Abstract
OBJECTIVE:
To investigate the effect and mechanism of mild moxibustion on the non-neuronal cholinergic system (NNCS) in rats with ulcerative colitis (UC).
METHODS:
UC rat model was established by administering 4% dextran sulfate sodium. After 7 d, mild moxibustion, α7 nicotinic acetylcholine receptors (α7nAchRs) antagonist (α-bungarotoxin, α-BGT), vesicular acetylcholine transport inhibitor (vesamicol hydrochloride, VH) and organic cation transporters inhibitor (quinine, Qu) treatments were performed once daily for 7 d. Haematoxylin and eosin staining was used for morphological evaluation of colon tissues. Enzyme-linked immunosorbent assay (ELISA) was used to measure the protein expressions of interleukin-1β (IL-1β) and choline acetyltransferase (ChAT) in colon tissue. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) was used to detect the mRNA expressions of IL-1β, carnosine acetyltransferase (CarAT), ChAT, and nuclear factor kappa-B p65 subunit (NF-κB p65) in colon tissue. Western blot was used to detect NF-κB p65 protein expression in colon tissue. Immunofluorescence was used to detect the expressions of neuronal acetylcholine (nAch) and non-neuronal acetylcholine (nnAch, released by NNCS) in colon tissue.
RESULTS:
Mild moxibustion inhibited colon inflammation and repaired mucosal damage to the colon in UC rats. Meanwhile, mild moxibustion could downregulate the expressions of IL-1β, NF-κB p65 protein and mRNA (P < 0.01), and upregulate the expressions of ChAT protein and CarAT mRNA (P < 0.05, P < 0.01). The α7nAChR antagonist α-BGT can reverse the protective effect of mild moxibustion on the UC and the inhibitory effect on the inflammatory factors. VH cannot affect the effect of mild moxibustion on the expressions of IL-1β and nnAch, while Qu can reverse the effect of mild moxibustion on the expression of IL-1β and nnAch.
CONCLUSIONS:
Mild moxibustion can inhibite colon inflammation in UC rats, which is closely related to the release of acetylcholine by NNCS and its mediated mechanism of cholinergic anti-inflammation pathway.
Keywords: colitis, ulcerative; non-neuronal cholinergic system; neuroimmunomodulation; mild moxibustion
1. INTRODUCTION
Ulcerative colitis (UC) is a chronic non-specific inflammatory bowel disease with unclear etiology. In 2000, Tracey et al 1 introduced the cholinergic anti-inflammation pathway (CAP), highlighting a pivotal mechanism for modulating bodily inflammation through immune pathway. Neuronal acetylcholine (nAch), originating in cholinergic nerve fibers, engages the α7 nicotinic acetylcholine receptors (α7nAchR) on macrophages. This results in the activation of downstream pathways, notably involving nuclear factor kappa B (NF-κB), which in turn inhibits the release of pro-inflammatory cytokines such as tumor necrosis factor-alpha, interleukin-1β (IL-1β), IL-17α, and high-mobility group box 1 (HMGB1).
Additional research has revealed a non-neuronal cholinergic system (NNCS) within human physiology, which locally synthesizes and releases acetylcholine (nnAch) to inhibit pro-inflammatory cytokines. Although nnAch and nAch share a similar biosynthetic pathway catalyzed by choline acetyltransferase (ChAT), they differ significantly in their release mechanisms: nnAch is primarily secreted via organic cation transporters (OCT), whereas nAch relies on the vesicular ACh transporter (VAChT).2
Moxibustion, a key component of Traditional Chinese Medicine, has demonstrated beneficial effects in UC treatment, substantiated by various clinical and basic research studies.3,⇓-5 Prior investigations indicate that acupuncture can activate the vagus nerve and CAP, reducing pro-inflammatory cytokine production.6,⇓-8 Moreover, electroacupuncture has been shown to alleviate colonic inflammation in UC rat models by modulating the expression of α7nAChR and downstream IL-1β within the CAP pathway.9 Despite these advances, the roles of the NNCS and the CAP’s anti-inflammatory mechanisms remain underexplored. This study focuses on the anti-inflammatory function of the intestinal CAP to assess the effects of mild moxibustion at the Zusanli (ST36) on UC rats. Further investigation aims to determine whether intestinal acetylcholine primarily originates from cholinergic nerve endings or the local NNCS.
