Effects of human-derived Gardnerella vaginalis and probiotics on menopausal symptoms in mice

국문 초록 (Abstract)

폐경기 여성에서 Gardnerella vaginalis로 인한 세균성 질염에 대한 연구는 거의 없는 상태이며, 생체내 위험 인자로서 역할도 입증되지 않았다. 본 연구에서는 폐경기 여성 마우스 모델에서 Gardnerella vaginalis로 인한 질염과 골다공증 사이의 관계와 Gardnerella vaginalis가 위험인자로 작용하는 메커니즘을 조사하고자 하였다. 인간유래 Gardnerella vaginalis로 세균성 질염 유도모델과 폐경기 상태인 난소절제술 마우스에서 세균성 질염을 유도하였다. 이 유도 모델에서 우리는 Gardnerella vaginalis가 RANK/RANKL 경로를 조절하여 골감소증을 촉진하고 골다공증을 악화시키며 신경정신 질환을 유발한다는 것을 관찰했습니다. 생체외 실험에서는 Gardnerella vaginalis가 분비하는 세포 외 소포체(EV)가 이러한 영향의 원인이라는 것을 확인했습니다. 또한, 우리는 질내 미생물군과 Gardnerella vaginalis에 의해 변형된 장내 미생물군과 질병이 유발되고 악화되는 상관관계를 관찰했습니다. 전반적으로, Gardnerella vaginalis유발 마우스 모델을 이용한 이번 연구는 Gardnerella vaginalis에 의한 세균성 질염이 질염 유발, 골감소증 유발, 골다공증 악화 및 신경정신병적 질환에 기여한다는 것을 뒷받침한다.

다국어 초록 (Multilingual Abstract)

There has been little research on bacterial vaginosis caused by Gardnerella vaginalis in postmenopausal women, and its role as a risk factor in vivo also remains to be proven. In this study, we sought to investigate the relationship between vaginitis caused by Gardnerella vaginalis and osteoporosis in a postmenopausal female mouse model and the mechanism by which Gardnerella vaginalis acts as a risk factor. Bacterial vaginitis was induced in a bacterial vaginosis induction model using human-derived Gardnerella vaginalis and ovariectomized mice in menopausal state. In this induction model, we observed that Gardnerella vaginalis regulates the RANK/RANKL pathway, promoting osteopenia and worsening of osteoporosis and also causing neuropsychiatric disorders. Our in vitro experiments confirmed that the extracellular vesicles (gEV) secreted by Gardnerella vaginalis is causative of this impact. Furthermore, we observed the correlation between vaginal microbiota and gut microbiota altered by Gardnerella vaginalis and the disease that is induced and worsened. Overall, this study with the Gardnerella vaginalis-induced mouse model supports that bacterial vaginosis caused by Gardnerella vaginalis contributes to the induction of vaginitis, osteopenia induction, osteoporosis worsening, and neuropsychiatric disease.

목차 (Table of Contents)

• 1. Abstract .1
• 2. Introduction .3
• 3. Materials and Methods .12
• 3.1 Culture of Gardnerella vaginalis and preparation of extracellular vesicles .12
• 3.2 Isolation and culture of probiotics P32 and P45 .13
• 3.3 Cell culture .13
• 3.4 Experimental animal models .15
• 3.5 Fecal microbiota transplantation .17
• 3.6 Behavioral experiments .17
• 3.7 Ovariectomy .22
• 3.8 Enzyme-linked immunosorbent assay (ELISA) .22
• 3.9 Immunostaining, hematoxylin & eosin (H&E) and full staining .22
• 3.10 Real-time quantitative polymerase chain reaction (RT-PCR) .24
• 3.11 FITC labeling of extracellular vesicles in vivo .2 6
• 3.12 Next-generation sequencing-assisted microbiota analysis .27
• 3.13 Whole-genome sequencing .28
• 3.14 Statistical analysis .29
• 4. Results .30
• 4.1 Gardnerella vaginalis is a risk factors for osteoporosis and neuropsychiatric disorders .30
• 4.1.1 Gardnerella vaginalis induces inflammation and promotes osteoclast formation in vitro .30
• 4.1.2 Gardnerella vaginalis induces bacterial vaginosis and alters the microbiota .34
• 4.1.3 Gardnerella vaginalis regulates hormone and bone factors in the blood, induces colitis, and alters the gut microbiota .49
• 4.1.4 Gardnerella vaginalis induces neuroinflammation and alters serotonin levels, contributing to neuropsychiatric disorder .65
• 4.1.5 Gardnerella vaginalis induces osteopenia in a bacterial vaginosis model and worsens osteoporosis in an ovariectomy model .74
• 4.1.6 Altered vaginal and gut microbiota are correlate with pro-inflammatory cytokines, osteoporosis, and neuropsychiatric disorders .78
• 4.2 Bacterial vaginosis fecal microbiota transplantation induces osteopenia and neuropsychiatric disorders in a Pseudo-Germ-Free model .84
• 4.2.1 Fecal microbiota transplantation induces inflammation and osteopenia in the vagina and bone .84
• 4.2.2 Fecal microbiota transplantation regulates hormone and bone factors in the blood and causes colitis .87
• 4.2.3 Fecal microbiota transplantation induces neuroinflammation and alters serotonin levels, contributing to neuropsychiatric disorders .89
• 4.3 Extracellular vesicles of Gardnerella vaginalis is a risk factor in osteoporosis and neuropsychiatric disorders .94
• 4.3.1 Extracellular vesicles induce lower cell viability and higher levels of RANK-RANKL and pro-inflammatory cytokines in vitro .94
• 4.3.2 Extracellular vesicles induce osteopenia and worsen osteoporosis .99
• 4.3.3 Extracellular vesicles induce bone factors in the blood and causes colitis .105
• 4.3.4 Extracellular vesicles induce neuroinflammation and alter serotonin levels, contributing to neuropsychiatric disorders .108
• 4.3.5 Extracellular vesicles originate in the vagina induce to the bone, gut, and brain through blood vessels .115
• 4.4 Effects of probiotics on osteoporosis and neuropsychiatric disorder improvement through the Microbiota-Vagina-Gut Axis .118
• 4.4.1 Effect of probiotics on inhibiting Gardnerella vaginalis growth and osteoclast formation in vitro .118
• 4.4.2 Effect of probiotics on Gardnerella vaginalis-induced bacterial vaginosis .125
• 4.4.3 Effect of probiotics for ovariectomy model .135
• 4.4.4 Effect of probiotics on the Gardnerella vaginalis-induced ovariectomy model 143
• 5. Discussion .154
• 6. Reference .165
• 7. Acknowledgments .179
• 8. 국문 초록 .181