Upregulation of microRNA-96 alleviates the neurological damage in acute seizure rats by down-regulating RAC1 and inhibiting the activation of RhoA/ROCK signaling pathway

Objective Today, the research about the involvement of non-coding RNAs on neurological disorders is still scarce. Hence, we aimed to investigate the effect of miR-96 targeted RAC1 to mediate RhoA/ROCK signaling pathway in neurological damage in acute seizure rats. The acute seizure rat model was constructed by using classical chlorinated-pilocarpine injection, which was treated with miR-96 mimics, RAC1-siRNA or their controls. The number of BrdU positive cells, neuronal changes and apoptosis, expression of NGF, BDNF and GFAP were detected by different staining assays. At the same time, the activity of SOD, the content of MDA and the protein contents of TNF-α and IL-6 in rat hippocampus were detected as well. Next, the expression of NGF, BDNF, GFAP, Bax, Bcl-2, caspase-3, RAC1, RhoA and ROCK were determined by RT-qPCR and western blot analysis. Then, the binding site between miR-96 and RAC1 was analyzed by bioinformatics software and luciferase assay. Finally, the altered expression of miRNAs was investigated in exosomes released from excitotoxic neurons with a customized rat miRNA chipset.

sedimented. After that, the sample was taken out in the coronal position, and 1 to 2 mm 3 hippocampus tissues were taken and 5% agar was used to make a concentration of 0.01 mol/L phosphate buffer saline (PBS). The prepared agar was used to fix the hippocampus tissue section, and each piece was continuously taken for 10 pieces with a thickness of about 50 μm.
The hippocampal tissue of each group was washed with PBS for 10 min × 3 times.
The antigen was repaired by 2N-Cl (37°C, 30 min), followed by 0.1 mol/L boric acid buffer (pH 8.5) rinsing for 10 min. Then, BrdU (1:300, Abcam, Cambridge, MA, USA) was added with 0.01 mol/L fetal bovine serum (BSA)-PBS, placed in a refrigerator (4°C, overnight), and then rinsed with PBS (10 min, 3 times). After that, 0.01 mol/L BSA-PBS was added to Cy3 monkey anti-rat IgG (1:200) and incubated (1 h, room temperature), rinsed with PBS, and glycerin was used for sealing. After that, the sections were placed under a laser confocal microscope, and observed from the X, Y, and Z axis, and the number of BrdU-positive cells was recorded. Images were processed by Image J (Oxford UK).

HE staining
The prepared paraffin sections were routinely dewaxed to water, then hematoxylin was used for staining (10 min). Then 1% hydrochloric acid was used to separate color (2 -5 s), followed by ammonium hydroxide for returning blue (20 -30 s), and eosin was added for staining (5 min). After rinsing (3 min), gradient alcohol was used for color separation followed by regular dehydration and clearance, then neutral gum was taken for sealing, and finally microscope (Nikon, Tokyo, Japan) was used for observation.

Electron microscopy specimen preparation and observation
The obtained diced tissues were fixed in 3% glutaraldehyde for 3 h, and rinsed with PBS. Subsequently, the diced tissues were fixed with 1% of osmic acid for 2 h, followed by dehydration with gradient alcohol, and resin was used to embed the tissues. Then, the LKBIII ultrathin slicer (Pharmacia LKB, Sweden) was used for sectioning, and double-electron staining by uranyl acetate and lead citrate, and observed with JE-OLJEM-2100F transmission electron microscope (Siemens, Berlin, Germany).

Toluidine blue staining
The above-mentioned hippocampal tissue was placed in xylene and immersed in gradient ethanol. Then, it was stained with 1% toluidine blue (40 min). After that, the color separation was performed, and sections were cleared by using xylene and gradient alcohol, and then sealed with a neutral gum. Finally, a microscope (Nikon, Tokyo, Japan) was used to count and analyze the Nissl positive cells.

