LncRNA Snhg5 Attenuates Status Epilepticus Induced Inflammation through Regulating NF-κΒ Signaling Pathway

Abstract

Status epilepticus (SE) induced inflammation plays an important role in the pathogenesis of SE. Long non-coding RNA small nucleolar RNA host gene 5 (lncRNA Snhg5) has been reported in various inflammatory diseases. However, the mechanism of Snhg5 regulated inflammation in SE remains unclear. Therefore, this study aimed to clarify the role and mechanism of Snhg5 in SE-induced inflammation in vitro and vivo. In vitro, lipopolysaccharide (LPS)-induced inflammation in microglia was used to mimic the inflammation after SE. In vivo, SE model was induced by lithium chloride and pilocarpine. The level of Snhg5, p65, p-p65, p-inhibitor of kappaB (IκB)α, IκBα and inflammatory factors (tumor necrosis factor (TNF)-α, interleukin (IL)-1β) were measured via quantitative real-time PCR or Western blot. The Nissl stain and immunohistochemical stain were performed to observe hippocampal damage and microglia proliferation. The results showed Snhg5 was up-regulated in the rat and microglia. Knockdown of Snhg5 inhibited LPS-induced inflammation and relative expression of p-65/p65, p-IκBα/IκBα. Moreover, down-regulation of Snhg5 attenuated SE-induced inflammation and reduced the number of microglia in hippocampus. These findings indicated that Snhg5 modulates the inflammation via nuclear factor-kappaB (NF-κB) signaling pathway in SE rats.

INTRODUCTION

Status epilepticus (SE) is a common neurological emergency with high morbidity and mortality. It is characterized by continual seizure activity that can vary widely in the intensity of convulsions.^1,2) Sustained seizures can cause extensive brain damage and even death.³⁾ Various neurological diseases, which include brain trauma, central nervous system infection, brain tumors, stroke, and irregular use of anti-epileptic drug (AED) can cause SE.^4,5) Over the past decade, many clinical and animal studies have focused on understanding the pathophysiology, treatment, and long-term complications of SE. A large number of studies have confirmed that SE causes a rapid and intense inflammatory cascade in the brain, which activated of microglia and released of inflammatory factors.^6,7) Inflammatory response after SE participates in the brain damage, especially in the hippocampus is vulnerable to damage.^8,9) A large number of studies have confirmed that the nuclear factor-kappaB (NF-κB) signaling pathway, as the core junction of multiple inflammatory signaling pathway, plays a vital role in SE-induced inflammation.^10,11) Currently studies have shown that inhibited the NF-κB signaling pathway can reduce the inflammatory response in the brain of SE rats and attenuate corresponding damage.^12,13) However, the exogenous inhibitors of NF-κB signaling pathway were limited clinical application for their toxicity and non-specificity.^14,15) Therefore, seeking specific molecules that endogenously regulated NF-κB signaling pathway may help solve this problem.

Long non-coding RNA (lncRNA) is a non-coding RNA with length of more than 200 nucleotides.¹⁶⁾ It is involved in many important regulatory processes in the cell, mainly from epigenetics, transcription and post-transcription three different levels to achieve gene regulation.¹⁷⁾ Many studies have shown that the expression of lncRNA small nucleolar RNA host gene 5 (Snhg5) was decreased in various inflammatory diseases such as ankylosing spondylitis, osteoarthritis and sepsis-related myocarditis.^18–21) However, there is no findings reveal the role of lncRNA Snhg5 acting on inflammatory response in SE. Therefore, in this study, we aimed to confirm whether lncRNA Snhg5 regulated the SE-induced inflammation through NF-κB signaling pathway in vitro and vivo.

In this study, we hypothesized that lncRNA might involve in regulating the inflammation by NF-κB signaling pathway in status epilepticus. Considering this, a status epilepticus rat model and lipopolysaccharide (LPS)-induced microglia cell were separately constructed to explore the mechanism of lncRNA Snhg5. According to these investigations, we hope to provide some new insights and theory basic information for understanding and intervention of status epilepticus.

