Moxibustion Inhibits the Expression of Colonic NLRP3 through miR7/RNF183/NF-κB Signaling Pathway in UC Rats

Background Moxibustion has been recognized as an effective approach for ulcerative colitis, yet its mechanism is not clear. The research aimed to investigate the influence of moxibustion on the activation of NLRP3 inflammasome and its mechanism in treating ulcerative colitis by observing miR7/RNF183 inducing IκB α ubiquitination to regulate NF-κB signaling pathway in an ulcerative colitis rat model. Methods An ulcerative colitis rat model was established by unlimited access to self-administration of 3.5% (w/v) dextran sulfate sodium solution. Mild moxibustion was applied to bilateral Tianshu points (ST25) in the moxa-stick moxibustion group; rats in the control group were intervened by intraperitoneal injection of ubiquitination inhibitor, MG132. The disease activity index was determined at the end of the intervention; colon injury was observed and scored after hematoxylin-eosin staining; the immunohistochemical method was adopted to detect the expressions of colonic IL-1β and NLRP3 proteins; Western blot determined the expressions of RNF183, IκB α, and NF-κB p65 proteins in the colon; the immunofluorescence test was used to observe the coexpression of IκB α/ubiquitin and IκB α/RNF183 proteins in the colon; immunoprecipitation assay was adopted to observe the interaction between IκB α and RNF183 proteins; and quantitative real-time polymerase chain reaction determined the expression of colonic miR7. Results Moxibustion lowered the disease activity index, manifesting as restored colonic tissue and reduced inflammatory reaction, and decreased expression levels of NLRP3 and IL-1β proteins, compared with the model group. It also reduced colonic expression of NF-κB p65 protein, together with the increased level of IκB α protein and weaker expression levels of ubiquitin and RNF183 proteins and mRNAs and stronger expression of miR7. There were no significant differences between the moxa-stick moxibustion group and the control group except the expressions of RNF183 protein and mRNA and miR7. Conclusion Moxibustion encourages the recovery of colon injury probably by regulating the expression of NLRP3 protein in ulcerative colitis rats through miR7/RNF183/NF-κB signaling pathway.

inflammasome activation is beneficial to the inhibition of inflammation and the reconstruction of colonic mucosal immune homeostasis [6]. Ubiquitination modification has been proved to be one of the effective ways to regulate NLRP3. e ubiquitinated cascade-related proteins in the nuclear factor kappa B (NF-κB) signaling pathway participate in the inflammatory response of IBD via regulating the transcription of NLRP3 and its downstream inflammatory factors [7]. Ring finger protein 183 (RNF183) promotes the degradation of ubiquitinated IκB α and boosts the nuclear translocation of NF-κB p65, triggering intestinal inflammation [8]. erefore, it may be one part of the mechanism to suppress the activation of NLRP3 inflammasome and alleviate colonic inflammatory injury in UC through regulating the ubiquitination modification in the NF-κB signaling pathway.
In recent years, there has been a surge in the clinical reports of moxibustion treatment of IBD [9][10][11], which all showed satisfactory improvements in symptoms like abdominal pain and diarrhea, and thus, moxibustion has been recommended as an adjuvant treatment in clinical practice [12]. However, it still remains a mystery how this treatment works. Our previous findings manifested that moxibustion downregulated the expression levels of NLRP3, IL-1β, and ubiquitin (Ub) proteins in the colon of UC rats so as to fulfill its anti-inflammatory function and repair inflammatory damage in the colon [13]. It requires in-depth studies to find out if RNF183 is involved for moxibustion to regulate the ubiquitination level of the NF-κB signaling pathway in colon tissue and inhibit the expression of NLRP3. e current study adopted a rat UC model established using dextran sulfate sodium (DSS) to observe the relationship between the RNF183-induced IκB α ubiquitination and the expression of NLRP3 in colon tissue and the regulatory effect of moxibustion, for elaborating the anti-inflammatory immune regulation of moxibustion in the treatment of UC from the perspective of the signal pathway.

Animals.
Forty-four clean male Sprague-Dawley (SD) rats weighing 150 ± 10 g were purchased from Shanghai SLAC Laboratory Animal Co. Ltd. (SYXK(hu)2014-0008) and were bred in the Laboratory Animal Center of Shanghai University of Traditional Chinese Medicine. After three-day adaptive feeding and exclusion of unhealthy rats by observing their diet, activity, posture, hair, etc., the experiment started. All experimental operations strictly followed the Guiding Opinions on the Treatment of Experimental Animals (National Science-Technology and Finance (2006).398).
is experiment has been approved by the Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine (PZSHUTCM18111612).

