Basic Study on the Application of Therapeutic Strategies for Complement C3 Deficiency-induced Constipation = Complement C3 결핍으로 유도된 변비의 치료제 적용 연구

다국어 초록 (Multilingual Abstract)

Complement component 3 (C3), a key player in various complement activation pathways essential for triggering immune responses, has recently emerged as a potential novel contributor to constipation. Noteworthy constipation phenotypes, such as a reduction in stool excretion parameters, decreased gastrointestinal (GI) transit, histological alterations in colon structure, and diminished mucin secretion, were observed in FVB/N-C3em1Hlee/Korl complement component knockout (C3 KO) mice at 16 weeks of age. Additionally, these mice exhibited fecal microbiota dysbiosis and dysregulation of the enteric nervous system (ENS) in the mid colon as a result of C3 deficiency-induced constipation. Despite these findings, there is currently a lack of fundamental research on strategies for treating constipation induced by C3 deficiency. Consequently, our study aimed to identify therapeutic drugs and novel genetic markers that could be employed in addressing C3 deficiency-induced constipation.
Initially, we explored the potential of uridine (Urd) and the aqueous extract of Liriope platyphylla L. (AEtLP), both previously recognized for their constipation-alleviating effects, in ameliorating symptoms associated with C3 deficiency-induced constipation. Our investigation focused on evaluating improvements in constipation-related parameters and the underlying molecular mechanisms in C3 KO mice following treatment with Urd and AEtLP, both known for their laxative properties.
Upon treatment with Urd and AEtLP, there was a substantial increase in stool parameters, including stool frequency (128% and 71% increase, respectively) and water contents (150% and 337% increase, respectively), compared to the vehicle (Veh)-C3 KO group. While the total length of the intestine remained unchanged, the length of the GI tract was extended in the Urd- and AEtLP-treated groups (36% and 18% increase, respectively). Histopathological analysis of mid colon tissue structure revealed significant enhancements in the mucosal layer's length (7% and 39% increase, respectively), the muscularis mucosae, and the expression levels of tight junction markers such as zonula occludens-1 (ZO-1), Occludin, Claudin-1, Claudin-4, and p120.
Moreover, Urd and AEtLP treatment led to significant increases in mucin-related markers, including Mucin (MUC)2, MUC1, and kruppel-like factor (Klf)4, indicative of improved mucin secretion ability. Water retention markers, including aquaporin (AQP)3 and AQP8, demonstrated a substantial increase after Urd or AEtLP treatments. Additionally, the density of neuronal cells and intestinal cajal cells (ICC) significantly increased in Urd- and AEtLP-treated C3 KO mice compared to the Veh-treated group, accompanied by improvements in the excitatory and inhibitory functions of the ENS. Furthermore, concentrations of two key hormones, cholecystokinin (CCK) and gastrin, were markedly enhanced in the Urd- and AEtLP-treated C3 KO groups. Overall, these findings suggest promising therapeutic potential for Urd and AEtLP in addressing C3 deficiency-induced constipation.
Second, we tried to identify the novel gene in the mid colon of C3 KO mice in response to the constipation caused by C3 deficiency. To achieve this, the total RNA extracted from the mid colon of C3 KO mice was hybridized to oligonucleotide microarrays, and the function of novel candidate gene was verified in in vitro and in vivo. C3 KO mice showed a successful reduction in the number of stools, GI transit, colon length, mucosal layer thickness, and muscle layer thickness. Overall, compared to the wild type (WT) (Fold change (FC)>=1.5), 1,237 genes were upregulated and 1,292 genes were downregulated in C3 KO mice with constipation phenotypes. Their number changes to 226 upregulated genes and 229 downregulated genes when the FC value is set to more than 2. Among them, the major genes in the downregulated categories included Eif2s3y, Uty, Kdm5d, Igkv6-32, Olfr870, Ddx3y, Endothelin-1 (Edn1) and Neurexophilin and PC-esterase domain family member 4 (Nxpe4), whereas the major genes in the upregulated categories were Pnlip, Cel, Cpb1, Ctrl, Pnliprp1, Reg1, Resistin like beta (Retnlb) and alkaline phosphatase of intestinal (Alpi). Especially, Alpi gene were selected as novel candidates because it was associated with loperamide (Lop)-induced constipation and inflammatory bowel disease (IBD). The upregulation of Alpi mRNA treated with acetate in epithelial cells derived from colon of C3 KO mice recovered the suppression of MUCs, Klf4 and AQPs mRNA detected during C3 deficiency. Also, the most constipation phenotypes including stool number, GI transit, histopathological structure of colon, mucin secretion ability and water retention capacity were significantly recovered in C3 KO mice after upregulation of Alpi gene treated by acetate.
In conclusion, our studies showed that Urd and AEtLP have the potential to alleviate C3 deficiency-induced constipation through increasing mucin secretion ability, enhancing water retention capacity, promoting ENS recovery and regulating GI hormones. The findings of the current investigation offer valuable scientific insights into various functional gene groups and specific genes serving as novel biomarkers responsive to constipation induced by C3 deficiency. Furthermore, the present results indicate that Alpi gene can play an important role as the new responsible gene for constipation.

