Effects of Complement 3 deciency on its receptor downstream signaling pathway-mediated inammatory response in knockout mice

The current study measured alterations in the inducible nitric oxide synthase (iNOS) ‐ mediated cyclooxygenase ‐ 2 (COX ‐ 2) induction pathway, inammasome pathway, NF-kB activation, and inammatory cytokine expressions in the transverse colon of C3 knockout (KO) mice, to determine whether complement component 3 (C3) deciency affects its receptor downstream-mediated inammatory response. Compared to wild type (WT) mice, the expression level of C3 protein was successfully suppressed in the transverse colon of C3 KO mice. Signicant enhancement was observed in expression levels of important members of the iNOS ‐ mediated COX ‐ 2 induction pathway, and in the phosphorylation of mitogen ‐ activated protein (MAP) kinase members. Also, a similar pattern of increase was observed in the expression levels of inammasome proteins in C3 KO mice. Moreover, compared to WT mice, C3 KO mice showed remarkably enhanced phosphorylation of NF-kB and IkB-a, which was reected in entirety as increased expressions of TNF-a, IL-6 and IL-1a. Taken together, results of the current study indicate that C3 deciency induces activation of the iNOS ‐ mediated COX ‐ 2 induction pathway, ASC-inammasome pathway, and NF-kB signaling pathway, resulting in the enhancement of inammatory cytokine expressions in the transverse colon of C3 KO mice. Serum concentrations of TNF-α and IL-6 cytokines were measured using a mouse TNF-α enzyme-linked immunosorbent assay kit (Biolegend, and was was


Introduction
The complement system plays a key role in the opsonization of pathogens and injured cells, induction of in ammation, and destruction of microorganisms, in the innate immune system of various organs including the heart, lung, liver, kidney and gut [1,2]. These responses of the complement are mediated via three routes, viz., the classical, lectin and alternative pathways, subsequently resulting in elimination of the antigenic agent and activation of the in ammatory response [2]. During activation of the classical and alternative pathways, C3 is important for regulating various innate immune responses including promotion of opsonic phagocytosis, regulation of humoral immune response, and some T-cell biology [3].
C3 convertase is formed with fragments produced in the classical and lectin pathways (C4bC2b, formerly C4b2a) or the alternative pathway (C3bBb), which subsequently splits the C3 protein into C3a and C3b via proteolytic activity [4]. Subsequently, C3a acts as an anaphylatoxin, while C3b participates to form C5 convertase, following which it contributes to formation of the membrane attack complex (MAC) comprising C5b, C6, C7, C8 and polymeric C9 [5]. Based on the key role of C3 within the complement cascade, this protein has received great attention as a target of complement-directed therapeutic intervention [6].
The correlation between C3 concentration and in ammatory response is known to play a signi cant role in several gastrointestinal in ammatory diseases. Patients with in ammatory bowel diseases (IBD), such as ulcerative colitis (UC) and Crohn's disease (CD), show enhanced catabolism and deposition of C3 as well as circulating C3 conversion products at the site of in ammation [7][8][9]. Stimulation of C3 and IL-6 production has also been detected in basolateral and apical membranes of Caco-2 cells treated with IL-1β [10]. Moreover, expressions of IL-17 and C3 mRNA were remarkably increased in UC and CD, whereas levels of these two cytokines in the same a ictions were suppressed by p42/44, Mitogen-activated protein kinase (MAPK) inhibitor, and p38 MAPK inhibitor [11]. Furthermore, complement de ciency signi cantly suppresses tumor development through activation of the intestinal IL-1β/IL-17A axis in the azoxymethane and dextran sulfate sodium (DDS)-induced colitis-associated colorectal cancer (CAC) model [12]. Furthermore, activation of the C3 receptors (C3R), including C3a and C3b receptor (C3aR and C3bR), on the cell membrane is linked to regulation of the in ammatory response within various cells. C3aR activation ampli es the MAPK signaling pathway, leading to enhanced expression of proin ammatory cytokines. However, the involvement of iNOS and COX-2 proteins are yet to be examined [13,14]. This activation signal induces NLRP in ammasome activation via an increase of ERK1/2 and interaction with the NF-κB activation [14,15]. Furthermore, addition of C3 stimulates increased expressions of iNOS, IL-6 and IL-1β, and decreases the TGF-β and TNF-α expressions [16]. However, alterations in the C3R downstream-mediated in ammatory response in transverse colon of mice during C3 de ciency is poorly understood, although the complement cascade has been considered a novel therapeutic target for IBD.
In the current study, we investigated alternative regulation of the C3R downstream-mediated in ammatory response during C3 de ciency. Our study especially focuses on regulation of the iNOS-mediated COX-2 induction pathway, ASC-in ammasome pathway, NF-κB phosphorylation, and in ammatory cytokine expression in the transverse colon of C3 KO mice.