UC Rat Modeling.
e UC rat model was prepared by unlimited access to self-administration of 3.5% (w/v) DSS solution (molecular mass 36,000-50,000) for 7 consecutive days [14]. At the end of modeling, one rat was randomly selected from each group for model identification. Intervention would begin given the success of the model.

Intervention.
e 44 rats were randomly divided into four groups: normal group (NG), model group (MG), moxastick moxibustion group (MSMG), and control group (CG), 11 each. Rats in the MSMG were given moxibustion at bilateral Tianshu (ST25) points [15], that is, a moxa stick was ignited and suspended over the acupoints by 2-3 cm away, 10 min each time, once a day, for a total of 8 days (Figure 1). Rats in the CG received intraperitoneal injection of ubiquitination inhibitor MG132, 2.1 mL/kg each time, once a day for 8 consecutive days [16]. Rats in the MG did not receive any intervention but the same grasping and fixing as in the MSMG. Rats in the NG were not subjected to modeling or treatment but only grasped and fixed in the same way as in the MSMG. During treatment, all the rats were provided with free access to 1% DSS solution to maintain the model stability except for those in the NG.

Colonic Morphology.
Hematoxylin-eosin (HE) staining was used to observe and score the changes in the colonic morphology under an optical microscope [19].

Immunohistochemistry (IHC).
After the colon tissue sections were deparaffinized, hydrated, antigen retrieved, and sealed, the primary antibody (NLRP3 1 : 300; IL-1β 1 : 50) was added dropwise and incubated overnight at 4°C. On the next day, the secondary antibody (1 : 200) was added dropwise and incubated at room temperature for 1 h; DAB developed. After hematoxylin-counterstained for 1 min, the sections were differentiated, dehydrated, transparentized, and mounted. e tan particles found in the cytoplasm were taken as the positive target. Five visual fields were randomly selected from each slice to be photographed, and the Image-Plus Pro 6.0 software was used to analyze and calculate the integral optical density (IOD) of each photo. e average value was taken as the IOD of the slice.

Western Blotting (WB)
. RIPA lysis buffer was added to colon tissues to extract the total protein, and protein quantification was performed by the BCA method. e proteins were separated by SDS-PAGE gel electrophoresis and transferred to the PVDF membrane. en, the PVDF membrane was blocked with 5% BSA for 1 h, and the primary antibody (RNF183 1 : 1000; IκB α 1 : 1000; NF-κBp65 1 : 1000) was added and incubated overnight at 4°C. On the next day, HRP + secondary antibody (1 : 1000) was added dropwise and incubated at room temperature for 1 h, and the expression of the target protein was detected by the ECL chemiluminescence method. e PVDF membrane was reblocked for 1 h; HRP + GAPDH primary antibody (1 : 1000) was added and incubated for 1 h, and the expression of internal reference protein was detected. e Image J software was adopted to analyze the gray value of the corresponding band and calculate the ratio of the target protein to the internal reference protein.

Immunofluorescence (IF) and Colocalization.
After deparaffinization, hydration, antigen retrieval, and sealing of the colon tissue sections, the primary antibody (Ub 1: 500) was added and incubated overnight at 4°C. On the next day, a fluorescent secondary antibody (1 : 200) was added in shade and incubated at 37°C for 45 min, counterstained with DAPI counterstain for 10 min, and then mounted. One visual field was chosen and activated with excitation light of the corresponding wavelength. DAPI-stained nucleus showed blue, and the positive expression of Ub protein was in red. Pictures were taken and merged. Five fields were randomly selected from each slice; the IOD of the positive target was calculated; and the average was taken for analysis.
Colocalization of different proteins is as follows: the first primary antibody (IκB α 1 : 200) was treated as above. Before counterstaining with DAPI counterstain, 5% BSA was used for reblocking and incubated for 1 h at room temperature. e second primary antibody (Ub 1 : 500; RNF183 1 : 200) was then added and incubated at 37°C for 2 h. Afterwards, the second fluorescent secondary antibody (1 : 200) was added and incubated at 37°C for 1 h. Finally, the nucleus was counterstained with DAPI for 10 min and then mounted. A field was chosen and activated with excitation light of the corresponding wavelength. DAPI-stained nucleus was in blue; positive Ub and RNF183 protein expression were in red; and positive IκBα protein was in green. Pictures were taken and merged.
2.11. Immunoprecipitation (IP). IP protein lysate was added to the colon tissues for extracting the total protein, and the protein concentration was determined by the BCA method. e cross-linking buffer and primary antibody were added into a spin column containing the resin, and they were incubated at room temperature to bind the antibody to protein A/G agarose. en, cross-linking buffer and DSS (disuccinimidyl suberate) were added to the spin column for cross-linking the combined antibody. Antigen immunoprecipitation was completed by adding the pretreated tissue lysate into the spin column containing the primary antibody and cross-linked resin and incubated at 4°C overnight. After the spin column was added the elution buffer, incubated, and centrifuged, the antigen eluent was obtained. And, the loading buffer was added into the eluted protein solution for SDS-PAGE analysis. Finally, Ub or RNF183 primary antibody was added for incubation, and the expression of target protein in elution protein solution was detected by WB in order to observe the ubiquitination of IκB α and interaction of RNF183-IκB α. Evidence-Based Complementary and Alternative Medicine 2.12. Statistical Analysis. All data were statistically processed using SPSS version 25.0 statistical software. e measurement data were firstly tested for distribution. ose conforming to normal distribution were expressed as X ± S, while those not were expressed as median (P 25 , P 75 ). Between-group differences were analyzed using one-way ANOVA when it was normal distribution and homogenous variance, and the least significant difference (LSD) test for pairwise comparisons. For heterogeneous variance, the Games-Howell test was applied. A nonparametric test was employed for data of skewed distribution. e test level was set as α � 0.05, and P < 0.05 was recognized as statistical significance.