TdT-mediated dUTP Nick-End Labeling (TUNEL) staining
The above-mentioned hippocampal tissues were detached in proteinase K (1 h, room temperature). Then, the tissue was routinely treated by the TUNEL reaction kit.
Finally, the apoptotic cells' number were recorded under a microscope (Nikon, Tokyo, Japan). TUNEL staining was used for staining positive cells and its color turned brown yellow or brown.

Immunohistochemical staining
The prepared paraffin tissue sections were baked at room temperature for 60 min.
After conventional xylene dewaxing and alcohol hydration, 0.3% fresh hydrogen peroxide solution (H 2 O 2 ) was prepared and sealed the tissue for 5-10 min. Next, sections were immersed in 0.01 mol/L citrate buffer (pH 6.0, 95°C), and the sections were heated for 10-15 min. After cooling, 5% goat serum was added (30 min, room temperature) followed by primary anti-nerve growth factor (NGF) (1:200, Millipore, Inc., Massachusetts, USA), brain-derived neurotrophic factor (BDNF) (1:500, Abcam, Cambridge, MA, USA), and glial fibrillary acidic protein (GFAP) (1:1000, Abcam, Cambridge, MA, USA) were added. Then the sections were incubated (2 h, 37°C) and placed overnight (4°C). According to the kit instructions, the corresponding secondary antibody was added, and together with the horseradish-labeled streptavidin incubated (2 h, 37°C). Finally, the diaminobenzidine (DAB) coloring solution was used for color development (The normal group was not added with primary antibody, replaced with 0.01 mol/L PBS, and the other steps were the same). Each immunohistochemical stained section was quantitatively analyzed by Image Pro Plus 6.0 image. Three fields of view were casually chosen for each measurement, and the average value was taken. At the same time, the number of NGF, BDNF, and GFAP immunoreactive cells was also observed.

Spectrophotometry detection
The hippocampal tissues were added with saline in a ratio of 1:9 to make a 10% brain tissue homogenate on ice. Subsequently, it was centrifuged (15 min, 4°C, 3000 r/min), and the supernatant was collected. After that, the activity of superoxide dismutase (SOD) and malonadialdehyde (MDA) were determined by chemical colorimetry based on the instructions of detection kit (Nanjing JianCheng Bioengineering Institute, Nanjing, China).

Enzyme-linked immunosorbent assay (ELISA)
According to the instruction of ELISA kit (Yixin Bioengineering Co., Ltd., Shanghai, China), the homogenate supernatant was assayed for measuring tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). The steps were as follows: first, the 50 μL coated antibody was added to each well, then incubated at 4°C. After that, the plate was washed with a washing solution, then 150 μL of blocking solution was poured into each well, and the plate was washed at 37 °C for 1 h. After a standard curve was established, 50 μL of the sample was added and mixed at room temperature (2 h). Subsequently, avidin was added to each well, and the tetramethyl benzidine (TMB) substrate working solution was poured into each well. The reaction was performed in the dark (room temperature, 10 min). Finally, H 2 SO 4 (2M) was added to each well to stop the liquid mixture. The microplate reader was used to measure the absorbance at 450 nm and 405 nm, respectively, and the concentration value was obtained by calibration.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
Total RNA from brain tissues were extracted with a one-step method from Trizol  Table 1). RT-qPCR was performed by using SYBR Green chimeric fluorescence. The PCR reaction was performed by using SYBR Premix Ex Taq (Perfect Real Time) kit reagent. U6 worked as an internal reference for miR-96, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) played an internal reference role in concerning NGF, BDNF, GFAP, Bax, Bcl-2, caspase-3, TNF-α, IL-6, RAC1, RhoA, and ROCK mRNA. 2 -△△Ct was used to obtain the relative expression level of the target gene [19] . The experiment was performed three times.