목차 (Table of Contents)

• Contents i
• List of Figures iv
• List of Tables vi
• Abbreviations vii
• Abstract ix
• I. INTRODUCTION 1
• 1. Complement component 3 1
• 1.1. Definition of complementary system 1
• 1.2. Roles of C3 in biological system 2
• 2. Novel role of C3 as a new cause of constipation 6
• 2.1. Possibility of constipation in C3 KO mice 6
• 2.2. Previous study in C3 deficiency-induced constipation 9
• 3. Constipation 12
• 3.1. Definition of constipation 12
• 3.2. Mechanism of constipation 21
• 4. Purpose of this study 25
• II. MATERIALS AND METHODS 29
• 1. Production and identification of C3 KO mice 29
• 2. Design of animal experiment 29
• 3. Preparation of Urd and AEtLP 32
• 4. Stool parameters analysis 33
• 5. Food and water parameter analysis 33
• 6. Histopathological analysis 34
• 7. Alcian blue staining 34
• 8. Western blot analysis 35
• 9. RT-qPCR analysis 38
• 10. AChE activity assay 38
• 11. NO assay 41
• 12. Measurement of GI hormone concentration 41
• 13. Microarray analysis 42
• 14. Isolation of intestinal epithelial cells from C3 KO mice and acetate treatment 43
• 15. Statistical analysis 44
• Ⅲ. RESULTS 45
• 1. Verification for C3 deficiency-induced constipation 45
• 1.1. Verification for C3 deficiency-induced constipation phenotypes 45
• 2. Evaluation of therapeutic substances for C3 deficiency-induced constipation 45
• 2.1. Effect of Urd and AEtLP on excretion parameters of C3 KO mice 45
• 2.2. Effect of Urd and AEtLP on GI transit of C3 KO mice 50
• 2.3. Effect of Urd and AEtLP on histopathological structure in mid colon of C3 KO mice 55
• 2.4. Effect of Urd and AEtLP on mucin secretion ability and water retention capacity in C3 KO mice 58
• 2.5. Effect of Urd and AEtLP on ENS regulation in mid colon of C3 KO mice 66
• 2.6. Effect of Urd and AEtLP on GI hormones regulation in mid colon of C3 KO mice 73
• 3. Identification of novel markers for C3 deficiency-induced constipation 80
• 3.1. Characterization of the changes in global genes expression in mid colon in response to C3 deficiency-induced constipation 80
• 3.2. Biological functions of genes that response to C3 deficiency-induced constipation 84
• 3.3. Reliability of the expression for differential genes detected from the microarray analysis 95
• 3.4. Selection of Alpi gene as a new treatment target for C3 deficiency-induced constipation and verification of their function in acetate-treated intestinal epithelial cells 95
• 3.5. Verification of Alpi genes as a new treatment target for C3 deficiency-induced constipation in acetate treated C3 KO mice 102
• IV. DISCUSSION 112
• V. REFERENCES 119
• SUMMARY (In Korean) 129
• Curriculum Vitae 132
• Acknowledgement 141