2. MATERIALS AND METHODS
2.1. Animals
Healthy adult male clean grade Sprague-Dawley rats, weighing (160 ± 20) g, purchased in Shanghai Slack Laboratory Animal Co., Ltd., animal certificate number: SYXK (Shanghai) 2014-0008, raised in the clean grade breeding room of the Laboratory Animal Center of Shanghai University of Traditional Chinese Medicine, temperature (20 ± 2) ℃, humidity 50%-70%, 12 h light and 12 h dark. This study was approved by the Ethics Committee of the Experimental Animal Center of Shanghai University of Traditional Chinese Medicine (No. SZY201706025) and the procedures were conformed to the Guide for the Care and Use of Laboratory Animals.
2.2. Dextran sulfate sodium-induced UC model preparation
For preparing the UC rat model, the adult male clean grade Sprague-Dawley rats received 4% dextran sodium sulfate (DSS, 36000-50000 Da, MP Biomedicals, Santa Ana, CA, USA) for 7 consecutive days, and maintaining the stability of the model with 1% DSS starting from the 8th day.10 After the modeling was completed, the model was identified, and the success of modeling in UC rats was judged by observing the general condition and colonic histopathology. Meanwhile, rats in the control group received normal drinking water.
2.3. Grouping and interventions
This study is divided into three experiments.
Experiment 1: the rats were randomly divided into 5 groups: the control (Con) group, the UC model (Mod) group, the mild-moxibustion (Mox) group, the alpha-bungarotoxin (α-BGT) group, and the alpha-bungarotoxin + mild-moxibustion (α-BGT + Mox) group, with 10 rats in each group.
Experiment 2: the rats were randomly divided into 5 groups: the control (Con) group, the UC model (Mod) group, the mild-moxibustion (Mox) group, the vesamicol hydrochloride (VH) group, and the vesamicol hydrochloride + mild-moxibustion (VH + Mox) group, with 10 rats in each group. However, due to model creation during the experiment, some rats died, resulting in 8 rats in the Mod group and 9 rats in the Mox, VH, and VH + Mox groups included in the final analysis.
Experiment 3: the rats were randomly divided into 5 groups: the control (Con) group, the UC model (Mod) group, the mild-moxibustion (Mox) group, the quinine (Qu) group, and the quinine + mild-moxibustion (Qu + Mox) group, with 10 rats in each group.
UC rat model was prepared by DSS except for the Con group. For mild moxibustion, a moxa stick of 5 mm in diameter was ignited and placed 2-3 cm above the bilateral Zusanli (ST36) for 10 min per day for 7 d of continuous treatment. In the α-BGT group, the rats were intraperitoneally injected with 1 μg/kg α-BGT. In the VH group, the rats were intraperitoneally injected with 0.5 mM VH (0.1 mL). In the Qu group, the rats were intraperitoneally injected with 100 μM Qu (0.1 mL).
The rats in the α-BGT + Mox group, VH + Mox group and Qu + Mox group were injected intraperitoneally with 1 μg/kg α-BGT, 0.5 mM VH (0.1 mL), 100 μM Qu (0.1 mL) half one hour before moxibustion intervention respectively. The rats in the Con group and the Mod group were given the same fixation as the Mox group without any intervention at 10 o’clock every day for a total of 7 d.
After the intervention, the rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital. The abdominal cavity was exposed, and the distal colon measuring 6-8 cm in length was dissected longitudinally along the mesentery. It was then divided into two parts, one of which was fixed in 4% paraformaldehyde, while the remaining portion was placed in liquid nitrogen for 1 h and transferred to a －80 ℃ freezer.
2.4. Histopathological observation of colon
Colon tissues fixed at 4% paraformaldehyde were dehydrated, embedded, and sliced into 4 μm sections for hematoxylin and eosin (HE) staining. Morphologic changes of colonic tissue were observed under an optical microscope.