MATERIALS AND METHODS

Cell Culture

Rat microglia was obtained from ScienCell (U.S.A.) and were cultured in Dulbecco’s modified Eagle’s medium (DMEM), a high glucose medium with 10% fetal bovine serum (FBS). The rat microglia were supported in a humid mixed atmosphere of 5% CO₂ and 95% air at 37 °C. When the cell coverage in the culture dish reached 65%, it was treated with LPS (10 µg/ mL). RNA and protein were extracted 48 h after treatment with the reagent.

Cell Transfection

To knockdown Snhg5, small interfering RNA (siRNA)-Snhg5 (si-Snhg5) were designed and synthesized by GenePharma (Shanghai, China). A scramble siRNA was used as negative control (si-NC). The related sequences were as follows: sense 5′-GCU UGG CUU CUA UUC U-3′; antisense 5′-AAA GGA UUC AGA AUA GAA GC-3′; si-NC: sense 5′-UUCUCCG AAC GUGUCAC G-3′; antisense 5′-ACG UGA CAC GUU-3′. Rat microglia were transfected with siRNAs using LipofectamineTM 2000 (Invitrogen) according to the manufacturer's instructions. Forty-eight hours after transfection, cells were harvested for real-time (RT)-PCR analysis to examine the knockdown efficiency.

Stereotactic Intrahippocampal Injection

Male Sprague-Dawley (SD) rats were randomly divided into three groups: control group, SH NC group and snhg5 group; Adeno associated virus 9 (AAV9) was injected into hippocampus of empty vector group and knockdown group. Aav9-sh-nc and aav9-sh-snhg5 viruses were separately packaged and thawed on ice. Rats were anesthetized by intraperitoneal injection of 10% chloral hydrate. Cut off the hair on the top of the rat’s head, cut off the scalp, peel off the subcutaneous tissue, expose the exposed seam clearly, mark the anterior fontanel and posterior fontanel gently with a pencil, find the position of hippocampus according to the stereotactic atlas of rat brain (the position used in this experiment is: LV AP = −5.2 mm, r = 5.0 mm, H = 4.8 mm), drill the hole, carefully place the micro syringe, and record the height. Each rat was injected with 2 sides of hippocampus, 2 UL/side. The injection was done slowly and evenly, every 2 UL of AAV9 was injected within 10 min to make it fully absorbed in the tissue. After 10 min, the injector was slowly pulled out, the wound was disinfected and sutured, and the rats were gently removed and put into the incubator until they woke up.

Animal Model of Status Epilepticus

All animal experiments were performed in compliance with institutional guideline and had been approved by the Ethics Committee of Jinshan Hospital of Fudan University (Certificate No. 20141601) and was operated according to the recommendations of the Chinese Academy of Medical Sciences Animal Experiment Guide. Male Wistar rats (160–180 g) were purchased from the Animal Center of Fudan University (Shanghai, China). The rats were housed in standard animal housing. Rats were housed at a controlled temperature (21–25 °C) and 12 h cycles of light and dark; they were free to move and feed in the cage. Twenty-four hours before pilocarpine injection, the rats were intraperitoneally injected with lithium chloride (127.3 mg/kg in 0.9% saline). Thirty minutes before pilocarpine injection, scopolamine (1 mg/kg in 0.9% saline) was intraperitoneally injected to reduce peripheral side effects. SE groups were intraperitoneally injected with 30 mg/kg pilocarpine, and normal saline was injected into the control group. Behavioral seizures were scored using the modified Racine scale.²²⁾ If the rats had seizures that did not reach grade IV–V, the rats were injected with 10 mg/kg pilocarpine every 30 min until the seizures reached grade IV–V. No rats received pilocarpine in excess of 60 mg/kg. Diazepam (10 mg/kg) was administered after 60 min to stop the seizures.

