Anticonvulsant Effect of both Ketogenic Diet and Synbiotics on Epileptic Seizure Rodent Model by Modulating Gut Microbiota
뇌전증 발작 유도 동물에서 케톤식이와 신바이오틱스 섭취 후 장내균총 변화를 통한 발작 완화 효과 연구
- 주제(키워드) Epilepsy , Seizure , Synbiotics , Ketogenic diet , Gut microbiota
- 발행기관 고려대학교 대학원
- 지도교수 김세헌
- 발행년도 2020
- 학위수여년월 2020. 8
- 학위구분 박사
- 학과 대학원 생명공학과
- 세부전공 식품미생물학 전공
- 원문페이지 201 p
- UCI I804:11009-000000231983
- DOI 10.23186/korea.000000231983.11009.0001180
- 본문언어 영어
초록/요약
The overall objective throughout the studies in this thesis were to find out the neuroprotective effect of both Lactobacillus fermentum MSK 408 and galactooligosaccharide (GOS) with ketogenic diet (KD) of the brain through modulating the gut microbiota having synergistic interactions against epileptic seizures. The subjects of this study includes cell, rodent and human. Firstly the probiotics, prebiotics and synbiotics were chosen by performing few studies step by step. The Lactobacillus strains went through the probiotics evaluation tests. The qualified probiotic strains as probiotics were ten strains out of 300 strains. In the end, 393 strain for FOS and 408 strain for GOS were then selected, respectively. The selected two kinds of synbiotics were then assessed by checking cell adhesion test. After that, human colon epithelial cell derived HT-29 cell, checked whether two different kinds of synbiotics have anti-inflammation effects against LPS. Secondly, the human brain derived neuroblastoma cell SH-SY5Y cell was chosen for brain protection against oxidative stress. The results suggested that synbiotic treatment significantly increased tight junction proteins, and decreased inflammatory cytokines in HT-29 cell. Moreover, synbiotic treatment also showed the attenuation of oxidative stress in SH-SY5Y cells. Collectively, this study highlight the potential of both MSK 408 strain and GOS as symbiotic which could be used in vivo studies. The second study is highlighting the present epilepsy-gut microbiota scenario and design of personalized KD targeting the altered gut microbiota in children with epilepsy. The results of this study are suggesting that one month of KD treatment in childhood epilepsy patients could modulate gut microbiota and change some metabolites in the body. And thus decrease the seizure symptoms and even cognition problem. In the second study, I investigated about anti-seizure effect of both ketogenic diet and single probiotic strain in chemical induced seizure rodent model. The study was initiated with thirty 3 week-old male ICR mice in 6 groups. 4:1 KD were chosen from previous pretest and finally screened Lactobacillus strain was orally gavaged (4 x 109 CFU/ml) with both diets for 4 weeks and Pentylenetetrazol (PTZ) (40 mg/kg) was injected intraperitoneally once 30 min before sacrifice. The results showed that KD treatment significantly reduced seizure compared to ND group. Four week administration of MSK 408 strain with both ND and KD restored colon morphology and tight junction protein mRNA expression of both gut and brain additionally, administration of MSK 408 strain showed independently different tendency in PCoA and fecal short chain fatty acids (SCFAs) and microbiota modulation. PICRUSt results suggested that the modulation of microbiota by KD increased GABA metabolism and MSK 408 strain also triggered increased GABA metabolism. The results suggested that supplementation of MSK 408 strain with KD showed no disturbing anti-seizure effects of KD and may improve brain and colon barrier function via gut microbiota and SCFA modulation. The final study was designed in a same way to assess the effect of synbiotics that was selected from previous steps, and the experimental period was elongated to 8 weeks to maximize the effect of KD. The results suggested that seizure attenuating effect of KD treatment showed more impact after eight week than four week administration. And co-administration of synbiotics with KD also didn’t interrupt the effect of KD against seizure. Additionally, synbiotics treated ND group also showed reduced seizure related score. To find out whether gut microbiota modulation could affect these kinds of results, I decided to perform NGS analysis and GC-MS analysis with PICRUSt analysis. SCFAs concentration in KDs was dramatically reduced compared to NDs, the ratio of KD groups acetate concentration decreased and propionate concentration increased compared to NDs. Interestingly, NDS showed independently different ratio compared to both NDs and KDs which may indicate the reduction of seizure symptoms in NDS group was also shown in a different way compared to KD groups. All KD treated groups showed higher Firmicutes and Proteobacteria level than ND groups. KD effect on gut microbiota was stronger than each treatments. Firstly, Acetatifactor, Anaerotaenia, Escherichia, Flintibacter, Oscillibacter and Erysipelatoclostridium were higher in KD groups. In contrast, Lactobacillus, Muribaculum and Alistipes were significantly higher in ND group than KD group. In KD group, results are suggesting that the gut microbiota is helping the host to generate energy, increase SCFAs such as butyrate and acetate. Furthermore, PICRUSt results also suggested same as other results such related to SCFAs, and independent pathway of NDS group against seizure. Finally, this series of study may indicate that KD could be used as supplement with KD and synbiotics could reduce seizure symptoms without KD which needs further study. However, to understand its mechanisms and clinical benefits in human all studies are not enough and seems limited, so further investigations have been required.