2.5. Detection of NF-κB p65 in colon tissue by western blot
The protein was extracted from colon tissue using radio immunoprecipitation assay lysis (RIPA) buffer and the protein concentration was determined by bicinchoninic acid protein concentration assay kit. After the protein bands were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the 5% skimmed milk powder was blocked at room temperature for 1 h, and NF-κB p65 antibody (ab19870, abcam, Cambridge, UK) and glyceraldehyde-3-phosphate dehydrogenase (ab181602, abcam, Cambridge, UK) were added and incubated overnight at 4 ℃. Tris-buffered saline with tween-20 (TBST) is washed 3 times for 5 min each, incubated at 37 ℃ for 1 h after adding 1∶1000 diluted horseradish peroxidase-labeled secondary antibody, and TBST washed 3 times for 5 min each. Enhanced chemiluminescence is configured for color development, the gel imaging system takes a band map, and the grayscale value is analyzed using Image Pro Plus software (Media Cybernetics, Bethesda, MD, USA).
2.6. Detection of CarAT, ChAT, NF-κB, IL-1β mRNAs in colon tissue by reverse transcription quantitative real-time polymerase chain reaction
Total RNA was extracted by trizol from an appropriate amount of frozen colon tissue. The RNA was separated by chloroform, purified by isopropanol, washed with 75% alcohol, and dissolved with ddH2O. The RNA purity and concentration were measured using a NanoDrop 1000 (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was synthesized using PrimeScript RT reagent Kit (Takara, Osaka, Japan) according to the manufacturer’s protocols.
Reaction mixtures were prepared with 2 μL cDNA, 10 μL 2 × SYBR Green Master Mix (Takara, Osaka, Japan) and primers. The sequences of the primers are listed in supplementary Table 1. The qPCR was conducted using a Roche PCR System (Roche, Basel, Switzerland) applying the following conditions: 95 ℃/30 s, 40 cycles of 95 ℃/10 s, 60 ℃/20 s, and 72 ℃/20 s. The results were calculated by the 2-ΔΔCt method.
2.7. Detection of ChAT and proinflammatory cytokine IL-1β in colon tissue by ELISA
The frozen colon tissue was cut into small fragments, add RIPA, homogenized with a homogenizer until completely lysed. Then the sample was centrifuged at 12 000 rpm at 4 ℃ for 15 min, the supernatant was taken, and the protein concentration was determined using the bicinchoninic acid assay according to the instructions. Rat ChAT (ER0530, Wuhan Fine Biotech Co., Ltd., Wuhan, China), and IL-1β ELISA kits (PH303, Beyotime Biotechnology, Shanghai, China) were used to detect the content of related proteins in colon tissue samples. The optical density values of each hole were measured at 450 nm wavelength by enzyme labeling instrument (Bio Tek, Winooski, VT, USA), and the content of corresponding protein was calculated after formula conversion.
2.8. Detection of nAch and nnAch in colon tissue by immunofluorescence
In experiment 2, nACh was labeled using ChAT and protein gene product 9.5 (PGP9.5) co-labeling, while nnACh was labeled using ChAT and organic cation transport 1 (OCT1) co-labeling. In experiment 3, nnACh was labeled using ChAT and OCT1 co-labeling. Colon slices were prepared using a frozen section method. The slices were then subjected to baking, dewaxing, hydration, antigen retrieval, primary antibody incubation (ChAT: ab181023, abcam, Cambridge, UK; PGP9.5: ab8189, abcam, Cambridge, UK; OCT1: ab51313, abcam, Cambridge, UK), secondary antibody incubation, slide sealing, and photography. The co-labeled regions were analyzed for integral optical density using Image Pro Plus software (Media Cybernetics, Bethesda, MD, USA).
2.9. Statistical analysis
The data were analyzed with SPSS 21.0 statistical software (IBM Co., Armonk, NY, USA). If the data were in accordance with the normal distribution and the variance was uniform, one-way analysis of variance (ANOVA) was performed, and the least significant difference (LSD) test for pairwise comparisons. The data that did not conform to a normal distribution were analyzed by the nonparametric Kruskal-Wallis H test. P < 0.05 was considered statistically significance.
3. RESULTS
3.1. Effect of mild moxibustion on DSS induced UC rats
In normal rats, the tissue and gland structure of the colon was intact without signs of inflammatory cell infiltration, congestion, or edema. In contrast, model rats showed significant destruction in colon tissue and glands accompanied by substantial inflammatory cell infiltration (Figure 1A). The observed histopathological changes confirmed the successful establishment of the UC model.