Western Blot (WB)

Rat hippocampus tissues and microglia cell proteins were extracted using 1% phenylmethylsulfonyl fluoride (PMSF). The protein (15 µg) was separated by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis and transferred to a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was blocked with 5% skim milk for 1 h at room temperature. The membrane was then washed thrice with TBST for 15 min each time and promote with the primary antibody (p-p65, p65, p-inhibitor of kappaB (IκB)α, IκBα, interleukin (IL)-1β and tumor necrosis factor (TNF)-α antibody) overnight at 4 °C. The membrane was washed with TBST and promote with horseradish peroxidase (HRP)-conjugated Affinipure goat anti-rabbit immunoglobulin G (IgG) (H + L) for 1 h at room temperature. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was the internal control for quantitative analysis of the relative expres-sion levels of the proteins.

RT-PCR

Rat hippocampus tissue or microglia were treated with Trizol; the tissues were separated thoroughly with a homogenizer, and the mRNA was extracted using chloroform and isoamyl alcohol. The RNA sediment was dissolved in diethylpyrocarbonate (DEPC) water (TaKaRa, Shanghai, China), quantified using a spectrophotometer, and reverse transcribed into cDNA by using Prime Script reagent kits (TaKaRa). PCR was performed using SYBR Green PCR kits (TaKaRa) by a Step One Real-Time PCR System. Relative RNA Expression was normalized to GAPDH and calculated. The primers used in the study are listed as follows: Snhg5 (5ʹ-GCC CAA TTC CAC CAA GGA AG-3ʹ) and (5ʹ-GCT GCU UUC ATT CTA TA-3ʹ) GAPDH (5ʹ-TTC GCC ATG GAT ATC-3ʹ and 5ʹ-TAG GAG TCC TTC TAT AC-3ʹ), IL-β (5ʹ-ATC TCA CAG CAG CAT CTC GAC AAG-3ʹ and 5ʹ-CAC ACT AGC AGG TCA TCC-3ʹ), CD11b (5ʹ-GCA TCA GTA GCC AGT A-3ʹ) and 5ʹ-CCC CGT CCA TTG TGA GAT G-3ʹ).

Nissl Stain

Sacrificed the rats in each group according to the Chinese Medical Sciences Animal Experiment Guide. After the rats recovered for a week, we removed the rat brain tissues on ice. The fresh rat brain tissue was fixed in 4% formalin solution and dehydrate. Sliced into 5 μm thick sections and put into Cresyl violet Stain, place the dyeing tank in incubator (56 °C) for 1 h, and heat with alcohol lamp (10 min). Put into Nissl Differentiation for 1 to 3 min and observed under a microscope until the background was nearly colorless. Dehydrated sections rapidly with Anhydrous ethanol and sealed with a neutral gum. Observe pyramidal neurons in hippocampus under microscope within a fixed area (400×) of the CA1 and CA3, as previous studies described.²³⁾ The images were processed by ImageJ software (U.S.A.), which calculated the number of neurons per 0.1 m² in CA1 and CA3 regions of stained hippocampus. The average number of positive cells was calculated: 3 rats in each group and one slice in each region.

Immunohistochemistry

After the rats recovered for a week, we extracted the brain tissue on ice. Follow the immunohistochemical staining kit instructions. The fresh brain tissue sections (5 µm) were heated at 60 °C. Then dewaxed and permeabilized the sections. Washing 3 times with PBS, goat serum was added for 20 min to block non-specific antigen binding. Rabbit anti-Iba1 primary antibody was added and incubated overnight at 4 °C and the sections were incubated with Goat Anti-Rabbit IgG (H + L) for 60 min. After hematoxylin staining, rinse with 0.1% HCl-ethanol, then dehydrate in graded ethanol and fix in resin. Magnify 400 times with a microscope, select CA1 and CA3 region of hippocampus to observe the number of microglia, as previous studies described.²⁴⁾ Photos are processed by ImageJ software (U.S.A.), which count the number of Iba1stained microglia in per 0.1 m² of CA1 and CA3 region of hippocampus. The average value was calculated for the number of positive cells, 3rats each group, one slice per rats.