Results
Suppression of C3 protein in the kidney, spleen and thymus of C3 KO mice To verify the suppression of C3 protein, expression levels of the C3 protein were measured in the kidney, spleen and thymus of C3 KO mice. Compared to levels obtained in WT mice, we determined successful decrease in expression levels of the protein in the kidney, spleen and thymus of C3 KO mice. (Fig. 1C).
These results indicate that the expression of C3 protein is successfully inhibited in the kidney, spleen and thymus of C3 KO mice, generated using the CRISPR/Cas9-mediated technique.
Suppression of C3 and C3R protein in the transverse colon of C3 KO mice To verify the suppression of C3 protein, expression levels of the C3 protein were measured in the transverse colon of C3 KO mice. Compared to levels obtained in WT mice, we determined successful decrease in expression levels of the protein and mRNA, whereas C3aR and C3bR protein levels were higher in the transverse colon of C3 KO mice ( Fig. 2A,B). Moreover, tissue distribution of the C3 protein was detected in the intestinal villus of transverse colon of WT mice, whereas no signi cant color change was observed for C3 proteins in sections obtained from C3 KO mice (Fig. 2C). These results indicate that the expression of C3 protein is successfully inhibited in the transverse colon of C3 KO mice, generated using the CRISPR/Cas9-mediated technique.
Regulatory effects of C3 de ciency on MAPK signaling pathway Since the MAPK signaling pathway mediates the transfer of signals derived from C3R [13,14], we rst investigated whether C3 de ciency affects regulation of the MAPK signaling of the C3R downstream pathways in transverse colon. To achieve this, alterations in the phosphorylation of ERK, JNK and p38 were measured in the colon of C3 KO mice. As shown in Fig. 3, phosphorylation levels of three members in the MAPK signaling pathway are remarkably enhanced in KO mice, although the increase rates differed for each protein. The highest protein phosphorylation level was observed in JNK and p38. These results indicate that the MAPK signaling pathway of C3R downstream pathways may be activated in the transverse colon during C3 de ciency.
To investigate whether activation of the MAPK signaling pathway in C3R downstream pathways is accompanied with changes in the iNOS-mediated COX-2 induction pathway during C3 de ciency, alterations in the expression levels of iNOS, COX-2 were measured in the transverse colon of C3 KO mice. A similar pattern of regulation was observed in all three mediators of the iNOS-mediated COX-2 induction pathway. The expression levels of COX-2, iNOS proteins were signi cantly increased in the transverse colon of C3 KO mice as compared with WT mice, although the rate of increase was varied (Fig. 4). These results indicate that activation of the MAPK signaling pathway and iNOS-mediated COX-2 induction pathway in C3R downstream pathways is tightly linked to C3 de ciency in the transverse colon of C3 KO mice.
Regulatory effects of C3 de ciency on the ASCin ammasome pathway To investigate whether C3 de ciency can affect regulation of the ASC-in ammasome pathway of the C3R downstream pathways in transverse colon, altered expressions of NLRP3, Cleaved cas1/Cas1 and ASC were measured in the transverse colon of C3 KO mice. The expression level of ASC was remarkably increased in KO mice compared with WT mice. A similar pattern was observed for the expressions of the three in ammasomal proteins (Fig. 5). These results indicate that C3 de ciency is associated with upregulation of the ASC-in ammasome pathway of the C3R downstream pathway.
Regulatory effects of C3 de ciency on the NF-κB signaling pathway NF-κB pathway is important for the transcriptional regulation of numerous genes involved in the host immune and in ammatory response, and also for the proliferation and survival of cells [20]. We examined whether upregulation of the ASC-in ammasome pathway and activation of iNOS-mediated COX-2 induction pathway is accompanied by changes in the NF-κB signaling pathway during C3 de ciency. To achieve this, we measured for altered phosphorylation levels of NF-κB and IκB in the colon of C3 KO mice. As shown in Fig. 6, phosphorylation levels of two members in the NF-κB signaling pathway were remarkably enhanced in KO mice, although the increase rates differed for both proteins. Especially, the highest phosphorylation level of protein was observed in IκB. These results suggest that activation of the NF-κB signaling pathway regulated by upregulation of the ASC-in ammasome pathway and iNOS-mediated COX-2 induction pathway is associated with C3 de ciency in the transverse colon of C3 KO mice.
Regulatory effects of C3 de ciency on in ammatory cytokines Finally, we examined whether activation of the NF-κB signaling pathway during C3 de ciency induces regulation of in ammatory cytokine expressions in the transverse colon. To achieve this, the transcript levels of NF-κB, TNF-α, IL-6 and IL-1α were evaluated by RT-qPCR of the transverse colon in C3 KO mice. A similar pattern of regulation was observed for all four in ammatory cytokines. The mRNA levels of NF-κB, TNF-α, IL-6 and IL-1α cytokines were remarkably increased in C3 KO mice, as compared with WT mice.
TNF-α exhibited the highest increase, while NF-κB showed the lowest rate of increase (Fig. 7A). A similar increase was observed for levels of the IL-6 protein, in transverse colon obtained from C3 KO mice ( Fig. 7B). Moreover, the ELISA assay determined that protein levels of TNF-α and IL-6 were constantly maintained in the serum of WT and C3 KO mice (Fig. 7C). These results indicate that upregulation of the in ammatory cytokines regulated by activation of the NF-κB signaling pathway is associated with C3 de ciency in the transverse colon of C3 KO mice.