Western blot assay
The total protein in the hippocampus was extracted, and the bicinchoninic acid protein concentration determination kit (Beyotime Biotechnology Research Institute, Shanghai, China) was used to determine the protein concentration, and the concentration of all group was adjusted uniformly. The total protein from each group was added with 80 μL 5× sodium dodecyl sulfate (SDS) protein loading buffer, and the protein was degenerated by boiling water for 5 min. After the transfer was completed, the polyvinylidene difluoride (PVDF) membrane was removed and placed in 5 mL blocking solution (room temperature, 1 h). Primary antibody: NGF, GAPDH

Luciferase activity assay
The target site of RAC1 and corresponding miR-96 binding was determined by online prediction software http://www.targetscan.org, and the primers were designed with the 3'-untranslated region (3'UTR) sequence of RAC1 gene. We also performed a PCR chip array (designed by WCgene Biotech, Shanghai, China) to obtain the top 10 target genes of miR-96. We compared the expression of different genes targeted by miR-96 mimics against the negative control and listed the top ten decreased genes.
The forward and reverse primers were introduced into restriction endonuclease Hind III and Spe I. The mutated sequence of the binding site was designed, and the target sequence fragment was synthesized by Nanjing Genscript Biotechnology Co., Ltd.

Exosomes isolation from cell cultures and NTA analysis
Cortical cultures were obtained from either E18 or P3 rat (P3 days for astrocytes and E18 for neurons) as previously reported [19]. The cultured medium from four groups Q1~Q4 were collected and stored in 4℃ fridge.
Exosome Isolation: The supernatant of cell medium was taken from the 4°C freezer, balanced with PBS, and centrifuged at 1500g, 4°C for 30 minutes; the supernatant was taken and for a next 10000g, 4 ° C, centrifugation for 60 minutes; after this, the supernatant was centrifuged again at 12000g, 4 ° C for 30 minutes; Next, the centrifugated supernatant was carefully moved to a single-use ultracentrifugation, at 110000g for 60 minute at 4°C; carefully discard the supernatant after centrifugation, and the trace liquid at the bottom of the obtained centrifuge tube is exosomes. Carefully blow the bottom of the centrifuge tube with 30-100 ul volume of 1×PBS and inhale into 0.5-1.5 ml centrifuge tube, gently pipet with a pipette to completely dissolve.
For the Transmission electron microscopy morphology investigation, 10 ml of exosomes pellet was placed on formvar carbon-coated 200-mesh copper electron microscopy grids, and incubated for 5 min at room temperature, and then was subjected to standard 1% uranyl acetate staining for 1 min at RT. The grid was washed with three times of PBS and allowed to semi-dry at room temperature before observation in transmission electron microscope (Hitachi H7500 TEM, Japan).
Analysis of absolute size distribution and concentration of exosomes were determined using Nanoparticle tracking analysis (NTA). Exosomes were diluted in 1ml PBS and mixed well, then the diluted exosomes were injected into the NanoSight NS300 instrument (Malvern, UK), particles were automatically tracked and sized based on Brownian motion and the diffusion coefficient. Filtered PBS was used as controls. The NTA measurement conditions 25 frames per second, measurement time 60s. The detection threshold was similar in all the samples.
Three recordings were performed for each sample.

MiRNA quantitative RT-PCR (qRT-PCR) array
The miRNA qRT-PCR array experiments were conducted at Wcgene Biotechnology Corporation, Shanghai. Total RNAs, including miRNAs, were isolated from 100 μl of liquid sample, using a 1-step acidified phenol/chloroform purification protocol.
Synthesized exogenous RNAs were spiked into each sample to control for variability in the RNA extraction and purification procedures. The purified RNAs were polyadenylated through a poly(A) polymerase reaction and was then reversedtranscribed into cDNA. Individual miRNAs were quantified in real-time SYBR Green RT-qPCR reactions with the specific MystiCq miRNA qPCR Assay Primers (Sigma-Aldrich). The protocol of miRNA qRT-PCR array analysis was as described in detail on the website of Wcgene (http://www.wcgene.com).

