Journal of Traditional Chinese Medicine ›› 2023, Vol. 43 ›› Issue (2): 286-294.DOI: 10.19852/j.cnki.jtcm.20220928.002
• Original articles • Previous Articles Next Articles
Efficacy and mechanisms of Dingxian pill (定痫丸) combined with valproic acid on pentylenetetrazol-induced chronic epilepsy in rats
QU Dongxiao1, GE Yiqin2, ZHUO Limin4, CHEN Liji1, XUE Yonghua1,3, CHENG Jiwei1,3, TAO Jie1,3, LI Guoyi1,3, ZHU Yudan1(
), XIAO Qian1()
- 1 Department of Neurology and Central Laboratory, Putuo Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200062, China
2 Department of Neurosurgery, Jiading Branch of Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 201803, China
3 Department of Neurology and Central Laboratory, Putuo Clinical Medical School, Anhui Medical University, Shanghai 200062, China
4 College of Sciences, Shanghai University, Shanghai 200442, China
-
Received:2022-01-29Accepted:2022-04-29Online:2023-04-15Published:2023-03-14 -
Contact:XIAO Qian, Department of Neurology, Putuo Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200062, China. emilyand208@126.com; ZHU Yudan, Department of Central Laboratory, Putuo Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200062, China. yudanzhu_putuo@foxmail.com. Telephone: +86-21-22233345 -
Supported by:National Natural Science Foundation of China: Molecular Mechanism Underlying the Intervention of Scorpion Extract BmK IT2 on Epileptogenesis via Voltage-gated Nav1.6 Channels in Hippocampal Neurons(81903995);Molecular Mechanism Underlying the Intervention of Scorpion Polypeptide MarTX on Temporal Lobe Epileptogenesis by Inhibiting Mechanosensitive Channel Piezo1(82074162);Youth Talent Promotion Project of China Association of Chinese Medicine(CACM-2019-QNRC2-C10);Project for Capacity Promotion of Putuo District Clinical Special Disease(Stroke, 2019tszb02);Science and Technology Innovation Project of Putuo District Health System: Molecular Mechanism of Aerobic Exercise Intervention on Post-epileptic Depression Through "Bone-brain Axis"(ptkwws202107);Antiepileptic Mechanism of Scorpion Active Extract BmK AS Regulating Nav1.6 Sodium Channel in Hippocampal Pyramidal Neurons(ptkwws201902);Evaluation of Microglial Lysosomal BK Channels as Molecular Targets for Clinical Treatment of Temporal Lobe Epilepsy(ptkwws201908);Molecular Mechanism of Sodium Channel SCN8A (Nav1.6) Mutation Mediating Epilepsy and Sodium Valproate Resistance in Children the Research Project of Putuo Hospital, Shanghai University of Traditional Chinese Medicine(2019308);Molecular Mechanism of Sodium Channel SCN8A (Nav1.6) Mutation Mediating Epilepsy and Sodium Valproate Resistance in Children the Research Project of Putuo Hospital, Shanghai University of Traditional Chinese Medicine(2019307)
Cite this article
QU Dongxiao, GE Yiqin, ZHUO Limin, CHEN Liji, XUE Yonghua, CHENG Jiwei, TAO Jie, LI Guoyi, ZHU Yudan, XIAO Qian. Efficacy and mechanisms of Dingxian pill (定痫丸) combined with valproic acid on pentylenetetrazol-induced chronic epilepsy in rats[J]. Journal of Traditional Chinese Medicine, 2023, 43(2): 286-294.
