Background/Objectives: Constipation can be induced in animal models through various factors such as loperamide (Lop) or complement component 3 (C3) deficiency. The effectiveness of therapeutic agents in the clinical management of constipation has been primarily evaluated within only one model, but between-model comparisons have not been performed so far. Therefore, we investigated whether the effectiveness of the laxative drugs for the clinical management is related to etiological factors. Methods: The changes in the key parameters for defecation were compared between C3 knockout (KO) mice with C3-deficiency-induced constipation and ICR mice with Lop-induced constipation after the oral administration of Uridine (Urd) and aqueous extract of Liriope platyphylla L. (AELP). Results: Similar effectiveness of Urd and AELP were detected on the stool frequency, intestinal epithelial barrier structure, and mucin secretion in both models. However, other parameters (namely gastrointestinal (GI) transit, water retention, and enteric nervous system (ENS) structure and function) showed higher effectiveness in C3 KO mice than in the Lop-induced model. Only the effectiveness of the two therapeutic agents on the histological structure of the mid-colon was greater in the Lop-induced mice model compared to the C3 KO mice model. Furthermore, these differences in the therapeutic effectiveness of Urd and AELP were partially reflected in alterations in the cyclic adenosine monophosphate (cAMP) downstream signaling pathway. Conclusions: The results suggest that the therapeutic effectiveness of Urd and AELP is sensitive to C3-deficiency-induced constipation and these differences may be linked to the alternative regulation of the cAMP downstream signaling.

Complement component 3 (C3) deficiency has recently been reported as one of the novel causes of constipation. To identify a unique gene specific to constipation caused by C3 deficiency, the total RNA extracted from the mid colon of C3 knockout (C3 KO) mice was hybridized to oligonucleotide microarrays, and the function of the candidate gene was verified in in vitro and in vivo models. C3 KO mice used for microarrays showed definite phenotypes of constipation. Overall, compared to the wild type (WT), 1237 genes were upregulated, and 1292 genes were downregulated in the C3 KO mice. Of these, the major genes included were lysine (K)-specific demethylase 5D (KDM5D), olfactory receptor 870 (Olfr870), pancreatic lipase (PNLIP), and alkaline phosphatase intestinal (ALPI). Specifically, the ALPI gene was selected as a novel gene candidate based on alterations during loperamide (Lop)-induced constipation and intestinal bowel disease (IBD). The upregulation of ALPI expression treated with acetate recovered the expression level of mucin-related genes in primary epithelial cells of C3 KO mice as well as most phenotypes of constipation in C3 KO mice. These results indicate that ALPI plays an important role as the novel gene associated with C3 deficiency-induced constipation.

Complement component 3d (C3d), the final cleavage product of complement component C3, interacts with CR2 and thus plays a crucial role in linking the innate and adaptive immune systems. Additionally, human C3d executes various functions in its dimeric form, which is more effective than its monomeric form. In this study, we aimed to explored whether chicken C3d (chC3d) exhibits similar characteristics, namely dimerization and binding of dimeric chC3d to chicken CR2 (chCR2). We investigated the interaction and co-localization of chC3d with itself using coimmunoprecipitation and confocal laser scanning microscopy, respectively. Then, dimeric chC3d was detected using native polyacrylamide gel electrophoresis and western blotting, and its equilibrium dissociation constant KD (827 nM) was determined using surface plasmon resonance. Finally, the interaction modes of dimeric chC3d were identified using molecular docking simulations, which revealed that dimeric chC3d could crosslink with chCR2 receptor. Overall, our findings will facilitate future explorations of the chicken complement system.

Recent reports suggest that complement system contributes to allograft rejection. However, its underlying mechanism is poorly understood. Herein, we investigate the role of complement component 3 (C3) in a single MHC-II molecule mismatched murine model of allograft rejection using C3 deficient mice (C3(-/-)) as skin graft donors or recipients. Compared with C3(+/+) B6 allografts, C3(-/-) B6 grafts dramatically prolonged survival in MHC-II molecule mismatched H-2(bm12) B6 recipients, indicating that C3 plays a critical role in allograft rejection. Compared with C3(+/+) allografts, both Th17 cell infiltration and Th1/Th17 associated cytokine mRNA levels were clearly reduced in C3(-/-) allografts. Moreover, C3(-/-) allografts caused attenuated Th1/Th17 responses, but increased CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cell expression markedly in local intragraft and H-2(bm12) recipients. Depletion of Treg cells by anti-CD25 monoclonal antibody (mAb) negated the survival advantages conferred by C3 deficiency. Our results indicate for the first time that C3 deficiency can prolong MHC-II molecule mismatched skin allograft survival, which is further confirmed to be associated with increased CD4(+) CD25(+) Treg cell population expansion and attenuated Th1/Th17 response.