gramine pre-treatment ( Figure 1H-K). These data demonstrated that LPS improved the higher expression of pro-inflammatory mediators in microglia, but Gramine pre-treatment can significantly attenuate its increase.

| Gramine inhibits LPS-induced microglia activation
Microglia exhibit branching morphology under normal condition, Our results demonstrated that iNOS and COX-2 were significantly enhanced by LPS stimulation and restored followed by Gramine pretreatment ( Figure 2C-E).

| Gramine inhibits activation of microglia and promotes axonal regeneration of neuron
In our study, we detected the Iba-1, which is a hallmark of activated microglia by immunofluorescence to evaluate the anti-inflammatory effect of Gramine. Our data show that the fluorescence intensity of Iba-1 was remarkably increased after LPS stimulation, whereas reversed by Gramine pre-treatment in a dose-dependent manner ( Figure 3A). Moreover, a co-culture system was used to assess the effects of activated microglia on neurons. We treated the microglia firstly using different dose of Gramine (10 and 20 μM). After that, treated microglia were harvested and added into a co-culture system, which worked with neurons. We used immunofluorescence staining to assess the apoptosis and axonal regeneration of neuron after co-culture with microglia.
As shown in Figure 3B, neurons performed axonal regeneration at a remarkably decreased rate (green fluorescence intensity) and apoptosis (red fluorescence intensity) at a significantly increased rate co-cultured with activated microglia. Interestingly, the axonal regeneration and apoptosis rate of neurons were restored by co-cultured with Gramine pre-treatment microglia.

| Gramine suppresses microglia activation via NF-κB signal pathway
NF-κB and IκBα were critical proteins in NF-κB signal pathway; the Western blot results show that these proteins were phosphorylated after LPS stimulation and dephosphorylated followed by Gramine pre-treatment in a dose-dependent manner ( Figure 4A-C). In order to investigate the affinity between NF-κB and Gramine, we used a molecular docking analysis according to diverse binding pockets of the antagonist. By studying all the models returned, we found that Gramine formed some favourable connections and docked nicely within the NF-κB binding sites ( Figure 4D). In Figure 4D  Furthermore, the nuclear translocation of NF-κB was detected by immunofluorescent showing that NF-κB protein would translocate to the nucleus after LPS stimulation which means the activation of microglia. Moreover, this phenomenon could be suppressed by Gramine pre-treatment, NF-κB protein performed at cytoplasm mostly ( Figure 4G and H).

| Gramine exerts neuroprotective effects on spinal cord injury
Behavioural experiments including BBB scores, the inclined plane test and footprint analysis were measured to assess the functional recovery of SCI. 1 Interestingly, our results show that Gramine treatment group could achieve a nice BBB scores compared with SCI group. Moreover, the BBB scores of SCI group could also tar-

| Gramine suppresses microglia/macrophage activation after SCI
As shown in Figure 6A, CD68 (Green) and GFAP (Red) were doubledyed to watch the number and distribution of activated microglia/ macrophage. Compared with the SCI group, the distribution of positive expression cells (CD68) in the Gramine treatment group significantly reduced ( Figure 6B).
Western blot analysis of CD68 and Iba-1 also confirmed the above results, showing that CD68 and Iba-1 proteins expression were prominently decreased after Gramine treatment ( Figure 6C-E) which suggested Gramine could reduce the inflammatory response after SCI in rats.

| Gramine improved axonal regeneration after SCI
Co-immunostaining was performed with anti-MAP-2 antibody to label neurons and anti-GFAP antibody to label astrocyte. As shown in Figure 7A, GFAP-positive astrocytes, which named glial scar, were distributed along the lesion border. Notably, Gramine treatment group showed a narrower lesion area compared to SCI group.
Furthermore, in Gramine treatment group, MAP-2-positive neurons remarkably increased compared with SCI group in the lesion border ( Figure 7A). In addition, we measured the fluorescence intensity of MAP-2, suggesting that the animals in Gramine treatment group significantly prevented robust loss of neurons ( Figure 7B). From the perspective of motor functional recovery, axon regeneration is vital.
Therefore, the extension of neurofilaments was evaluated by NF-

| D ISCUSS I ON
SCI is a serious disabling disease, and its incidence is about 12,000 new cases every year. The main feature is the obvious loss of sensory and motor functions. 28 Therefore, it is imperative to find effective strategies for treating SCI. At present, increasingly studies have proved that Chinese herbal medicine, especially its bioactive compounds can also produce obvious therapeutic effects on spinal cord injury. 1,[29][30][31] Recently, extraction of herb performed nice neuroprotection effects in central nervous system, such as Parkinson's disease, 27 Alzheimer's disease 32 and SCI. 33    Gramine pre-treatment microglia. Thus, Gramine is identified as an anti-inflammatory medicine on microglia, which could promote axonal regeneration of neurons; however, the underlying mechanism of the anti-inflammation effects of Gramine is still unclear.
There are numerous pro-inflammatory mediators involved in initiating inflammation, and NF-κB is a key protein, which could regulate the expression of iNOS and COX-2. 47,48 The complex formed by NF-κB and IκBα resides in the cytoplasm of resting cells. When the NF-κB signalling pathway is stimulated and activated by extracellular factors such as LPS and IL-1β, the complex formed by IκBα and NF-κB will be separated into monomers and phosphorylated, respectively. Finally, phosphorylated NF-κB will translocate to the nucleus. In our study, a docking analysis demonstrated that NF-κB has an active binding domain for attracting Gramine. Consistent with our Western blot and immunofluorescence results that the expression of phosphorylated NF-κB and IκBα was decreased after Gramine treatment. It suggested that Gramine could combine with NF-κB and then perform suppressing of NF-κB activation and nuclear translocation.

| CON CLUS ION
To sum up, our research shows that Gramine inhibits the high expression of LPS-induced pro-inflammatory mediators and neuronal apoptosis by regulating NF-κB signalling pathways. We observed in both that Gramine can exert neuroprotective effects by inhibiting excessive activation of microglia in vivo and in vitro. The above research proves that Gramine may become a potential drug for SCI therapy.

This research was supported by Wenzhou Municipal Science and
Technology Bureau Foundation (No. Y20180027, Y20190022).

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets generated and/or analysed during the current study are not publicly available but are available from the corresponding author on reasonable request.