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Table 1 Effect of each group on the latency of seizures in rats induced by PTZ
| Animal group | n | Latency of seizures on the 1th day | Latency of seizures on the 7th day | Latency of seizures on the 14th day | Latency of seizures on the 21th day | Latency of seizures on the 28th day |
|---|---|---|---|---|---|---|
| CTRL | 10 | 207.0±8.3 | 117.9±13.0 | 168.5±23.0 | 126.9±17.1 | 177.6±21.2 |
| DXP | 12 | 199.8±6.0 | 156.4±29.3a | 242.2±34.2a | 389.2±37.0a | 420.7±24.3b |
| VPA | 8 | 202.1±5.0 | 195.6±27.9a | 297.4±31.1a | 295.1±35.0a | 395.4±27.2b |
| D+V | 8 | 207.1±6.3 | 347.7±49.5bc | 435.6±53.7bc | 510.4±74.8bc | 449.8±45.2b |
Table 1 Effect of each group on the latency of seizures in rats induced by PTZ
| Animal group | n | Latency of seizures on the 1th day | Latency of seizures on the 7th day | Latency of seizures on the 14th day | Latency of seizures on the 21th day | Latency of seizures on the 28th day |
|---|---|---|---|---|---|---|
| CTRL | 10 | 207.0±8.3 | 117.9±13.0 | 168.5±23.0 | 126.9±17.1 | 177.6±21.2 |
| DXP | 12 | 199.8±6.0 | 156.4±29.3a | 242.2±34.2a | 389.2±37.0a | 420.7±24.3b |
| VPA | 8 | 202.1±5.0 | 195.6±27.9a | 297.4±31.1a | 295.1±35.0a | 395.4±27.2b |
| D+V | 8 | 207.1±6.3 | 347.7±49.5bc | 435.6±53.7bc | 510.4±74.8bc | 449.8±45.2b |
Figure 1 Comparison of anticonvulsant effects among different groups on seizure-like behavior induced by PTZ A-E: the percentage of seizure stage before treatment about each group; F: EEG waveforms of each group after 28 d of treatment; F1: CTRL group; F2: DXP group, F3: VPA group; F4: D+V group; G: average power spectral density (PSD) of each group; H: the energy of each single waveform after rats’ treatment. CTRL group: PTZ model rats without treatment (n = 6); DXP group: PTZ model rats treated with Dingxian pill (2.4 g/kg) (n = 5); VPA group: PTZ model rats treated with Valproic acid (0.2 g/kg) (n = 5); D + V group: PTZ model rats treated with Dingxian pill (2.4 g/kg) combined with Valproic acid (0.2 g/kg) (n = 5). PTZ: pentylenetetrazol. EEG: electroencephalograms. DXP: Dingxian pill. VPA: valproic acid. PSD: average power spectral density. aP < 0.001, DXP group vs CTRL group; bP < 0.001, D + V group vs CTRL group; cP < 0.05: VPA group vs D + V group; dP < 0.05, VPA group vs CTRL group; eP < 0.05, D+V group vs CTRL group; fP < 0.01, D + V group vs CTRL group. Differences among groups analyzed by one-way analysis of variance.
Figure 1 Comparison of anticonvulsant effects among different groups on seizure-like behavior induced by PTZ A-E: the percentage of seizure stage before treatment about each group; F: EEG waveforms of each group after 28 d of treatment; F1: CTRL group; F2: DXP group, F3: VPA group; F4: D+V group; G: average power spectral density (PSD) of each group; H: the energy of each single waveform after rats’ treatment. CTRL group: PTZ model rats without treatment (n = 6); DXP group: PTZ model rats treated with Dingxian pill (2.4 g/kg) (n = 5); VPA group: PTZ model rats treated with Valproic acid (0.2 g/kg) (n = 5); D + V group: PTZ model rats treated with Dingxian pill (2.4 g/kg) combined with Valproic acid (0.2 g/kg) (n = 5). PTZ: pentylenetetrazol. EEG: electroencephalograms. DXP: Dingxian pill. VPA: valproic acid. PSD: average power spectral density. aP < 0.001, DXP group vs CTRL group; bP < 0.001, D + V group vs CTRL group; cP < 0.05: VPA group vs D + V group; dP < 0.05, VPA group vs CTRL group; eP < 0.05, D+V group vs CTRL group; fP < 0.01, D + V group vs CTRL group. Differences among groups analyzed by one-way analysis of variance.