Statistical Analysis

All data were analyzed using GraphPad Prism 8. The results are expressed as mean ± standard deviation (S.D.). Comparisons between groups were performed using one-way ANOVA and t-test. Pearson correlation analysis was used for two-variable correlation analysis. The p < 0.05 was considered to be statistically significant. All experiments were repeated at least three times.

RESULTS

Knockdown of lncRNA Snhg5 Attenuated LPS-Induced Inflammation through Regulating the NF-κB Signaling Pathway in Microglia

In vitro microglia culture is powerful tools to study specific molecular pathways involved in inflammation. Thus, we used LPS-induced inflammation in microglia partly mimic the SE-induced neuroinflammation to explore the effect of Snhg5 on inflammation. As shown in Fig. 1a, it was demonstrated that Snhg5 expression were greatly increased in the LPS dose of 10 µg/mL. We thus chose a concentration of 10 µg/mL treated the microglia. And in the Fig. 1b, the Snhg5 expression was the most markedly increased at 48 h after LPS-treated. On the basis of this result, we extracted the protein and RNA 48 h after LPS-stimulation. In Fig. 1c, all three siRNAs could knock down the snhg5 in varying degrees, among which the si-1 was the most obvious. The Snhg5 were successfully knockdown in LPS-induced microglia (differential fold >1,5) in Fig. 1d. As seen in Fig. 1e, the M1 microglia markers (CD86 and inducible nitric oxide synthase (iNOS)) were decrease in LPS + si-Snhg5 group compared to LPS + si-NC group (# p < 0.05). By contrast, the M2 microglia markers (CD206 and Arg1) increased in LPS + si-Snhg5 group in Fig. 1f (# p < 0.05vs. LPS + si-NC). Moreover, the LPS treated increased the protein level of inflammatory factors TNF-α and IL-1β (* p < 0.05 vs. control). And the LPS + si-Snhg5 group compared with the LPS + si-NC group, the relative expression of TNF-α and IL-1β were decreased (# p < 0.05) in Fig. 1g. The expression of p-65 and p-IκΒα were increased in LPS-treated group in Fig. 1h-i. And p-65 and p-IκΒα were decreased in LPS + si-Snhg5 group compared to LPS + si-NC group (#p < 0.05). The results in Fig. 1 suggested that knockdown of Snhg5 inhibited the M1/M2 polarization of microglia and attenuated LPS-induced inflammation by regulating NF-κB signaling pathway.

Fig. 1. Snhg5 Inhibited the Inflammation in LPS-Treated Microglia

(a) The expression of Snhg5 in microglia with different LPS doses. (b) The time course of Snhg5 expression in microglia. (c) The si-RNA mediated knockdown of Snhg5 in microglia. (d) The relative expression of lncRNA Snhg5 in microglia. (e) The mRNA of M1-associated markers CD86 and iNOS in microglia cells. (f) The mRNA of M2-associated markers CD206 and Arg1 in microglia. (g) The protein expression of TNF-α and IL-1β in microglia. (h) The protein expression of p65, p-p65, p-IκBα and IκBα in cells. (i) The relative expression of p-p65 and p-IκBα in microglia. The relative expression of protein and mRNA were normalized to GAPDH expression. Values are presented at the mean ± S.D. (* p < 0.05, ** p < 0.01 vs. Control; # p < 0.05, ## p < 0.01 vs. LPS + si-NC group).