Figure 1. Histopathological manifestations of colonic tissues in rats in each group.
A: the colonic histopathological manifestations of normal and model rats (hematoxylin-eosin staining, × 200). A1: normal rats; A2: model rats. B: the colonic histopathological manifestations of rats in experiment 1 (hematoxylin-eosin staining, × 200). B1: Con group; B2: Mod group; B3: Mox group; B4: α-BGT group; B5: α-BGT + Mox group. C: the colonic histopathological manifestations of rats in experiment 2 (hematoxylin-eosin staining, × 200). C1: Con group; C2: Mod group; C3: Mox group; C4; VH group; C5: VH + Mox group. D: the colonic histopathological manifestations of rats in experiment 3 (hematoxylin-eosin staining, × 200). D1: Con group; D2: Mod group; D3: Mox group; D4: Qu group; D5: Qu + Mox group. Con group: drink and eat freely + the same fixation as the Mox group; Mod group: drink 4% dextran sodium sulfate for 7 d, and 1% DSS starting from the 8th day + the same fixation as the Mox group; Mox group: mild moxibustion at the bilateral Zusanli (ST36) for 10 min per day for 7 d; α-BGT: injected with 1 μg/kg α-BGT; α-BGT + Mox: injected with 1 μg/kg α-BGT + mild moxibustion at the bilateral Zusanli (ST36) for 10 min per day for 7 d; VH: injected with 0.5 mM VH (0.1 mL); VH+ Mox: injected with 0.5 mM VH (0.1 mL) + mild moxibustion at the bilateral Zusanli (ST36) for 10 min per day for 7 d; Qu group: injected with 100 μM Qu (0.1 mL); Qu + Mox: injected with 100 μM Qu (0.1 mL) + mild moxibustion at the bilateral Zusanli (ST36) for 10 min per day for 7 d. Con: control group; Mod: UC model group; Mox: mild moxibustion group; α-BGT: alpha-bungarotoxin group; α-BGT + Mox: alpha-bungarotoxin + mild moxibustion group; VH: vesamicol hydrochloride group; VH + Mox: vesamicol hydrochloride + mild moxibustion group; Qu: quinine group; Qu + Mox: quinine + mild moxibustion group. Arrows indicate ulcerated (Red) or healed mucous membranes (Blue).
The control group exhibited well-preserved colon epithelial cells and gland structures without visible congestion, edema, ulceration, or inflammatory cell infiltration. In the UC model group, α-BGT group, VH group, Qu group, α-BGT + mild moxibustion group and Qu + mild moxibustion group, the colon epithelial structure was compromised; glands were partially absent, mucosal structure damaged, and inflammatory cells infiltrated both mucosa and submucosa. Conversely, the mild moxibustion group and the VH + mild moxibustion group showed repair in colon epithelial structure, improved glandular architecture, and reduced inflammatory cell presence (Figures 1B-1D).
Mild moxibustion was found to decrease IL-1β protein and mRNA expression in the colon of UC rats. However, this anti-inflammatory effect was nullified after α-BGT blocked α7nAchRs. Even when nAch release was inhibited with VH, mild moxibustion still improved colon inflammation and reduced IL-1β protein levels. Conversely, blocking nnAch release with Qu prevented mild moxibustion from alleviating colon inflammation (supplementary Figure 1).
3.2. Effect of mild moxibustion on colonic acetylcholine-related molecules in UC rats
In experiment 1, compared to the control group, NF-κBp65 protein and mRNA expression significantly increased in the colon tissue of the UC model group (P = 0.000).
Mild moxibustion treatment significantly decreased NF-κBp65 protein and mRNA levels compared to the UC model group (P = 0.000) (Figures 2A-2C). In the UC model group, ChAT protein expression was significantly reduced compared to the control group (P = 0.001). Mild moxibustion significantly elevated ChAT protein content and CarAT mRNA expression in comparison to the UC model group (P = 0.001, P = 0.002). Blocking α7nAchRs with α-BGT significantly decreased ChAT protein and mRNA expressions even with mild moxibustion (P = 0.001, P = 0.013) (Figures 2D-2F).