Figure 2 Comparation of cognitive impairments in MWM A-B: the swimming pathways and the latency of different groups at the end of training; C-D: The number of times each group crosses the platform area and the percentage of distance in the target quadrant after the platform is dismantled; E: Average swimming speed of each group; G1: CTRL group; G2: DXP group; G3: VPA group; G4: D + V group; D1: first day of training; D2: second day of training; D3: third day of training; D4: fourth day of training. CTRL group: PTZ model rats without treatment (n = 6); DXP group: PTZ model rats treated with Dingxian pill (2.4 g/kg) (n = 5); VPA group: PTZ model rats treated with Valproic acid (0.2 g/kg) (n = 5); D + V group: PTZ model rats treated with Dingxian pill (2.4 g/kg) combined with Valproic acid (0.2 g/kg) (n = 5). MWM: Morris water maze; DXP: Dingxian pill; VPA: valproic acid. PTZ: pentylenetetrazol. aP < 0.001, DXP group vs CTRL group. bP < 0.05, compared with the CTRL group. cP < 0.01, D + V group vs CTRL group. dP < 0.01, D + V group vs VPA group. eP < 0.05, D + V group vs VPA group. fP > 0.05, no significant difference.
Figure 2 Comparation of cognitive impairments in MWM A-B: the swimming pathways and the latency of different groups at the end of training; C-D: The number of times each group crosses the platform area and the percentage of distance in the target quadrant after the platform is dismantled; E: Average swimming speed of each group; G1: CTRL group; G2: DXP group; G3: VPA group; G4: D + V group; D1: first day of training; D2: second day of training; D3: third day of training; D4: fourth day of training. CTRL group: PTZ model rats without treatment (n = 6); DXP group: PTZ model rats treated with Dingxian pill (2.4 g/kg) (n = 5); VPA group: PTZ model rats treated with Valproic acid (0.2 g/kg) (n = 5); D + V group: PTZ model rats treated with Dingxian pill (2.4 g/kg) combined with Valproic acid (0.2 g/kg) (n = 5). MWM: Morris water maze; DXP: Dingxian pill; VPA: valproic acid. PTZ: pentylenetetrazol. aP < 0.001, DXP group vs CTRL group. bP < 0.05, compared with the CTRL group. cP < 0.01, D + V group vs CTRL group. dP < 0.01, D + V group vs VPA group. eP < 0.05, D + V group vs VPA group. fP > 0.05, no significant difference.
Figure 3 Effects of the combination group and the single group on c-Fos expression in the hippocampus region elicited by PTZ A: these sections illustrated the c-Fos expression in hippocampus regions after treatment (G1/G2/G3/G4×100, A1/A2/A3/B1/B2/B3/C1/ C2/C3/D1/D2/D3×200); B: histograms about the statistical analysis of the number of labeled FLI neurons in hippocampal region after administrating saline, DXP, VPA and D + V. G1: CTRL group. G2: DXP group. G3: VPA group. G4: D + V group. CTRL group: PTZ model rats without treatment (n = 6); DXP group: PTZ model rats treated with Dingxian pill (2.4 g/kg) (n = 6); VPA group: PTZ model rats treated with Valproic acid (0.2 g/kg) (n = 6); D + V group: PTZ model rats treated with Dingxian pill (2.4 g/kg) combined with Valproic acid (0.2 g/kg) (n = 6). A1/B1/C1/D1: the CA1 subregion of hippocampus; A2/B2/C2/D2: the CA3 subregion of hippocampus; A3/B3/C3/D3: the DG subregion of hippocampus. PTZ: pentylenetetrazol; FLI: Fos-like protein immunoreactivity; DXP: Dingxian pill; VPA: valproic acid. aP < 0.001, compared with the Control group; bP < 0.05, D + V group vs VPA group; cP < 0.0001, D + V group vs VPA group; dP < 0.01, D + V group vs VPA group. Differences among groups analyzed by one-way analysis of variance.