Statistical analysis
All the data were processed by SPSS 21.0 statistical software (IBM Corp. Armonk, NY, USA). The measurement data were showed in the form of mean ± standard deviation. The data with normal distribution between pairwise groups were conducted by the t test and one-way analysis of variance (ANOVA) was used for comparison among various groups. The least significant difference (LSD) test was taken for pairwise comparison. P < 0.05 indicated the difference was statistically significant.

Modeling of acute seizure
After introduction of lithium chloride-pilocarpine in rats, we found that: after injection of pilocarpine for 5-30 min, acute seizure rats began to behave mechanical chewing, continuous nodding, unilateral forelimb clonic, and alternate clonus of forelimb. After that, there were bilateral forelimbs clonus, retreats, falls, and finally the rats began screaming and running, reaching the state of spasticity. 12-15 SD rats in the SE group, mimics NC group, siRNA-NC, miR-96 mimics and siRNA-RAC1 groups were successfully induced, and the successful rate was about 60-75%. SE indicated level III or above.

Upregulated miR-96 or silenced RAC1 reduces Racine score and prolongs latency of epilepsy in rats
The Racine scores of SD rats in each group within 3 hours were (Fig. 1A): facial clonus + rhythmic nodding + forelimb clonus + hind limb standing scored 4 points, facial clonus + rhythmic nodding + forelimb clonus + hind limb standing + falling scored 5 points, accompanied by hoarseness and galloping scored 6 points, spasticity scored 7 points. No significant difference in Racine scores was found among the SE, mimics NC and siRNA-NC groups (P > 0.05). Compared with the mimics NC group, the Racine score of the miR-96 mimics group was lower (P < 0.05); in contrast to the siRNA-NC group, the Racine score of the siRNA-RAC1 group was also lower (P < 0.05).
Considering the latency of each group of rats, we found (Fig. 1B) that the average latency period of the SE group was 50.15 ± 2.32 minutes. If taken SE group as the comparison group, there was no obvious difference in latency period compared with the mimics NC group (50.55 ± 2.12 min) and the siRNA-NC group (50.12 ± 2.42 min) (P > 0.05). In contrast to the mimics NC group, the latency period of the miR-96 mimics group (58.98 ± 2.45 min) was prolonged (P < 0.05); compared with the siRNA-NC group, the latency period of the siRNA-RAC1 group (57.32 ± 3.82 min) was stretched as well (P < 0.05).

Upregulated miR-96 or silenced RAC1 reduces the high amplitude spike wave and frequency of electroencephalogram (EEG)
The EEG of rats in each group was monitored. We found (Fig. 2) that in the normal group, all rats mainly showed α wave and β wave, the basic wave scattered in a small amount of θ wave, and no seizure wave was released. High amplitude spikes were observed in the SE, mimics NC and siRNA-NC groups. The amplitude and frequency of spikes in the miR-96 mimics group were dramatically lower than those in the mimics NC group, which in the siRNA-RAC1 group were obviously lower than those in the siRNA-NC group. ±3.12) was also obviously reduced (P < 0.05).