Figure 2. Expression of acetylcholine-related molecules in the colon of rats in each group.
A: representative western blot bands of NF-κB p65 expression in the colon; B: the protein expressions of NF-κB p65 in the colon; C: relative mRNA expression of NF-κB p65 in the colon; D: results of ELISA of ChAT in the colon in experiment 1; E-F: relative mRNA expressions of ChAT and CarAT in the colon in experiment 1, respectively; G-H: results of ELISA of ChAT in the colon in experiment 2 and experiment 3, respectively. Con group: drink and eat freely + the same fixation as the Mox group; Mod group: drink 4% dextran sodium sulfate for 7 d, and 1% DSS starting from the 8th day + the same fixation as the Mox group; Mox group: mild moxibustion at the bilateral Zusanli (ST36) for 10 min per day for 7 d; α-BGT: injected with 1 μg/kg α-BGT; α-BGT + Mox: injected with 1 μg/kg α-BGT + mild moxibustion at the bilateral Zusanli (ST36) for 10 min per day for 7 d; VH: injected with 0.5 mM VH (0.1 mL); VH+ Mox: injected with 0.5 mM VH (0.1 mL) + mild moxibustion at the bilateral Zusanli (ST36) for 10 min per day for 7 d; Qu group: injected with 100 μM Qu (0.1 mL); Qu + Mox: injected with 100 μM Qu (0.1 mL) + mild moxibustion at the bilateral Zusanli (ST36) for 10 min per day for 7 d. Con: control group; Mod: UC model group; Mox: mild moxibustion group; α-BGT: alpha-bungarotoxin group; α-BGT + Mox: alpha-bungarotoxin + mild moxibustion group; VH: vesamicol hydrochloride group; VH + Mox: vesamicol hydrochloride + mild moxibustion group; Qu: quinine group; Qu + Mox: quinine + mild moxibustion group. ELISA: enzyme-linked immunosorbent assay; NF-κB p65: nuclear factor kappa-B p65 subunit; ChAT: choline acetyltransferase; CarAT: carnosine acetyltransferase. B-D, F-H: data were presented as mean ± standard deviation (in experiment 2, Con group: n = 10, Mod group: n = 8, Mox, VH, and VH + Mox groups: n = 9. Other groups: n = 10). Statistical analyses were measured using one-way analysis of variance followed by least significant difference test for pairwise comparisons. E: data were presented as median (P25, P75) (n = 10). Statistical analyses were measured using the nonparametric Kruskal-Wallis H test. Compared with the control group, aP < 0.01, dP < 0.05; compared with the UC model group, bP < 0.01, fP < 0.05; compared with the mild moxibustion group, cP < 0.01, eP < 0.05; compared with the vesamicol hydrochloride group, gP < 0.05.
In experiment 2, the UC model group showed a marked reduction in ChAT protein expression in colon tissues compared to the control group (P = 0.000). Mild moxibustion and VH + mild moxibustion groups significantly increased ChAT protein levels compared to UC model group (P = 0.015, P = 0.020). VH treatment alone significantly decreased ChAT protein expression compared to mild moxibustion (P = 0.007). VH + mild moxibustion group significantly increased ChAT protein compared to VH alone (P = 0.01) (Figure 2G).
In experiment 3, ChAT protein levels were significantly lower in UC model group than in control group (P = 0.002). Mild moxibustion effectively raised ChAT protein expression compared to UC model (P = 0.031). Both Qu treatment alone and Qu + mild moxibustion significantly reduced ChAT protein compared to mild moxibustion alone (P = 0.002 for both) (Figure 2H).
In Experiment 2, compared to the control group, nAch and nnAch protein expressions in the UC model group’s colon tissue were significantly decreased (P = 0.012, P = 0.000) (Figure 3, supplementary Figures 2A, 2B and 3). Mild moxibustion significantly increased nnAch protein expression in colon tissue compared to the UC model group (P = 0.000). Similarly, the VH + mild moxibustion group showed increased nnAch protein expression (P = 0.011). However, nAch protein expression was significantly lower in both the VH group and VH + mild moxibustion group compared to the mild moxibustion group (P = 0.014, P = 0.004, P = 0.000), and nnAch protein expression was significantly lower in the VH group compared to the mild moxibustion group (P = 0.000). Notably, the VH + mild moxibustion group had a significant increase in nnAch protein expression compared to the VH group (P = 0.008).