Discussion
Chronic gastrointestinal in ammation is the most common in ammatory response linked to the development of various IBDs, including CD and UC [21]. Complications of long-term in ammation are proposed to be a major contributor to the development of colorectal cancer (CRC). During the in ammatory response, diverse physiological events occur in the gastrointestinal mucosa and lumen, including cell activation, cytokine production, complement activation and tissue damage [22,23]. The regulatory factors assessed in this study are considered important for the balance between pro-and antiin ammatory responses, which is the driving force behind in ammation and immune response to treat these diseases [21]. We therefore undertook to investigate the effects of C3 de ciency on the C3R downstream-mediated in ammatory response of the gastrointestinal tract, by analyzing alterations in the iNOS-mediated COX-2 induction pathway, ASC-in ammasome pathway, NF-κB signaling pathway, and in ammatory cytokine expression, in the transverse colon of C3 KO mice. Results of the present study provide rst evidence that C3 de ciency may be tightly linked with upregulation of the in ammatory response of the C3R downstream signaling pathway in transverse colon of C3 KO mice. Furthermore, these results indicate that activation of C3 can be considered as one of the important factors during IBD. However, more studies are required to verify the molecular mechanism of C3 and C3 receptors in the transverse colon of C3 KO mice.
Several split products of complement, including C3a and C5a, play a major role in regulating the immune system activity including degranulation, extravasation and chemotaxis, as well as activation of the immune cells and non-myeloid cells [24,25]. Considering all factors of the complement, C3a is known to have con icting functions on the in ammatory response. Brie y, this product contributes to the proin ammatory response, including enhancement of the G-protein coupled C3aR expression in immune cells and non-myeloid cells, induction of oxidative burst in macrophages, and stimulation of histamine release from basophils and mast cells [25][26][27][28]. However, the anti-in ammatory role was also investigated in ischemia-reperfusion injury and in the sepsis model showing acute phase of in ammation [29,30]. Especially, C3a prevents the migration and degranulation of neutrophils, although other granulocytes are activated [31]. Furthermore, some signi cant alterations on the expression level of in ammatory cytokines were observed in C3 KO mice. The cytokine ratio between IL-10 and interferon (IFN)-γ or IL-17 levels shows a shift in the jejunum of ovalbumin challenged C3 KO mice [32]. The increase of TNF-α and IL-12 levels was greater in the colon of DSS-treated C3 KO mice than DSS-treated WT mice [33]. In the current study, the iNOS-mediated COX-2 induction pathway and in ammatory cytokine expressions were upregulated in the transverse colon of C3 KO mice. These results provide additional evidence that C3 de ciency plays an alternative role in the in ammatory response of transverse colon, although further studies are required to determine the molecular mechanism.
In the current study, we measured the mRNA level of some cytokines in the transverse colon of C3 KO mice. Enhanced levels of TNF-α, IL-6 and IL-1α transcripts were observed in the transverse colon during C3 de ciency, although the rate of increase varies widely. It is impossible to directly compare our ndings with previous results because the disease model used in each study was different. However, few studies revealed a correlation between C3 and in ammation in the gastrointestinal tract. Previous studies also reported signi cant alterations in the levels of cytokines and C3 protein in IBD. C3 metabolism (including cleavage, circulation and deposition) was signi cantly increased in IBD, while levels of IL-17 and C3 mRNA were enhanced in UC and CD [7][8][9]11]. Furthermore, IL-1β-induced C3 and IL-6 production was observed at the apical or basolateral membrane or chamber of CaCo-2 cells [10]. A similar response was also observed in macrophages after treatment with C3 peptides, where increased iNOS, IL-6 and IL-1β expressions and decreased TGF-β and TNF-α expressions were determined [16].
Oxidative stress is a condition in which the balance of oxidative stimulators and inhibitors in the body is disturbed by events such as in ammation. This ultimately causes oxidative damage to cells and the human body [34]. During this balancing, the iNOS-mediated COX-2 induction pathway is considered a key regulatory mechanism, since expressions of iNOS and COX-2 proteins are induced by a variety of pro-in ammatory stimuli (such as LPS and TNF-α) in various diseases [35,36]. The overexpression and activation of iNOS promotes the production of NO, which stimulates the activation of COX-2 [37]. This process, mediated by the NF-κB and MAPK signaling pathways, has a critical role in the regulation of cell growth and differentiation, as well as in the control of cellular responses to cytokines and stresses [38][39][40]. Also, the above in ammatory pathway regulated by the iNOS-mediated COX-2 induction pathway was observed to improve with diet components, including bers, polyphenols, and poly-unsaturated fatty acids, as well as lifestyle changes including fasting and physical exercise [41]. In the current study, we measured whether the iNOS-mediated COX-2 induction pathway of the C3R downstream pathway can be activated by C3 de ciency in the transverse colon. We observed signi cantly enhanced expression levels of COX-2 and iNOS in the transverse colon of C3 KO mice, as compared to WT mice. These results provide additional evidence that the role of C3 in transverse colon is associated with the iNOS-mediated COX-2 induction pathway, although we were unable to directly compare our ndings to previous studies.
Taken together, our study undertook to investigate the effects of C3 de ciency on the C3R downstream signaling pathway-mediated in ammatory response in the transverse colon. Our results indicate that C3 de ciency induces the upregulation of in ammatory cytokines through activation of the iNOS-mediated COX-2 induction pathway, ASC-in ammasome pathway, and NF-κB signaling pathway, in the transverse colon of C3 KO mice (Fig. 8). Furthermore, we provide additional evidence for the role of C3 in in ammatory responses and mucosal damage to the colon. However, this study provides limited information on the correlation between C3 de ciency and C3R downstream-mediated in ammatory response, since the functional validation for C3 de ciency was not analyzed in the transverse colon of C3 KO mice. Therefore, more experimental evidence is required to clarify the functional mechanism of the in ammatory response during C3 de ciency.