Upregulated miR-96 or silenced RAC1 inhibit the pathological changes of hippocampus in acute seizure rats
For HE staining, we found (Fig. 4A) that the hippocampal tissues of SD rats in all group were homogeneously stained, the neurons in the normal group were wellorganized, well-arranged, with clear layers and clear nuclei. In contrast to the normal group, the SE group showed uneven staining, severe degeneration, shrinkage and loss of a great number of neurons, extensive loose tissue edema, with a great number of vacuoles, disordered arrangement of remaining neurons and unclear hierarchy. The morphological structure of hippocampus was similar in the SE group, mimics NC group and siRNA-NC group. The hippocampal neuronal condition in the miR-96 mimics group and siRNA-RAC1 group was between the SE group and the normal group. Some neurons showed pyknosis, the vacuole area narrowed, the cell arrangement was slightly disordered, the layers were still identifiable, and most of the nuclei were clear.
The ultrastructure of hippocampal tissue of SD rats in each group was observed by an electron microscopy. We found (Fig. 4B) that the structure of hippocampal neurons in the normal group was basically normal with complete morphology, clear outline and boundary. There was no decrease in the number of neurons, no deformation or necrosis. In the SE group, the hippocampal structure was obviously damaged, with obvious cell swelling, incomplete cell morphology, blurred outline, unclear boundaries, disordered arrangement, cell deformation, necrosis and neuronal reduction. The morphological structure of hippocampal neurons in the SE group, mimics NC group and siRNA-NC group was similar. The hippocampal tissues of the miR-96 mimics group and siRNA-RAC1 group showed mild edema in cytoplasm and nucleus of neurons. The number of organelles decreased, the proliferation degree was lighter, the number of cell ruptures was less, the shape was more complete, the outline and boundary were clearer, the arrangement was more orderly, the deformation and necrosis of neurons were lighter, and the neuron did not decrease dramatically.
After Nissl's staining, we found (Fig. 4C): In the hippocampus of the normal group, most cell were survived. In the SE group, mimics NC group and siRNA-NC group, pyramidal cells were obviously disordered, and survival neurons decreased. The morphology and staining of hippocampal neurons in the miR-96 mimics and the siRNA-RAC1 groups were similar to those in the normal group. The number of hippocampal neurons decreased in SE group compared to control group, with a similar reduced trend in SE group with miRNA-96 NC and siRNA NC group (Fig. 4D).
In contrast to the mimics NC group, the number of Nissl positive cells in the miR-96 mimics group increased. In contrast to the siRNA-NC group, the number of Nissl positive cells in the siRNA-RAC1 group also increased (P < 0.05).

Upregulated miR-96 or silenced RAC1 reduces TUNEL-positive cells, inhibits
Bax and caspase-3, and promotes Bcl-2 in acute seizure rats TNUEL assay was used to detect apoptosis in hippocampus of SD rats in each group.
We found (Fig. 5A) that the number of TUNEL positive cells in the SE group increased in contrast to that in the normal group (P < 0.05). In contrast to the SE group, there was no obvious disparity in the number of TUNEL positive cells between the mimics NC group and siRNA-NC group (P > 0.05). In contrast to the mimics NC group, the number of TUNEL positive cells in the miR-96 mimics group decreased. In contrast to the siRNA-NC group, the number of TUNEL positive cells in the siRNA-RAC1 group decreased (P < 0.05).
The Bax, caspase-3 and Bcl-2 expression in the hippocampus of SD rats in all group was determined by RT-qPCR and western blot analysis. We found (Fig. 5B, C) that Bax and caspase-3 expression in the SE group was higher than that in the normal group, while the expression of Bcl-2 in the SE group was lower than that in the normal group (all P < 0.05). No obvious disparity was found in Bax, caspase-3, Bcl-2 expression among the SE, mimics NC and siRNA-NC groups (P > 0.05). Compared with the mimics NC group, Bax and caspase-3 expression levels decreased and Bcl-2 level increased in the miR-96 mimics group (P < 0.05); compared with the siRNA-NC group, Bax and caspase-3 expression levels decreased in the siRNA-RAC1 group, while Bcl-2 level increased (P < 0.05).

Upregulated miR-96 or silenced RAC1 inhibits NGF and promotes BDNF expression in acute seizure rats
NGF and BDNF expression in hippocampus of SD rats in all group were determined by immunohistochemical staining. We found (Fig. 6A, B) The expression of NGF and BDNF in hippocampus of SD rats in each group were determined further by RT-qPCR and western blot analysis. We found (Fig. 6C, D): NGF and BDNF expression increased in the SE group compared with normal group (P < 0.05). No dramatic difference in the expression of NGF and BDNF was found among the SE group, the mimics NC group and the siRNA-NC group (P > 0.05). In contrast to the mimics NC group, the expression of NGF in the miR-96 mimics group decreased, and BDNF expression increased (P < 0.05). In contrast to the siRNA-NC group, the NGF expression in the siRNA-RAC1 group was decreased, and the BDNF expression increased (P < 0.05).

