LncRNA H19 contributes to hippocampal glial cell activation via JAK/STAT signaling in a rat model of temporal lobe epilepsy

Abstract

Background: Astrocyte and microglia activation are well-known features of temporal lobe epilepsy that may contribute to epileptogenesis. However, the mechanisms underlying glia activation are not well understood. Long non-coding RNA (lncRNA) H19 has diverse functions depending on physiological or pathological state, and its role in epilepsy is unknown. We previously demonstrated that H19 was significantly upregulated in the latent period of epilepsy and may be associated with cell proliferation and immune and inflammatory responses. We therefore speculated that H19 is involved in the hippocampal glial cell activation during epileptogenesis.

Methods: H19 was overexpressed or knocked down using an adeno-associated viral vector delivery system. A rat status epilepticus model was induced by intra-amygdala kainic acid injection. Astrocyte and microglia activation were assessed by immunofluorescence and western blot analyses. Expression of proinflammatory cytokines and components of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathways were evaluated with western blotting.

Results: H19 overexpression induced the activation of astrocytes and microglia and the release of proinflammatory cytokines (interleukin-1β and interleukin-6 and tumor necrosis factor-α) in the hippocampus, whereas H19 knockdown inhibited status epilepticus-induced glial cell activation. Moreover, H19 activated JAK/STAT signaling by promoting the expression of Stat3 and c-Myc, which is thought to be involved in astrocyte activation.

Conclusions: LncRNA H19 contributes to hippocampal glial cell activation via modulation of the JAK/STAT pathway and could be a therapeutic tool to prevent the development of epilepsy.

Keywords: Astrocytes; Inflammatory response; Microglia; Temporal lobe epilepsy; lncRNA H19.

Fig. 1

Astrocytes and microglia are activated in the hippocampus of epileptic rats. Representative fluorescence micrographs of GFAP and OX42 expression in the hippocampus of rats 30 days after SE. High-magnification images correspond to the labeled boxes in the left panels. Scale bar = 200 μm

Fig. 2

H19 is involved in astrocyte activation in the hippocampus of epileptic rats. a, b Top: experimental timeline. Middle: representative fluorescence micrographs of GFAP expression in the hippocampus of H19 overexpression (a) and H19 knockdown (b) rats with or without KA treatment for 7 days. Scale bar = 50 μm. Bottom panels show counts of cells in the CA3 regions of the hippocampus ipsilateral to the KA injection side (n = 3). c, d Western blot analysis of GFAP protein levels in the CA3 subfield of the hippocampus of H19 overexpression (c) and H19 knockdown (d) rats with or without KA treatment for 7 days (n = 3–4). Protein bands were quantified by densitometry and normalized to the level of GAPDH. Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. NC rats injected with empty AAV vectors, Scr rats injected with scrambled AAV vectors, ShRNA rats injected with AAV vectors containing short hairpin RNA targeting H19

Fig. 3

H19 is involved in the activation of microglia in the hippocampus of epileptic rats. a, b Top: experimental timeline. Middle: representative fluorescence micrographs of OX42 expression in the hippocampus of H19 overexpression (a) and H19 knockdown (b) rats with or without KA treatment for 7 days. Scale bar = 50 μm. Bottom panels show counts of cells in the CA3 regions of the hippocampus ipsilateral to the KA injection side (n = 3). c, d Western blot analysis of OX42 protein level in the CA3 subfield of the hippocampus of H19 overexpression (c) and H19 knockdown (d) rats with or without KA treatment for 7 days (n = 3–4). Protein bands were quantified by densitometry and normalized to the level of GAPDH. Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. NC rats injected with empty AAV vectors, Scr rats injected with scrambled AAV vectors, ShRNA rats injected with AAV vectors containing short hairpin RNA targeting H19

Fig. 4

H19 induces pro-inflammatory cytokine expression. a, b Protein levels of IL-1β and IL-6 and TNF-α in the CA3 subfield of the hippocampus of H19 overexpression (a) or H19 knockdown (b) rats with or without KA treatment for 7 days (n = 3–4). Protein bands were quantified by densitometry and normalized to the level of GAPDH. Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. NC rats injected with empty AAV vectors, Scr rats injected with scrambled AAV vectors, ShRNA rats injected with AAV vectors containing short hairpin RNA targeting H19

Fig. 5

H19 stimulates p-Stat3 and c-Myc expression. a, b Protein levels of p-Stat3 and c-Myc in the hippocampal tissue samples from patients with TLE (n = 4) (a) and the hippocampus of epileptic rats 7 days after KA injection (n = 3) (b), as determined by western blotting. c, d Quantification of p-Stat3 and c-Myc protein levels in the CA3 subfield of the hippocampus of H19 overexpression (c) and H19 knockdown (d) rats with or without KA treatment for 7 days (n = 3–4). Protein bands were quantified by densitometry and normalized to the level of GAPDH. Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. NC rats injected with empty AAV vectors, Scr rats injected with scrambled AAV vectors, ShRNA rats injected with AAV vectors containing short hairpin RNA targeting H19