High Expression of Snhg5 Was Consistent with the Inflammation in SE Rats

As shown in Fig. 2a that the expression of Snhg5 increased gradually 6h after SE compared to the normal group (* p < 0.05), and reached the highest level on 1w after SE (* p < 0.05 vs. Normal). The expression of NF-κB signaling pathway related proteins (p-p65, p65, p-IκBα, IκBα, IL-1β and TNF-α) were shown in Fig. 2b. From Fig. 2c the statistical analysis revealed the relative expression of p-p65 (p-65/p65) was increased at 1w after SE (* p < 0.05 vs. Normal). The result of Fig. 2e showed that the relative expression of p-IκBα (p-IκBα/IκBα) was increased mostly at 24h after SE (* p < 0.05 vs. Normal). Figures 2d and f showed that the expression of inflammatory factors (IL-1β and TNF-α) were both increased obviously at 48 h after SE in hippocampus tissues (* p < 0.05 vs. Normal). A positively correlation was found between Snhg5 expression and p-p65/p65 in Fig. 2g (r = 0.6080, p < 0.05), which indicated Snhg5 expression is related to NF-κB signaling pathway. The results from Fig. 2h suggested Snhg5 expression was also positively correlated with inflammatory factors TNF-α (r = 0.5014, p < 0.05). These results demonstrated that high expression of Snhg5 in SE rats may be related to SE-induced inflammation.

Fig. 2. The Expression of Snhg5 Was Consistent with NF-κB Signaling Pathway Mediated Inflammation in Rats Hippocampus

(a) The expression of lncRNA Snhg5 in rat hippocampus after SE (n = 6–7). (b) The protein p-p65, p65, p-ΙκΒα, ΙκΒα, IL-1β and TNF-α in hippocampus. (c–f) The relative protein levels of p-p65/p65, p-ΙκΒα/ΙκΒα, IL-1β and TNF-α in hippocampus. (g) Pearson correlation analysis of lncRNA Snhg5 and p-p65/p65 in hippocampus of SE 2w group. (h) Pearson correlation analysis of lncRNA Snhg5 and TNF-α mRNA in hippocampus of SE 1w group. The relative expression of protein and RNA were normalized to GAPDH expression. Values are presented as the mean ± S.D. (n = 5–6, * p < 0.05 vs. Normal).

Knockdown of Snhg5 Alleviated Hippocampal Neuron Injury in SE Rats

To further verify the results in vitro, we injected adeno-associated virus 9 (AAV9) into the hippocampus of rats to explored the effect of lncRNA Snhg5 on inflammation in vivo. As shown in Figs. 3a and b, compared with the normal group the number neurons in CA1 area of SE group were decreased (* p < 0.05). And the number of neurons in CA3 area was significantly reduced (* p < 0.05 vs. normal). Furthermore, in SE group the arrangement of neurons in CA3 was sparser than the normal group. And in Snhg5 knockdown group (SE + sh-snhg5) the number of hippocampal neurons were increased compared to the SE + sh-NC group (* p < 0.05). In addition, compared with SE + sh-NC group, the number of CA1 cells in SE group decreased slightly. These results indicated that the hippocampal neurons of SE group were damaged and Knockdown of lncRNA Snhg5 decreased the damage of CA3 hippocampal neurons.

Fig. 3. Effect of Snhg5 Knockdown on Hippocampal Neurons in SE Rats

(a)The Nissl stain of rat hippocampus. (b) The number of Nissl stain neurons. The general morphology of the hippocampus in rat magnification 40×. The CA1 and CA3 regions of hippocampus are marked in squares. CA1 and CA3 region magnification 400×. Positively stained cells are marked with black arrows. The cell density per square millimeter was measured by ImageJ software. (* p < 0.05 vs. Control; ** p < 0.01 vs. Control by one-way ANOVA (n = 3).