Upregulated miR-96 or silenced RAC1 inhibits GFAP expression in acute seizure rats
The expression of GFAP in hippocampus of SD rats in all group were detected by immunohistochemical staining (Fig: 7A), RT-qPCR and western blot analysis (Fig. 7B,   C). We discovered that GFAP expression in hippocampus of the SE group was higher than that in the normal group (P < 0.05). Compared with the SE group, GFAP expression in hippocampus of rats in the mimics NC group and siRNA-NC group had no obvious difference (P > 0.05). In contrast to the mimics NC group, the expression of GFAP in hippocampus of the miR-96 mimics group decreased (P < 0.05).
Compared with the siRNA-NC group, GFAP expression in hippocampus of rats in the siRNA-RAC1 group also decreased (P < 0.05).

MDA content in acute seizure rats
The SOD activity and MDA content in hippocampus of rats in each group were tested. We found (Fig. 8A, B) that compared with the normal group, SOD activity in the SE group decreased and MDA content increased (P < 0.05). In contrast to the SE group, no major difference was found in SOD activity and MDA content between the mimics NC group and siRNA-NC group (P > 0.05). Compared with the mimics NC group, SOD activity increased, and MDA content decreased in the miR-96 mimics group (P < 0.05); compared with the siRNA-NC group, SOD activity also increased and MDA content decreased in the siRNA-RAC1 group (P < 0.05).

Upregulated miR-96 or silenced RAC1 inhibits TNF-α and IL-6 expression levels in acute seizure rats
TNF-α and IL-6 expression in hippocampal homogenate of rats in all group was detected by RT-qPCR and ELISA. We found (Fig. 9A, B) that TNF-α and IL-6 expression was low in the normal group. Compared with the normal group, TNF-α and IL-6 levels in the SE group were higher (P < 0.05). In contrast to the SE group, the levels of TNF-α and IL-6 in the mimics NC group and siRNA-NC group had no obvious difference (P > 0.05). Compared with the mimics NC group, the levels of TNF-α and IL-6 in the miR-96 mimics group decreased (P < 0.05). Compared with the siRNA-NC group, the levels of TNF-α and IL-6 in the siRNA-RAC1 group were also lower (P < 0.05).

Upregulated miR-96 or silenced RAC1 inhibits RhoA and ROCK expression levels in acute seizure rats
RT-qPCR was used to detect the expression of miR-96 in hippocampus of rats in each group. We found (Fig. 10A): In contrast to the normal group, miR-96 expression level in the SE group decreased, suggesting that the expression of miR-96 was down-regulated in SE rats (P < 0.05). Compared with the SE group, no major disparity in miR-96 expression between the mimics NC group and siRNA-NC group (P > 0.05). In contrast to the mimics NC group, miR-96 expression level of the mimics group increased (P < 0.05). Compared with the siRNA-NC group, no dramatic change in miR-96 expression level in the siRNA-RAC1 group (P > 0.05).
RT-qPCR and western blot analysis were applied to determine the expression of RAC1, RhoA and ROCK in hippocampus of each group. We found (Fig. 10B) that RAC1, RhoA, and ROCK expression levels were increased in the SE group compared with the normal group. In contrast to the SE group, mimics NC group and siRNA-NC group had no dramatic difference in ROCK, RAC1 and RhoA expression (P > 0.05). In contrast to the mimics NC group, RAC1, RhoA and ROCK expression levels in the miR-96 mimics group decreased (all P < 0.05). In contrast to the siRNA-NC group, the expression levels of RAC1, RhoA and ROCK in the siRNA-RAC1 group also decreased (all P < 0.05).

