Periodontal disease is a prevalent chronic inflammatory condition affecting the tissues surrounding and supporting the teeth, leading to significant oral health issues, including tooth loss. This disease poses a substantial public health challenge due to its high prevalence and the complex interplay of risk factors such as smoking, poor oral hygiene, and systemic conditions like diabetes and cardiovascular diseases. The current treatment options for periodontal disease, which primarily focus on controlling the infection, stopping the progression, and trying to restore the damaged tissues, are limited in their effectiveness. The multifactorial nature of periodontal disease necessitates innovative therapeutic approaches to manage inflammation and bone loss effectively.
This thesis investigates the potential of betulin, a naturally occurring triterpene with well-documented anti-inflammatory, antimicrobial, and antioxidant properties, as a novel therapeutic agent for periodontal disease. The primary objective of this study is to explore betulin's efficacy in modulating inflammatory responses and preventing bone resorption, which are critical in the pathogenesis of periodontal disease.
Through a comprehensive array of cellular and molecular analyses, including: qPCR, ELISA, and Western Blotting, the study demonstrates that betulin significantly reduces the expression of key pro-inflammatory cytokines, thereby mitigating the inflammatory processes contributing to periodontal tissue destruction. Furthermore, betulin's ability to inhibit osteoclastogenesis, the process by which bone-resorbing cells form, highlights its potential in preserving alveolar bone integrity, which is often compromised in advanced stages of periodontal disease.

The findings from this research underscore betulin's dual role in addressing both inflammation and bone loss, positioning it as a promising candidate for periodontal therapy. However, it is crucial to note that this study is a stepping stone, advocating for further in vivo research and clinical trials. These are necessary to validate these findings and establish betulin's safety and efficacy in human subjects. The integration of betulin into existing treatment protocols can potentially enhance the management of periodontal disease, ultimately improving patient outcomes and quality of life.
In conclusion, this thesis provides compelling evidence for betulin's therapeutic potential in periodontal disease, highlighting its multifaceted benefits in reducing inflammation and preventing bone loss. The research paves the way for future studies to translate these findings into clinical practice, offering hope for more effective and holistic treatment options for individuals suffering from periodontal disease.

Abstract iv
Contents vi
Abbreviations ix
Chapter 1 Introduction 1
1.1. Epidemiology of periodontal disease 1
1.2. Periodontal disease diagnosis and treatment 1
1.3. Stages of periodontal disease 2
1.4. Periodontal disease progression and complications 3
1.5. Impact of periodontal disease on overall health 4
1.6. Understanding periodontal disease pathogenesis 4
1.7. Changing the paradigm of inflammation’s role in periodontal disease 6
1.8. Need for new treatments to reduce bone loss 8
1.9. Betulin and its properties 8
1.10. The aim of this study 10
Chapter 2 Materials and methods 11
2.1. Materials 11
2.1.1. Equipment 11
2.1.2. Cells 12
2.1.3. Cell culture 12
2.1.4. Real-time polymerase chain reaction (qPCR) 12
2.1.5. Western blot reagents 13
2.1.6. Western Blot antibodies 14
2.1.7. Enzyme-linked immunosorbent assay (ELISA) 14
2.1.8. Activators and other chemical compounds 15
2.1.9. Luciferase Assay 15
2.1.10. Osteoclastogenesis 15
2.1.12. Real-Time PCR Primer 16
2.2. Methods 16
2. Cell culture 16
2.1.1. MTT-assay 18
2.1.2. Analysis of the Gene Expression Omnibus DataSet 19
2.1.3. Isolation of RNA and quantitative real-time reverse transcription PCR 21
2.1.4. Enzyme-linked immunosorbent assay (ELISA) 23
2.1.5. IPA analysis 24
2.1.6. Western blot analysis 26
2.1.7. Transfection and luciferase reporter assay 27
2.1.8. Osteoclastogenesis assay 29
2.1.9. Immunofluorescence staining assay for c-Jun translocation into the nucleus in RAW 264.7 Cells 29
2.2.11. Statistical analysis 31
Chapter 3 Results 32
3.1. Betulin shows no impact on HGF viability 32
3.2. GEO DataSet GSE59932: Interleukin expression in periodontal disease 32
3.3. Betulin inhibits pro-inflammatory interleukin expression in LPS-induced HGF 33
3.4. Betulin suppresses inflammatory response in LPS-induced HGF 34
3.5. Identification of pathways for western blot analysis in HGF using IPA 34
3.6. Betulin inhibits JNK, ERK, p38, and c-Jun phosphorylation in LPS-induced HGF 35
3.7. Reversal of betulin's impact on pro-inflammatory interleukins in HGF by MAPK pathway activators 36
3.8. Reversal of betulin's impact on inflammatory response in LPS-induced HGF by MAPK pathway activators 37
3.9. Betulin's inhibition and reversal of AP-1 transcription factor complex activity in HGF by MAPK pathway activation 37
3.10. Betulin shows no impact on RAW 264.7 cell viability 38
3.11. Betulin inhibits RANKL-induced osteoclast differentiation 39
3.12. Identification of pathways for western blot analysis in RAW 264.7 cells using IPA 40
3.13. Betulin inhibits JNK, ERK, p38, and c-Jun phosphorylation in RANKL-induced RAW 264.7 cells 41
3.14. Betulin inhibits RANKL-induced c-Jun translocation into the nucleus in RAW 264.7 cells: Reversal by pathway-specific activators 42
Chapter 4 Discussion 43
Conclusions 47
Figures 48
Appendix 64
References 66

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