HDAC4 gene silencing alleviates epilepsy by inhibition of GABA in a rat model

Figures & data

Table 1 The primer sequences for reverse transcription quantitative PCR

Gene	Primer	Sequence	Tm (°C)
GABAARα1	Upstream	AGCCGAATGCCCCATGCACT	60
	Downstream	CAACCACTGAGCGGGCTGGC
GABAARα4	Upstream	5′-CCTCACGATGACCACCCTAA-3′	60
	Downstream	5′-GCGATGCGGCAGACGAAA-3′
GAD65	Upstream	TCGAGCCGAAGCAGCTTGCCC	50
	Downstream	GCCCGTGAACTTTTGGGCCAC
GAT-1 mRNA	Upstream	5′-TGCAAACACGTACGCACATAGAA-3′	65
	Downstream	5′-AGATGCCTCAGCCACACCAC-3′
GAT-3 mRNA	Upstream	5′-CGGTCACTGGAACAACAAGGTG-3′	65
	Downstream	5′-AACACCACGTAAGGAATCAGGAATG-3′
β-actin	Upstream	GTGGGGCGCCCCAGGCACCA	50
	Downstream	CTTCCTTAATGTCACGCACGATTTC

Abbreviation: Tm, temperature.

Figure 1 The relative mRNA levels of GABAARα1 and GABAARα4 increased and levels of GAD65, GAT-1, and GAT-3 decreased in epilepsy rats treated with si-HDAC4.

Notes: (A) The mRNA level of GABAARα1 detected by RT-qPCR; (B) the mRNA level of GABAARα4 detected by RT-qPCR; (C) the mRNA level of GAD65 detected by RT-qPCR; (D) the mRNA level of GAT-1 detected by RT-qPCR; (E) the mRNA level of GAT-3 detected by RT-qPCR; (F and G) the silencing efficacy of si-HDAC4 detected by Western blot analysis. *P<0.05 vs the control group at the same time point; ^#P<0.05 vs the epilepsy group at the same time point; P<0.05 vs the first day of administration in the same group.
Abbreviation: RT-qPCR, reverse transcription quantitative PCR.

Figure 2 si-HDAC4 elevates the number of positive cells of GABAARα1 (A) and GABAARα4 (B), while reduces that of GAD65 (C), GAT-1 (D), and GAT-3 (E) of the hippocampus tissues of epilepsy rats after treatment with si-HDAC4.

Notes: *P<0.05 vs the control group at the same time point; ^#P<0.05 vs the epilepsy group at the same time point; ^▲P<0.05 vs the first day of administration in the same group.

Figure 3 The protein levels of GABAARα1 (A) and GABAARα4 (B) enhanced, while that of GAD65 (C), GAT-1 (D), and GAT-3 (E) reduced in the hippocampus tissues of epilepsy rats after treatment with si-HDAC4.

Notes: *P<0.05 vs the control group at the same time point; ^#P<0.05 vs the epilepsy group at the same time point; ^▲P<0.05 vs the first day of administration in the same group.

Figure 4 si-HDAC4 inhibits the occurrence of epilepsy using electroencephalogram.

Table 2 Comparisons of rats’ behavior observation among three groups

Time	Group	Latency (s)	Percentage of incidence (≥grade 3) (%)	Seizure duration (h)	Percentage of incidence (≥ten times) (%)
Day 1	si-HDAC4 group	18.32±1.88	90	1.55±0.17	90
	Epilepsy group	21.63±2.17	90	1.89±0.17	90
	Control group	–	–	–	–
Day 14	si-HDAC4 group	16.29±1.64	80	1.07±0.14	90
	Epilepsy group	19.47±1.95	90	1.46±0.15	90
	Control group	–	–	–	–
Day 30	si-HDAC4 group	7.65±0.77*,#	50*,#	0.21±0.07*,#	40*,#
	Epilepsy group	14.37±1.34	80	0.84±0.08	80
	Control group	–	–	–	–

Notes:

* P<0.05 vs the epilepsy group at the same time point;

# P<0.05 vs the first day of administration in the same group; n=10. The data were presented as mean ± SD.

Figure 5 Silence of HDAC4 promotes cognitive function.

Notes: (A) Escape latency of rats in each group during localized navigation experiment; (B) residence time of rats in the target quadrant of the original platform after removal of the platform; *P<0.05 vs the control group at the same time point; ^#P<0.05 vs the epilepsy group at the same time point; ^▲P<0.05 vs the first day of administration in the same group.