DAI.
Before treatment, the DAI score was significantly higher in the MG, MSMG, and CG compared with the NG (all P < 0.01). After treatment, the DAI score was significantly higher in the MG than in the NG (P < 0.01); compared with the MG, the DAI score dropped notably in the MSMG and CG (both P < 0.01); there was no significant difference between the CG and MSMG (P > 0.05; Figures 2(a) and 2(b)).

Morphological Observation of the Colon and Pathological
Damage Score. Under the microscope, the colonic mucosal epithelium of rats in the NG was rather complete, with regularly arranged well-formed glands, and the submucosal connective tissues were not infiltrated by inflammatory cells, congested, or swollen. However, rats in the MG had severe colon injury, demonstrating incomplete tissue structure, mucosal epithelium, and gland defects, continuous ulcer formation, some crypt abscesses, notable swelling in connective tissues, and significant inflammatory cell infiltration. Compared with the NG, the colonic pathology score was significantly higher in the MG (P < 0.01). In the MSMG and CG, the structure of colonic mucosa was substantially intact, with new epithelial cells and gland hyperplasia, ulcers basically healed, and the submucosal connective tissues had mild inflammatory cell infiltration. Compared with the MG, the score of colonic pathological damage was significantly reduced in both MSMG and CG (both P < 0.01; Figures 2(c) and 2(d)).

Expression of NLRP3 and IL-1β Proteins in the Colon.
NLRP3 and IL-1β proteins were mainly expressed in intestinal mucosal epithelial cells, lymphocytes in the mucosal stroma, and monocyte/macrophage cytoplasm. Compared with the NG, the expression of colonic NLRP3 and IL-1β proteins increased significantly in the MG (P < 0.05 and P < 0.01, respectively). Compared with the MG, the expression of colonic NLRP3 and IL-1β proteins showed a notable decrease in both MSMG and CG (P < 0.05 and P < 0.01, respectively). ere were no significant differences in the expression of colonic NLRP3 and IL-1β proteins between CG and the MSMG (both P > 0.05; Figures 2(e)-2(h)).

Expression of IκB α, Ubiquitinated IκB α, and NF-κB p65
Proteins in the Colon. e colonic IκB α protein was separated by IP, and then the eluent was incubated with the ubiquitin primary antibody for WB verification. Compared with the NG, the expression of ubiquitin in IP eluent had strengthened when the expression of IκB α protein was downregulated in the MG (P < 0.01). Compared with the MG, the expression of ubiquitin in IP eluents weakened, and the expression of IκB α protein in the colon of rats in the MSMG and CG was upregulated (all P < 0.01; Figures 3(a), 3(b), and 3(d)). Compared with the NG, the expression of colonic NF-κB p65 protein was significantly upregulated in the MG (P < 0.01). Compared with the MG, the expressions of NF-κB p65 protein decreased in the MSMG and CG (both P < 0.01). e differences in the expression of colonic NF-κB p65 and IκB α proteins were statistically insignificant between the CG and the MSMG (both P > 0.05; Figures 3(c) and 3(e)).