Figure 3. Comparison of nAch protein expression in colon tissues of rats in each group in experiment 2 (immunofluorescence staining, × 200).
A: Con group. A1: Merge; A2: DAPI; A3: ChAT; A4: PGP9.5. B: Mod group. B1: Merge; B2: DAPI; B3: ChAT; B4: PGP9.5. C: Mox group. C1: Merge; C2: DAPI; C3: ChAT; C4: PGP9.5. D: VH group. D1: Merge; D2: DAPI; D3: ChAT; D4: PGP9.5. E: VH + Mox group. E1: Merge; E2: DAPI; E3: ChAT; E4: PGP9.5. Con group: drink and eat freely + the same fixation as the Mox group; Mod group: drink 4% dextran sodium sulfate for 7 d, and 1% DSS starting from the 8th day + the same fixation as the Mox group; Mox group: mild moxibustion at the bilateral Zusanli (ST36) for 10 min per day for 7 d; VH: injected with 0.5 mM VH (0.1 mL); VH + Mox: injected with 0.5 mM VH (0.1 mL) + mild moxibustion at the bilateral Zusanli (ST36) for 10 min per day for 7 d. Con: control group; Mod: UC model group; Mox: mild moxibustion group; VH: vesamicol hydrochloride group; VH + Mox: vesamicol hydrochloride + mild moxibustion group; DAPI: 4',6-diamidino-2-phenylindole; ChAT: choline acetyltransferase; PGP9.5: protein gene product 9.5.
In experiment 3, compared with the control group, the UC model group significantly decreased the expression of colonic tissue nnAch protein (P = 0.000) (Figure 4, supplementary Figure 2C). Compared with the UC model group, the mild moxibustion group significantly increased the expression of colonic tissue nnAch protein (P = 0.006), while the combination of quinine and mild moxibustion did not increase the expression of colonic tissue nnAch protein. Compared with the mild moxibustion group, the quinine group significantly decreased the expression of nnAch protein (P = 0.002).
Figure 4. Comparison of nnAch protein expression in colon tissues of rats in each group in experiment 3 (immunofluorescence staining, × 200).
A: Con group. A1: Merge; A2: DAPI; A3: ChAT; A4: OCT1. B: Mod group. B1: Merge; B2: DAPI; B3: ChAT; B4: OCT1. C: Mox group. C1: Merge; C2: DAPI; C3: ChAT; C4: OCT1. D: Qu group. D1: Merge; D2: DAPI; D3: ChAT; D4: OCT1. E: Qu + Mox group. E1: Merge; E2: DAPI; E3: ChAT; E4: OCT1. Con group: drink and eat freely + the same fixation as the Mox group; Mod group: drink 4% dextran sodium sulfate for 7 d, and 1% DSS starting from the 8th day + the same fixation as the Mox group; Mox group: mild moxibustion at the bilateral Zusanli (ST36) for 10 min per day for 7 d; Qu group: injected with 100 μM Qu (0.1 mL); Qu + Mox: injected with 100 μM Qu (0.1 mL) + mild moxibustion at the bilateral Zusanli (ST36) for 10 min per day for 7 d. Con: control group; Mod: UC model group; Mox: mild moxibustion group; Qu: quinine group; Qu + Mox: quinine + mild moxibustion group; DAPI: 4',6-diamidino-2-phenylindole; ChAT: choline acetyltransferase; OCT1: organic cation transport 1.
These results demonstrate that mild moxibustion’s beneficial effects on colonic inflammation in UC rats involve a mechanism linked to nnAch release, which is crucially disrupted when nnAch pathways are blocked.