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TABLE OF CONTENTS
Page
ABSTRACT OF THESIS ………………………….……………………… i
TABLE OF CONTENTS …………………………..………………………. v
LIST OF TABLES ……………………………………………………… xviii
LIST OF FIGURES ……………………………………………………… xiv
LIST OF ABBREVIATION ……………….……………………….xix
CHAPTER 1.
Literature Review ………….……………………………………………. 1
1.1. PROBIOTICS ………………………….………………………………. 2
1.1.1. Definition of probiotics ……………………………….………… 2
1.1.2. Lactic acid bacteria……………………………………………… 2
1.2. PREBIOTICS AND SYNBIOTICS ………………………….……… 4
1.2.1. Definition of prebiotics …………………………………………… 4
1.2.2. Types and sources of prebiotics ………………………………… 5
1.2.3. Fructo Oligo Saccharide (FOS)………………………………… 5
1.2.4. Galacto Oligo Saccharide (GOS)…………………………………. 6
1.2.5. Synbiotics …………………………………………………….…… 7
1.3. GUT MICROBIOTA ………………………….………………………. 7
1.3.1. Gut microbiota of human …………………………………….…… 7
1.3.2. Role of gut microbiota in epilepsy ………………………………… 8
1.4. EPILEPSY…………………………………………………….……… 10
1.4.1. Definition of epilepsy …………………………………………… 10
1.4.2. Epileptogenesis…………………………………………………… 11
1.4.3. Epileptic seizure ………………………………………………… 12
1.5. Ketogenic Diet (KD) …………………………………………….……… 12
1.5.1. Definition and types of ketogenic diet ……………………….…… 12
1.5.2. Functions of ketogenic diet on epileptic seizure ………………… 13
1.5.3. Role of gut microbiota in epilepsy ……………………………… 13
1.6. REFERENCES ……………………………………………………… 15
CHAPTER 2.
The effect of ketogenic diet on childhood epilepsy patients……. 21
2.1. ABSTRACT …………………………………………………………….. 22
2.2. INTRODUCTION ……………………………………………… 23
2.3. MATERIALS AND METHODS …………………………………… 25
Sample collection …………………………………………………… 25
Gut microbiota analysis …………………………………………………. 25
Fecal SCFAs analysis ………………………………………………… 26
Assessment of KD on seizure reduction ………………………………… 27
Statistical analysis ……………………………………………………… 27
2.4. RESULTS AND DISCUSSION…………………………………… 28
Alpha diversity analysis ………………………………………………… 28
PCA analysis …………………………………………………… 31
Fecal SCFAs analysis …………………………………………………… 32
Seizure reduction ratio……………………………………………… 34
2.5. REFERENCES……………………………………………………… 36
CHAPTER 3.