Expression of Ub Protein and Its Colocalization with IκB α.
Ub protein was mainly distributed in the intestinal lamina propria and weakly expressed in the mucosal epithelium (Figure 4(a)). Compared with the NG, the expression of Ub protein in the colon was significantly stronger in the MG (P < 0.01); compared with the MG, the expression of Ub protein in colon tissue was notably weaker in the MSMG and the CG (both P < 0.01). ere was no significant difference in the expression of Ub protein in the colon between the MSMG and CG (P > 0.05; Figures 4(a) and 4(b)). e immunofluorescence colocalization results revealed positive expression of Ub and IκB α proteins in the mucosal epithelium, lamina propria, and colonic submucosa in each group. e positive expression of Ub protein was in green fluorescence, and the IκB α protein was in red. e co-localized expression of the two proteins was in a color approaching yellow, mostly in the colonic mucosal lamina propria, which indirectly indicated their binding effect (Figure 4(c)).

Expression of RNF183 Protein and mRNA and Its
Colocalization with IκB α. Compared with the NG, the expression of colonic RNF183 protein and mRNA increased in the MG (both P < 0.01); compared with the MG, the expression of colonic RNF183 protein and mRNA reduced significantly in both the MSMG and the CG (all P < 0.05).
ere were also significant differences in the expression of RNF183 protein and mRNA in colon tissues between the MSMG and the CG (both P < 0.01; Figures 5(a)-5(c)). e immunofluorescence colocalization results showed positive expression of RNF183 and IκB α proteins in the mucosal epithelium, lamina propria, and submucosa of the colon. e positive expression of RNF183 protein was in red fluorescence, and IκB α protein was in green. e co-localized expression of the two was colored nearly yellow, more often found in the lamina propria ( Figure 6). e results of IP probably further affirmed their binding effect (Figure 4(d)).

Expression of miR7 in the Colon.
Compared with the NG, the expression of miR7 in the colon was significantly downregulated in the MG (P < 0.01); compared with the MG, the expression of miR7 in the colon was markedly upregulated in the MSMG and the CG (P < 0.05, P < 0.01). A significant difference was also found between the MSMG and the CG in comparing the expression of miR7 in colon tissue (P < 0.05; Figure 5(d)).