RAC1 is a direct target gene of miR-96
Target Scan was used to recognize the target sites of RAC1 binding to corresponding miR-96, and the sequence of 3'-UTR of RAC1 binding to miR-96 was shown in Fig. 11A. By using luciferase activity detection (Fig. 11B), we cotransfected the recombinant plasmids of miR-96 mimics and Wt-miR-96/RAC1 or Mut-miR-96/RAC1 in PC12 cells. The results indicated that the luciferase activity of Mut-miR-96/RAC1 was not dramatically affected by miR-96 mimics, but the luciferase activity of Wt-miR-96/RAC1 was obviously decreased (P < 0.05). We also confirmed the up-regulation of miR-96 and down-regulation of Rac1 after miR-96 mimic or Rac1 siRNA treating in primary cultured neurons (Fig. 11C). Based on the target prediction from Targetscan, we did a PCR array for selected targets of miR-96. Then, we analyzed their expression based on the weight and selected top 10 genes shown in the bubble figure (Fig. 11D). Next, we confirmed the dosedependent relationship between miR-96 mimic and relative expression of Rac1. With the increased concentration of miR-96, the Rac1 expression decreased gradually with a linear relation (R square=0.6108 and P < 0.05).

Decreased expression of miR96 in exosomes released from excitotoxic neurons
To investigate the mechanism that downregulation of miR-96 in epilepsy, we applied a customized rat miRNA chipset to explore the miRNA alterations in exosomes from excitotoxic neurons. We found 57 miRNAs increased in exosomes from excitotoxic neurons treated with Kainic acid, and 47 miRNAs decreased their expression when co-cultured with astrocytes, while 41 had this increased-decreased pattern as shown in Figure 12. In this case, we looked at the expression of miR96, which was decreased in KA treated neurons and the co-cultured astrocytes could increase its expression.