4. DISCUSSION
Studies suggest that moxibustion can enhance colon mucosal repair and downregulate inflammatory mediators such as IL-8 and NF-κB p65 in UC rats.11,12 This study indicates that mild moxibustion at Zusanli (ST36) acupoint substantially alleviates inflammation in UC model rats by reducing IL-1β expression in colon tissues. Additionally, it was observed that mild moxibustion decreases NF-κB mRNA and NF-κB p65 protein levels while enhancing expression of colonic Ach-related proteins like CarAT mRNA and ChAT mRNA/protein. The CAP pathway requires Ach binding to α7nAchRs for anti-inflammatory action.13 Using α-bungarotoxin to block α7nAchRs resulted in inhibition of their function which subsequently impeded reduction of IL-1β and NF-κB expression by mild moxibustion, confirming its reliance on Ach activity mediated through α7nAchRs for anti-inflammatory effects.
Research on CAP focuses on Ach expression, which is critical for its functionality. In this study, we observed that ChAT expression was significantly decreased in the colonic tissue of UC model rats, while mild moxibustion at Zusanli (ST36) enhanced its expression. Acetylcholine synthesis involves the catalysis by ChAT from choline and acetyl coenzyme A. Recent research has identified CarAT as an additional rate-limiting enzyme crucial for acetylcholine synthesis.14 Therefore, detecting the levels of ChAT indirectly reflects Ach expression. Our results showed that mild moxibustion significantly increased CarAT expression, indicating that it can enhance acetylcholine levels in colonic tissue. CAP encompasses multiple signaling pathways, with NF-κB being central.15 We found that mild moxibustion reduced NF-κB expression in colonic tissue. To further explore the mechanism, we used α-bungarotoxin to block α7nAchRs. The combination of α-bungarotoxin and mild moxibustion did not reduce IL-1β and NF-κB expression, suggesting that while mild moxibustion increases Ach levels, it requires α7nAchRs to inhibit NF-κB signaling and exert anti-inflammatory effects.
The NNCS, first proposed by Sastry et al 16 in the 1970s, releases nnAch, contrasting with cholinergic nerve fibers that release nAch.17 Although both forms are synthesized via ChAT catalysis, their release mechanisms differ: nnAch is mediated by OCT, whereas nAch uses VAChT. As Ach can be sourced from both neurons and non-neuronal systems, this study sought to clarify its origin.18 In Experiment 2, vesamicol hydrochloride was used to block nAch release by cholinergic neurons, revealing that mild moxibustion still reduced inflammation and promoted intestinal Ach expression. Immun-ofluorescence results confirmed increased nnAch expression in the VH + mild moxibustion group, suggesting regulation of inflammatory responses via NNCS-mediated nnAch release. In Experiment 3, we employed quinine, an nnAch release blocker. The quinine + mild moxibustion group did not lower IL-1β expression or increase ChAT levels in colonic tissue, further substantiated by immunofluorescence indicating increased nnAch expression. This confirms that mild moxibustion elevates Ach via NNCS synthesis and release.
In conclusion, our study indicates that mild moxibustion ameliorates colon inflammation in UC rats through local acetylcholine release and CAP-mediated anti-inflammatory mechanisms, primarily involving NNCS-derived Ach.
5. SUPPORTING INFORMATION
Supporting data to this article can be found online at http://journaltcm.com.
Funding Statement
Supported by National Natural Science Foundation of China: Study for the Mechanism of Moxibustion in Ulcerative Colitis based on the α7 Nicotinic Acetylcholine Receptor Mediated Cholinergic Anti-inflammatory Pathway (No. 82205293); National Natural Science Foundation of China: Study of the Central Nervous System Regulatory Mechanism of Moxibustion Repair of Ulcerative Colitis Gut Vascular Barrier Based on Ubiquitin Specific Peptidase 14 Deubiquitination (No. 82274641); National Natural Science Foundation of China: Moxibustion Regulates P300-mediated Histone H3K27 Acetylation Modification in the Treatment of Crohn's Disease (No. 82205262); National Natural Science Foundation of China: Study on the Protective Mechanism of Moxibustion on Intestinal Mucosal Barrier in Ulcerative Colitis based on GABAergic System (No. 82105012); Shanghai Sailing Program: to Study the Protective Effect of Moxibustion on Intestinal Mucosal Barrier in Crohn's Disease Based on Histone H3 Acetylation Modification (No. 22YF1444100); State Administration of Traditional Chinese Medicine High-level Key Discipline Construction Project (No. zyyzdxk-2023068)
Contributor Information
Qin QI, Email: 719928895@qq.com.
Huangan WU, Email: wuhuangan@126.com.
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