Knockdown of Snhg5 Inhibited the Proliferation of Hippocampal Microglia in SE Rats

Iba1 is a specific marker of microglia, thus in present study we used Iba1 to label microglia and investigated the effect of lncRNA Snhg5 on microglia proliferation. As shown in Figs. 4a and b, compared with the normal group, the number of microglia in CA1 and CA3 area of hippocampus were increased in SE group (* p < 0.05). In the knockdown group (SE + sh-snhg5), the number of microglia in both CA1 and CA3 were significantly decreased (* p < 0.05 vs. SE + sh-NC). The results showed that the proliferation of microglia in hippocampus of rats was not obvious one week after SE. Unexpectedly, the number of microglia in SE + sh-NC group were increased significantly both in CA1 and CA3 area of hippocampus (* p < 0.05 vs. SE). These results showed that the proliferation of microglia in hippocampus of SE rats was obvious. The increased number of microglia in hippocampus induced by exogenous AAV injection was significantly higher than SE. Knockdown of lncRNA Snhg5 could inhibit the proliferation of microglia in hippocampus of rats.

Fig. 4. Snhg5 Knockdown Inhibited the Proliferation of Microglia in the Hippocampus of SE Rats

(a) The immunohistochemistry stained of Iba1 in rat hippocampus. (b) The number of microglia. The general morphology of the hippocampus in rat magnification 40×. The CA1 and CA3 regions of hippocampus are marked in squares. CA1 and CA3 region magnification 400×. Positively stained cells are marked with black arrows. The cell density per square millimeter was measured by ImageJ software. (* p < 0.05 vs. Control; ** p < 0.01 vs. Control by one-way ANOVA (n = 3).

Knockdown of Snhg5 Reduced SE-Induced Inflammation through Regulating the NF-κB Signaling Pathway in Rats

As shown in Fig. 5a, the expression of Snhg5 was successfully knocked down in SE + sh-Snhg5 group (* p < 0.05 vs. SE + sh-NC) by AAV9. And in Figs. 5b and c, the expression of p-p65 and p-IκBα in SE group were significantly higher than those in control group (* p < 0.05). Compared with SE + sh-NC group, the expression of p-p65 and p-IκBα in SE +sh-Snhg5 group were decreased (#p < 0.05). These results suggest that down-regulation of lncRNA Snhg5 could inhibited the Phosphorylation of p65 and IκΒα in hippocampus of SE rats, which regulated the NF-κB signaling pathway. In addition, compared with the normal group, the expression of TNF-α and IL-1β in SE group was significantly higher than that in the normal group (* p < 0.05). The protein expression of inflammatory factors (TNF-α and IL-1β) were decreased in SE + sh-Snhg5 group (#p < 0.05 vs. SE + sh-NC). And these results were consistent with the results of cell verification in vitro. These results suggested that lncRNA Snhg5 knockdown inhibited inflammation by regulating NF-κB signaling pathway in SE rats.

Fig. 5. Snhg5 Attenuated the Inflammation in Hippocampus of SE Rats

(a) The expression of lncRNA Snhg5 in the hippocampus of rats. (b) The protein expression of NF-κB signaling pathway related proteins (p-65, p65, p-IκBα and IκBα) and Inflammatory factors (TNF-α, IL-1β). (c) The relative protein level of p-p65/p65, p-ΙκΒα/ΙκΒα, IL-1β and TNF-α in hippocampus. (* p < 0.05 vs. Control; ** p < 0.01 vs. Control; # p < 0.05 vs. SE + sh-NC; ## p < 0.01vs. SE + sh-NC).

DISCUSSION

In the past few decades, a strong relationship between inflammation and SE has been reported both in animal models and patients with SE.^25,26) Increasing evidence has noted that the inflammation during SE plays a decisive role in persistent seizure and the long-term sequelae.^13,27) And the activated microglia in the brain were considered to be the main cells involved in the inflammatory response during SE.^28,29) Therefore, in the current study we used lithium chloride and pilocarpine induced SE rat model and LPS-treated microglia to study the inflammation in vivo and vitro. The evidence of inflammation was detected in the hippocampus of SE rats, which consistent with those of other studies.^30,31) And there existed apparently inflammation in LPS-treated microglia to partly mimic the inflammation during SE in vitro. Based on these findings, early anti-inflammatory treatment of SE may represent a promising strategy to improve the brain damage caused by seizure, as previous research supported.^32,33) However, several anti-inflammation drugs were failed to terminate the recurrent seizure for non-specificity. Therefore, it is necessary to find drugs that specifically regulate inflammation in SE.