Animal care and use
All experiments and methods were performed in accordance with relevant guidelines and regulations. The experiments complied with the ARRIVE guidelines. Protocol for the C3 KO mice study was approved by the Pusan National University-Institutional Animal Care and Use Committee (PNU-IACUC; Approval Number PNU-2020-2657). The C3 KO and WT mice were handled at the Pusan National University-Laboratory Animal Resources Center, which is accredited by the Korea Food and Drug Administration (FDA) (Accredited Unit Number: 000231), and Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International (Accredited Unit Number: 001525). Seven-week-old C3 KO (n = 7) and WT (n = 7) mice having the Friend Virus B Type NIH (FVB) genetic background were kindly provided by the Department of Laboratory Animal Resources at the National Institute of Food and Drug Safety Evaluation (NIFDS, Chungju, Korea). The C3 KO mice have an 11-nt deletion mutation in exon 2 of the C3 gene, which is achieved using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) design tool (crispor.tefor.net) (Fig. 1A) [17]. Throughout the experimental period, all animals were provided ad libitum access to water, and a standard irradiated chow diet (Samtako BioKorea Co., Osan, Korea) consisting of moisture (12.5%), crude protein (25.43%), crude fat (6.06%), crude ber (3.9%), crude ash (5.31%), calcium (1.14%), and phosphorus (0.99%). All animals were maintained in a speci c pathogen free (SPF) state at 23 ± 2°C and 50 ± 10% relative humidity under a strict light cycle (lights on at 08:00 h and off at 20:00 h). After adaptation for 1 week, the mice were euthanized using a chamber lled with CO 2 gas, and transverse colons were subsequently harvested for further analysis.