Discussion
About 20%-40% of patients with epilepsy are refractory to medical treatment, presurgical evaluation for these patients including detailed history and examination, video electroencephalography (EEG) telemetry, advanced epilepsy protocol magnetic resonance imaging (MRI) researches, and neuropsychological and psychiatric evaluation [20]. The most common type of epilepsy is temporal lobe epilepsy (TLE), neuropsychological impairments in adult patients with TLE include deficits in a wide range of cognitive dysfunction and psychological disorders [21]. In the study, we determined to focus on the effect of miR-96 in acute seizure rats. We found that miR-96 alleviated the neurological damage in these rats by downregulating RAC1 and inhibiting the activation of RhoA/ROCK signaling pathway.
First, we learned that miR-96 was down-regulated in SE rats, and up-regulation of miR-96 inhibited the pathological changes of hippocampus in acute seizure rats.
Small noncoding RNAs, namely miRNAs, exert their functions in epileptogenesis, with potential contributions by acting as valuable biomarkers and targets for epilepsy therapy [22]. Consistent with our study that autophagy-related miR-96 was down-regulated in the hippocampus of SE rats, and SE-induced brain injury was attenuated by up-regulating miR-96 [23]. Also, it has been proved miR-96 was down-regulated in pancreatic cancer [24], and over-expression of miR-96 in pancreatic cancer inhibited cell proliferation, migration and invasion [25]. In addition, we found that RAC1 was upregulated in SE rats. Also, a recent study has shown increased expression of RAC1 in epilepsy patients and animal models [15].
Still, RAC1 expression is also up-regulated in testicular, gastric, breast and oral squamous cell carcinoma and plays a significant role in cell proliferation and tumor survival in vitro [ 26]. Furthermore, RAC1 has been found to be activated during muscle contraction in murine and human skeletal muscle [27], and it owns the capacity of stimulating actin cytoskeleton reorganization [28]. Additionally, we found that RAC1 was a direct target gene of miR-96 and it remained at the top of target genes after miR-96 mimics treatment. Evidence has suggested that RAC1 was co-regulated by miR-182 and miR-96 in the mouse retina [29]. Furthermore, we also found that upregulation of miR-96 could downregulate RAC1 and inhibit the activation of RhoA/ROCK signaling pathway. Inhibition of RAC1 is suggested to contribute to the Rho/ROCK-mediated maintenance of Cdc42-dependent protrusion [30].
BrdU often incorporates into denovo-synthesized DNA as the replacement for thymidine and hence permanently labels proliferation and daughter cells until it is diluted out through several rounds of cell division [31]. epilepsy, which could possibly delay or even prevent epileptogenesis [38].
As reported, antioxidant therapies targeting oxidative stress have received considerable attention in epilepsy treatment [39]. PTZ-induced reductions in total SOD activity and lipid antioxidant (a-tocopherol) content were observed in rat brain homogenates [40]. Meanwhile, the levels of MDA in the epilepsy patient were found to be higher compared to those of the control group [41]. These findings were consistent in our studies showing that SOD activity decreased, and MDA expression increased in the hippocampus in epilepsy. And both miR-96 mimics and RAC1 knockdown could reverse these pathologies.
At last, several studies have reported the miRNA changes in epilepsy, and we did an exosomal miRNAs chip-array from cell culture mediums. The exosomes released from these cells were confirmed by the size with NTA analysis and shape in EM. Importantly, we found the decreased expression of miR-96 in excitotoxic neurons treated with KA and this was prevented in cocultured excitotoxic neurons with astrocytes. Meanwhile, we also found the increased expression of exosomal miR-103 in KA treated neurons and this could be reversed by cocultured astrocytes as well, which is consistent with our previous study showing miR-103 is involved in the pathology of epilepsy [42].
However, in our studies, there are several limitations need to be addressed. One is the rescue study for the association between of miR-96 and Rac1 and their roles in epilepsy-related neurodegeneration. Based on the Target scan website, we found rat miR-96 has a target of 967 transcripts with conserved sites, containing a total of 1085 conserved sites and 251 poorly conserved sites. In future studies, we will design a series of rescue study to investigate the exact targets of miR-96. The other is the cell-specific changes in epilepsy. We found acute seizure rats have increased astrocytic marker GFAP and inflammatory marker (TNF-α and IL-6) in hippocampal homogenate and miR-96 mimics could reduce these pathologies. However, we did not do a further flowcytometry to separate these changes to individually compare the results in neurons, astrocytes and even microglias. It would be promising to compare these pathological markers in a single-cell profiling style. Last but not least, since the early publications [43], researchers have used the pilocarpine model to address acute seizure mechanisms and variables associated with SE attenuation, in addition to mechanisms that relate to the development and profile of chronic epilepsy in the post-SE period. The epilepsy after brain insults have been divided into two stages: acute and chronic stage. In our study, we were focusing on the pathology in acute stage which might be involved in the latency period and chronic epileptic stage.

Conclusion
In conclusion, our study revealed that upregulation of miR-96 could down-regulate RAC1 gene and inhibit the activation of RhoA/ROCK signaling pathway, thus alleviate the neurological damage in acute seizure rats. The further investigation of the cellspecific mechanism should be more scrupulously and profoundly performed with a larger cohort to provide a promising clinical application in treatment for patients with epilepsy.
ZP & CW conceived, designed the experiments and performed cell cultures. WZH and JW ran the animal study and molecular tests. CW & ZP did the cell culture and exosome isolation. RDB, & ZP analyzed the data and wrote the manuscript. All authors read and approved the final manuscript.

Competing interests
The authors declare that they have no competing interests.

Figure 1
Racine score is reduced and latency of epilepsy is prolonged by upregulated miR-96 or decre Figure 2 The amplitude of spike wave and frequency of EEG is reduced by upregulated miR-96 or decr The pathological changes of hippocampus in acute seizure rats are recovered by upregulated The expression of GFAP is inhibited by upregulated miR-96 or decreased RAC1. A: Expression  TNF-α and IL-6 expression levels are inhibited by upregulated miR-96 or decreased RAC1. A: Figure 10 RhoA and ROCK expression levels are inhibited by upregulated miR-96 or decreased RAC1. A: Figure 12 The heat map demonstrates the miRNA alterations in exosomes from primary cultured neuro