There were several inflammatory signaling pathway activated following prolonged seizure, which mainly include NF-κB signaling pathway, mammalian target of rapamycin (mTOR) signaling pathway and mitogen-activated protein kinase (MAPK) signaling pathway.^34,35) Many studies have confirmed that the NF-κB signaling pathway plays a central role in inflammation during SE.^36,37) Hence, we detected the NF-κB signaling pathway related protein family (p65, p-p65 and p-IκBα) both in SE rats and LPS-treated microglia. We found that the level of p-p65/p65, p-IκBα/IκBα, IL-1β and TNF-α were increased in SE rats and LPS-treated microglia. Our findings further support the idea of the inflammation during SE was mediated through the NF-κB signaling pathway as prior studies reported.^34,38) In addition, results in vitro confirmed that LPS stimulated can activate the NF-κB signaling pathway, release the p-p65 enter the nucleus and combine with proinflammatory genes to initiate transcription of target genes, as previous findings.^39,40)

LncRNA as an efficiently endogenous molecule can regulate the inflammation through NF-κB signaling pathway in various diseased.^16,41) The latest research found that lncRNA Carlr induce the inflammation in macrophages through inhibiting the NF-κB signaling pathway in celiac patients.⁴²⁾ Similarly, another study showed that lncRNA NKILA promoted the inflammation in breast cancer through the NF-κB signaling pathway.^43,44) Additionally, the expression of lncRNA Snhg5 has been reported to change in many inflammatory diseases.⁴⁵⁾ Our study found that the expression of Snhg5 was increased in SE rats and LPS-treated microglia. Expectedly, the level of Snhg5 was up-regulated in SE rats and positively correlated with the inflammatory factor TNF-α. Subsequently, Snhg5 knockdown was performed both in vitro and vivo. Snhg5 knockdown caused a reduction in the levels of p-p65 and p-IκBα. Correspondingly, the level of inflammatory factors (TNF-α and IL-1β) were reduced in LPS-treated microglia and rat hippocampus. Further, Snhg5 knockdown was sufficient to inhibit the LPS-induced inflammation, which can be explained by the M1 microglia decreased and M2 microglia increased. These findings indicated Snhg5 may regulated the inflammation through NF-κB signaling pathway. Therefore, as an endogenous molecule with pro-inflammatory effect, lncRNA Snhg5 may become a new treatment for inflammation in SE.

There are, however, some deficiencies in the present study. We did not observe a marked change of the microglia number in 24–48 h of LPS-treated. We considered this based on the following reasons. In vivo, the Iba1 labeled hippocampus slices were taken after one week of SE, which was different from the time in vitro. Besides, the chemotaxis of microglia to sites of tissue damage is one of the reasons for the obvious changes of microglia in SE rat hippocampus.⁴⁶⁾ Furthermore, the precise mechanisms between Snhg5 and NF-κB signaling pathway still unclear. Such considerations warrant further studies. Therefore, in the future experiments, we should set to verify and refine these problems.

CONCLUSION

In conclusion, our study revealed that lncRNA Snhg5 modulated inflammation in SE rats via regulating NF-κΒ signaling pathway. Our findings indicated that anti-inflammatory effect of Snhg5 might provide a promising treatment for SE.

Acknowledgments

This research was funded by the National Natural Science Foundation of China, Grant No. 81971209.

Author Contributions

MW and YC conceived and designed the study. YX contributed to data analysis. MW performed the experiments and drafted the manuscript. YC and YS reviewed and edited the manuscript. All authors read and approved the final manuscript.

Conflict of Interest

The authors declare no conflict of interest.

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