Quantitative Real-Time PCR Analysis (RT-qPCR)
RT-qPCR was applied to assess the relative quantities of in ammatory cytokine mRNAs. Brie y, total RNA molecules were isolated from frozen transverse colon tissues using RNA Bee solution (Tet-Test Inc., Friendswood, TX, USA). After quanti cation of RNA, complement DNA (cDNA) was synthesized using a mixture of oligo-dT primer (Thermo Fisher Scienti c Inc., Waltham, MA, USA), dNTP and reverse transcriptase (Superscript II, 18064-014, Thermo Fisher Scienti c Inc.). RT-qPCR was then achieved using a cDNA template and 2⋅ Power SYBR Green (TOYOBO Co., Osaka, Japan), as described in a previous study [18]. The primer sequences used to evaluate the mRNA levels were as follows:

Histopathological analysis
Transverse colons collected from mice of the subset group were xed in 10% formalin for 48 h, embedded in para n wax, and then sectioned into 4 µm thick slices. The colon sections were collected on glass slides and stained with Hematoxylin & Eosin (H&E) (Sigma-Aldrich Co., St. Louis, MO, USA), after which they were examined by light microscopy for histopathology, at 400× magni cation.
Tissue distribution of the C3 protein was detected by IHC staining using light microscopy, as previously described [19]. Brie y, the transverse colon tissue samples were xed in 10% formalin for 12 h, embedded in para n, and sliced into 4 µm thick sections. These sections were subsequently depara nized with xylene, rehydrated, and pretreated for 30 min at room temperature with 1× PBS blocking buffer containing 10% goat serum (Vector Laboratories, Burlingame, CA, USA). The sections were then incubated with primary anti-C3 antibody (Abcam Com., Cambridge, UK), diluted 1:300 in 1× PBS blocking buffer. The antigen-antibody complexes were visualized with biotinylated secondary antibody (goat anti rabbit)conjugated horseradish peroxidase (HRP) streptavidin (Histostain-Plus Kit, Zymed, South San Francisco, CA, USA), at a dilution of 1:300 in PBS blocking buffer. Finally, C3 proteins were detected using stable diaminobenzidine (DAB) (Invitrogen Co., Carlsbad, CA, USA) and the Leica Application Suite (Leica Microsystems, Wetzlar, Germany).

Western blot
Total homogenate proteins were extracted from the transverse colon of C3 KO and WT mice using the Pro-Prep Protein Extraction Solution (Intron Biotechnology Inc., Seongnam, Korea). Following centrifugation of tissue homogenates at 13,000 rpm for 5 min, protein concentrations were determined using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scienti c Inc.). Proteins (30 µg Enzyme-linked immunosorbent assay (ELISA) for evaluating TNF-α and IL-6 cytokines Serum concentrations of TNF-α and IL-6 cytokines were measured using a mouse TNF-α enzyme-linked immunosorbent assay kit (Biolegend, San Diego, CA, USA) and IL-6 ELISA kit (Biolegend), according to the manufacturer's protocols. Brie y, serum was isolated from whole blood of each mouse, diluted 1:75,000, and pipetted into designated wells of the kit. The plate was incubated for 20 min at room temperature, followed by washing and addition of 1× enzyme-antibody conjugate. After incubation for 20 min at room temperature and additional washing, tetramethylbenzidine (TMB) substrate was added to each well, and the color alteration was determined using a Vmax plate reader (Molecular Devices, Sunnyvale, CA, USA) at 450 nm.