Screening of effective combination of synbiotics in two different cell lines ………………………………………………………… 39
3.1. ABSTRACT …………………………………………………………… 40
3.2. INTRODUCTION ………………………………………………… 42
3.3. MATERIALS AND METHODS …………………………………… 44
Preparation of bacterial strains, and growth culture……………………… 44
Preparation of prebiotics and minimum medium………………………….44
Acid and bile tolerance test……………………………………………44
Safety evaluation test …………………………………………………… 45
Cell adhesion assay ……………………………………………………… 45
Cell viability assay (MTT assay) …………………………………… 45
Determination of synbiotics………………………………………… 46
Assessment of synbiotics antioxidant effect …………………………… 46
Analysis of the protective effect of synbiotics in cell cultures ………… 47
Quantitative real-time polymerase chain reaction (qRT-PCR) analysis … 47
Statistical analysis ……………………………………………………… 48
3.4. RESULTS AND DISCUSSION …….….….….……………………… 51
Probiotic characteristic evaluation……………………………………… 51
Cell adhesion test……………………………………………………… 54
Cell viability assay………………………………………………… 56
Two prebiotic substrates utilization evaluation…………………………. 58
Antioxidant effect of synbiotics ………………………………………. 61
Anti-inflammatory effect of synbiotics in the HT-29 cell line …………. 63
Anti-oxidative effect of synbiotics in the SH-SY5Y cell line …………. 66
3.5. REFERENCES ………………………………………………….… 69
CHAPTER 4.
Neuroprotective effect of Lactobacillus fermentum MSK 408 in pentylenetetrazol-induced epileptic seizure rodent model…… 74
4.1. ABSTRACT …………………………………………………………… 75
4.2. INTRODUCTION ………………………………………………… 77
4.3. MATERIALS AND METHODS …………………………………… 79
Animals ………………………………………………………………… 79
Diets …………………………………………………………79
Probiotic strain preparation …………………………………………. 79
Preparation of pentylenetetrazol(PTZ) reagent………………… 79
Treatment protocol ………………………………………… 80
Body weight change and feed intake ………………………………… 80
Serum biochemical analysis ……………………………………… 80
Morphologic analysis of the colon ……………………………………… 81
quantitative real time polymerase chain reaction (qRT-PCR) analysis… 81
Seizure video recording analysis ………………………………………. 82
Fecal SCFAs measurement …………………………………………. 82
Fecal gDNA extraction and sequencing analysis ……………………… 83
Taxonomy classification, diversity analysis, and metagenomic analysis. 84
Statistical analysis ……………………………………………………. 84
4.4. RESULTS AND DISCUSSION ….….….….….……………………… 87
Body weight change throughout the experiment………………………… 87
Serum biochemical analysis …….………………………….…………… 89
Analysis of morphology and mRNA expression levels in colon ………… 92
mRNA expression of brain tight junction proteins and GABA related marker ………………………………………………………………………94
Seizure analysis…………………………………………………… 96
Fecal SCFA analysis…………………………………………………… 98
Gut microbiota analysis……………………………………………… 100
PICRUSt analysis……………………………………………… 107
4.5. REFERENCES ………………………………………………………. 110
CHAPTER 5.
Neuroprotective effect of synbiotics and ketogenic diet in pentylenetetrazol-induced epileptic seizure rodent model…… 117
5.1. ABSTRACT …………………………………………………………… 118
5.2. INTRODUCTION ………………………………………………… 120
5.3. MATERIALS AND METHODS …………………………………… 122
Animals ………………………………………………………………… 122
Diets ……………………………………………………………………122
Treatment sample preparation …………………………………………. 122
Preparation of pentylenetetrazol(PTZ) reagent………………… 123
Treatment protocol ………………………………………… 123
Body weight change and feed intake …………………………………… 123
Serum biochemical analysis ……………………………………… 124
Morphologic analysis of the colon and hippocampal CA1 region……… 124
quantitative real time polymerase chain reaction (qRT-PCR) analysis… 125
Seizure video recording analysis ……………………………………. 125
Fecal SCFAs measurement ………………………………………….125
Fecal gDNA extraction and sequencing analysis …………………… 126
Taxonomy classification, diversity analysis, and metagenomic analysis. 127
Statistical analysis ……………………………………………………128
5.4. RESULTS AND DISCUSSION ….….….….….……………………… 131
Body weight change throughout the experiment……………………….. 132
Serum biochemical analysis …….………………….…………… 133
Colon length and morphologic analysis ………………………………… 135
mRNA expression levels in colon ……………………………………… 138
Physiological analysis of mouse brain hippocampus …………………140
mRNA expression of brain tight junction proteins and hippocampal receptor subunit markers ………………………………………………………… 144
Seizure analysis…………………………………………………… 148
Fecal SCFA analysis………………………………………………… 150
Gut microbiota analysis……………………………………………… 152
PICRUSt analysis……………………………………………… 158
5.5. REFERENCES ………………………………………………………. 160
CONCLUSION REMARKS …………………………………………… 164
ABSTRACT IN KOREAN ….……………………………………………171