Figure 3 Effects of the combination group and the single group on c-Fos expression in the hippocampus region elicited by PTZ A: these sections illustrated the c-Fos expression in hippocampus regions after treatment (G1/G2/G3/G4×100, A1/A2/A3/B1/B2/B3/C1/ C2/C3/D1/D2/D3×200); B: histograms about the statistical analysis of the number of labeled FLI neurons in hippocampal region after administrating saline, DXP, VPA and D + V. G1: CTRL group. G2: DXP group. G3: VPA group. G4: D + V group. CTRL group: PTZ model rats without treatment (n = 6); DXP group: PTZ model rats treated with Dingxian pill (2.4 g/kg) (n = 6); VPA group: PTZ model rats treated with Valproic acid (0.2 g/kg) (n = 6); D + V group: PTZ model rats treated with Dingxian pill (2.4 g/kg) combined with Valproic acid (0.2 g/kg) (n = 6). A1/B1/C1/D1: the CA1 subregion of hippocampus; A2/B2/C2/D2: the CA3 subregion of hippocampus; A3/B3/C3/D3: the DG subregion of hippocampus. PTZ: pentylenetetrazol; FLI: Fos-like protein immunoreactivity; DXP: Dingxian pill; VPA: valproic acid. aP < 0.001, compared with the Control group; bP < 0.05, D + V group vs VPA group; cP < 0.0001, D + V group vs VPA group; dP < 0.01, D + V group vs VPA group. Differences among groups analyzed by one-way analysis of variance.
Figure 4 Transcriptome analysis of VPA and Dingxian pill combined with VPA groups A: each column represents an experimental condition Each row represents a log2 Ratio value of a gene or log10 (FPKM + 0.01), and different expression variants or expression levels are expressed in different colors; B: the analysis of GO function of differentially expressed genes; B1: the first 30 genes with significantly up-or down-regulated gene expression; B2: functional and location classification of the top 30 genes; C: KEGG pathway enrichment analysis of differential expression genes; D: the comparison of differential gene expression between VPA group and DXP + VPA group by real time PCR analyze. VPA group, PTZ model rats treated with Valproic acid (0.2 g/kg), DXP + VPA group: PTZ model rats treated with Dingxian pill (2.4 g/kg) combined with Valproic acid (0.2 g/kg). VPA: valproic acid; FPKM: fragments per kilobase of transcript per million mapped reads; PTZ: pentylenetetrazol; KEGG: Kyoto Encyclopedia of Genes and Genomes; DXP: Dingxian pill; PCR: polymerase chain reaction; BDNF: brain-derived neurotrophic factor; GDNF: glial cell line-derived neurotrophic factor; NTF4: neurotrophin-4; Cav3: Caveolin-3; DRD5: Dopamine D5 receptor. aP < 0.05, bP < 0.01, VPA group vs DXP + VPA group. Differences between VPA group and DXP + VPA group analyzed by two-sample t-test.
Figure 4 Transcriptome analysis of VPA and Dingxian pill combined with VPA groups A: each column represents an experimental condition Each row represents a log2 Ratio value of a gene or log10 (FPKM + 0.01), and different expression variants or expression levels are expressed in different colors; B: the analysis of GO function of differentially expressed genes; B1: the first 30 genes with significantly up-or down-regulated gene expression; B2: functional and location classification of the top 30 genes; C: KEGG pathway enrichment analysis of differential expression genes; D: the comparison of differential gene expression between VPA group and DXP + VPA group by real time PCR analyze. VPA group, PTZ model rats treated with Valproic acid (0.2 g/kg), DXP + VPA group: PTZ model rats treated with Dingxian pill (2.4 g/kg) combined with Valproic acid (0.2 g/kg). VPA: valproic acid; FPKM: fragments per kilobase of transcript per million mapped reads; PTZ: pentylenetetrazol; KEGG: Kyoto Encyclopedia of Genes and Genomes; DXP: Dingxian pill; PCR: polymerase chain reaction; BDNF: brain-derived neurotrophic factor; GDNF: glial cell line-derived neurotrophic factor; NTF4: neurotrophin-4; Cav3: Caveolin-3; DRD5: Dopamine D5 receptor. aP < 0.05, bP < 0.01, VPA group vs DXP + VPA group. Differences between VPA group and DXP + VPA group analyzed by two-sample t-test.
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