Statistical Analysis
Statistical signi cance was evaluated using the one-way analysis of variance (ANOVA) (SPSS for Windows, Release 10.10, Standard Version, Chicago, IL, USA) followed by Tukey's post hoc t-test for multiple comparisons. Data are presented as mean ± standard deviation (SD). p < 0.05 is considered to indicate a statistically signi cant difference.

Competing interests
The authors declare that they have no competing interests. blot analysis using anti-C3 antibody and HRP-labeled anti-rabbit IgG antibody. Band intensities were determined using an imaging densitometer, and expressions of the proteins were calculated relative to the intensity of β-actin. Three to ve mice per group were used for the preparation of tissue lysates, and Western blots were assayed in duplicate for each sample. Data are reported as the mean ± SD. * indicates p < 0.05 compared to the WT mice. control. Three to ve mice per group were used for preparing total RNA, and RT-PCR analysis was assayed in duplicate for each sample. (B) The expressions of C3aR, C3bR protein in the transverse colon were measured with Western blot analysis using anti-C3aR, C3bR antibody. After determining the intensity of each band using an imaging densitometer, relative levels of the C3aR, C3bR protein were calculated, based on the intensity of β-actin. Three to ve mice per group were used for preparation of the tissue lysates, and Western blots were assayed in duplicate for each sample. Data are reported as the mean ± SD. * indicates p < 0.05 compared to the WT mice. (C) Tissue distribution of C3 protein was analyzed in the transverse colon of WT and C3 KO mice. The C3 protein-speci c antibody-stained sections of the transverse colon from the WT and KO mice were observed at 400× magni cation using a light microscope. The large image in the right column is a magni ed image of the rectangle in the left column.
H&E-stained sections (low rectangle in left corner) were observed at 400× magni cation using a light microscope.

Figure 3
Expression Levels of members in the MAPK signaling pathway. (A) Expression levels of ERK, p-ERK, JNK, p-JNK, p38 and p-p38 proteins were determined by Western blot analysis using speci c primary antibody and HRP-labeled anti-rabbit IgG antibody. (B) Band intensities were determined using an imaging densitometer, and expressions of the proteins were calculated relative to the intensity of β-actin. Three to ve mice per group were used for the preparation of tissue lysates, and Western blots were assayed in duplicate for each sample. Data are reported as the mean ± SD. * indicates p < 0.05 compared to the WT mice.

Figure 4
Expression levels of members in the iNOS-mediated COX-2 induction pathway. (A) Expression levels of COX-2 and iNOS proteins were determined by Western blot analysis using speci c primary antibody and HRP-labeled anti-rabbit IgG antibody. (B) Band intensities were determined using an imaging densitometer, and expressions of the proteins were calculated relative to the intensity of β-actin. Three to ve mice per group were used for the preparation of the tissue lysates, and Western blots were assayed in duplicate for each sample. Data are reported as the mean ± SD. * indicates p < 0.05 compared to the WT mice.

Figure 5
Expression levels of members in the ASC-in ammasome pathway. (A) Expression levels of NLRP3, Cleaved cas1/Cas1 and ASC proteins were determined by Western blot analysis using the speci c primary antibody and HRP-labeled anti-rabbit IgG antibody. (B) Band intensities were determined using an imaging densitometer, and protein expressions were calculated relative to the intensity of β-actin. Three to ve mice per group were used for the preparation of tissue lysates, and Western blots were assayed in duplicate for each sample. Data are reported as the mean ± SD. * indicates p < 0.05 compared to the WT mice. Figure 6 Expression levels of members in the NF-κB signaling pathway. (A) Expression levels of NF-κB and IκB proteins were determined by Western blot analysis using speci c primary antibody and HRP-labeled antirabbit IgG antibody. (B) Band intensities were determined using an imaging densitometer, and protein expressions were calculated relative to the intensity of β-actin. Three to ve mice per group were used for the preparation of the tissue lysates, and Western blots were assayed in duplicate for each sample. Data are reported as the mean ± SD. * indicates p < 0.05 compared to the WT mice.

Figure 8
Suggested mechanism of in ammatory response in the C3R downstream pathway during C3 de ciency. In this scheme, the downregulation of C3 concentration is thought to activate the iNOS-mediated COX-2 induction pathway, MAPK signaling pathway, ASC-in ammasome pathway, and NF-κB signaling pathway. Finally, activated NF-κB translocate into nucleus, and promotes the expression of in ammatory cytokines in the transverse colon.