Discussion
UC is a common digestive disease that may severely affect the quality of life and mood of the patients due to long-term abdominal pain, diarrhea, bloody stools, and so on [20,21]. Moxibustion therapy obtains its curative effect by thermal stimulation to acupoints generated by the burning of moxa. Known for its gentle action and safe operation, moxibustion works well in treating intestinal diseases mainly manifested by symptoms such as abdominal pain and diarrhea [22,23]. In recent years, many studies have found that moxibustion can improve the clinical symptoms and mucosal damage in UC patients [24,25], and it is an effective method worthy of attention and recommended in the treatment of UC [10]. An in-depth understanding of the mechanism of moxibustion in the treatment of UC will help provide a scientific basis for its clinical application. e current study demonstrated that moxibustion reduced DAI and the pathological score of the colon in UC rats, indicative of colonic mucosal repair and reduced inflammation, which is consistent with the previous research results [26,27].
Although the pathogenesis of UC is still unclear, colon inflammation is recognized as the main pathological manifestation and underlying pathogenic mechanism, which makes anti-inflammation and promoting mucosal healing become the fundamental principles in UC treatment [28].
Under normal physiological conditions, NLRP3 inflammasome protects intestinal mucosa. However, under pathological conditions, an increased level of NLRP3 will cause inflammation. e study also showed that the inflammatory response induced by abnormally activated NLRP3 played an important role in the onset, progression, and prognosis of IBD [29]. Various pathogenic factors act on the intestinal mucosa in UC patients, inducing abnormal activation of NLRP3 inflammasome and elevating the expression of IL-1β and other inflammatory factors as well, subsequently causing topical intestinal mucosal damage. Knockout or inhibition of NLRP3 activation will help alleviate colon inflammation [30]. It is reported by a few studies that acupuncture can produce a certain regulatory effect on NLRP3 in a variety of diseases [31][32][33]. Our previous research also showed that moxibustion can inhibit the expression of colonic NLRP3 in IBD rats [13,34]. In this study, we found that the expression of NLRP3 and IL-1β proteins in UC rat colon declined after moxibustion intervention, which may be part of the mechanism in anti-inflammation and colonic mucosa repair.
NLRP3 inflammasome is one of the most studied inflammasomes so far and has been found involved in many negative regulatory mechanisms [35]. Among them, NF-κB is a crucial transcription factor that affects the expression of NLRP3 protein.
e activation of NLRP3 inflammasome usually requires dual signals. e first signal, also the priming stage, is that the toll-like receptor (TLR) or tumor necrosis factor receptor (TNFR) induces the activation of NF-κB, thereby upregulating the expression of NLRP3 and IL-1β/IL-18 precursor. ere are two NF-κB binding sites in the gene promoter region of NLRP3, which are necessary for NLRP3 gene transcription [36,37], considered to be the key rate-limiting step in NLRP3 inflammasome activation. e results of this study showed that the expression of NF-κB p65 protein was significantly upregulated in UC rat colon, accompanied by severe inflammatory damage to the colon; the abnormally increased NF-κB p65 protein was reduced, and the colon damage was repaired after moxibustion, which were in line with the previous reports [38,39]. erefore, we can say that moxibustion may play its anti-inflammatory function by inhibiting the NF-κB signaling pathway, reducing NLRP3 transcription, and suppressing NLRP3 activation.
In addition to phosphorylation, ubiquitination is another important way of protein modification that affects cell signaling pathway networks. Ubiquitination modification refers to the process of Ub covalently binding to the target protein under the catalysis of a series of enzymes. Its main function is protein degradation, clearance, and activity regulation. In IBD patients, mucosal Mφ is the activated phenotype, and the expression of ubiquitin protease is significantly upregulated, manifested by antigen presentation and inflammatory response [40]. It can be seen that abnormal ubiquitination modification is involved in the onset and development of IBD, but the details are still under exploration. Research revealed that ubiquitination modification played an important role in regulating the activation of the NF-κB signaling pathway [41], thus getting involved in the colonic inflammation [42]. E3 ligase is a key factor in ubiquitination modification. A variety of RING E3s, including RNF183, have been confirmed closely related to IBD [43]. Yu et al. [8] found that RNF183 can promote the degradation of ubiquitinated IκB α, leading to nuclear translocation of NF-κB p65, thereby inducing intestinal inflammation. At the same time, microRNA7 (miR7) can regulate RNF183   Evidence-Based Complementary and Alternative Medicine [44], so that inhibiting the activity of miR7 will help repair the TNBS-induced mucosal damage in IBD [45]. In this study, the expression of colonic miR7 increased; the expression of Ub and RNF183 proteins decreased; and the expression of IκB α increased in the MSMG, together with notable improvement in colonic inflammation. It is suggested that moxibustion may inhibit the expression of RNF183 protein by regulating miR7, prevent the binding of IκB α and RNF183, and carry out ubiquitination modification and degradation, thereby reducing the expression of NF-κB p65, downregulating the transcription and activation of NLRP3, which may be one part of the mechanism in moxibustion treatment of UC. However, there still exist some shortcomings in this study. Firstly, since we failed to find an appropriate RNF183 IP antibody, so we only did one-way IP in the co-IP test between RNF183 and IκB α proteins in this experiment. Meanwhile, the immunofluorescence colocalization method showed certain interaction between the two proteins though the evidence was not sufficient. erefore, based on the previous studies [8,46] and the preliminary results of our experiment, we speculate that moxibustion can regulate IκB α possibly through RNF183. Available RNF183 IP antibody, pull-down, mass spectrometry, and quantitative proteomics can help confirm the hypothesis and assess the effect of moxibustion, and the relevant experiments will be carried out in our future research. Secondly, the experiment used MG132 as the control for inhibiting ubiquitination. is drug can selectively inhibit proteasome, for example, it can inhibit the degradation of IκB α mediated by the proteasome and effectively inhibit the activation of NF-κB induced by TNF-α in A549 cells [47]. Hence, although this study suggested that MG132 may adjust the degradation of IκB α by modulating miR7 and RNF183, it could also be a direct inhibition on the degradation, which may be one of the reasons for increased expression of IκB α in the CG during the IP experiment. ere is no evidence in this study but can only expect in vivo application of miR7 or RNF183 RNAi to confirm it, so as to provide a more comprehensive and reliable basis for further revealing the mechanism of moxibustion in treating UC.

Data Availability
e research data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare no conflicts of interest.

Authors' Contributions
Li XY, Yang YT, Zhao Y, and Yang G performed the animal experiment and detection of all indexes. Li XY and Kong XH analyzed the data. Li XY and Zhang D wrote this manuscript. Hong J and Ma XP revised this manuscript. Zhang D designed and supervised this research. Ma XP devoted to guidance. All authors approved the final version of this article, including the author list.