Chronic Electroacupuncture With High-Frequency at ST-36 Promotes Gastrointestinal Motility by Regulating Bone Morphogenetic Protein 2 Secretion of Muscularis Macrophages.

BACKGROUND: Electroacupuncture (EA) at Zusanli (ST36) is an alternative treatment for several gastrointestinal motility disorders; however, the exact mechanism is unconfirmed. We aimed to show the potential effects of EA on muscularis macrophages (MMφ), the bone morphogenetic protein (BMP)/BMP receptor (BMPR)-Smad signal pathway, and enteric neurons in diabetic mice. This may provide fresh insight into ways EA affects gastrointestinal motility. MATERIALS AND METHODS: C57BL/6J healthy adult male mice were randomly divided into five groups: regular control group, diabetes group, diabetes with sham EA group (acupuncture only), diabetes with low-frequency EA group (10 Hz), diabetes with high-frequency EA group (HEA) (100 Hz). The stimulation lasted eight weeks. Gastrointestinal motility was assessed. We identified M2-like MMφ in the layer of colonic muscle by flow cytometry. Western Blot, real-time polymerase chain reaction, and immunofluorescent staining were also used to determine the MMφ, molecules in the BMP2/BMPR-Smad pathway, and PGP9.5, neuronal nitric oxide synthase (nNOS) expression of enteric neurons in the colon of each group. RESULTS: 1) HEA improved the gastrointestinal motility (gastrointestinal transit time, defecation frequency) of diabetic mice. 2) HEA reversed the decreased proportion of M2-like MMφ and expression of the CD206 in the colon of diabetic mice. 3) HEA restored the downregulations of BMP2, BMPR1b, and Smad1 in the BMP2/BMPR-Smad pathway and increased downstream enteric neurons marked by PGP9.5, nNOS in the colon of diabetes mice. CONCLUSIONS: HEA might promote gut dynamics by upregulating M2-like MMφ in the colon of diabetic mice, which in turn leads to the accumulation of molecules in the BMP2/BMPR-Smad signaling pathway and downstream enteric neurons.

Electroacupuncture reduces blood glucose by regulating intestinal flora in type 2 diabetic mice.

BACKGROUND: The development of diabetes is closely related to the gut microbiota in recent studies, which can be influenced by intestinal motility. A few studies report that electroacupuncture (EA) can lower blood glucose. EA can promote colonic motility and influence gut microbes. In this study, we explored the effect of the EA on blood glucose level in mice with type 2 diabetes (T2D) and its mechanism. METHODS: The T2D mice model, fecal microbiota transplantation mice model, and KitW/Wv mice model （Point mutation of mouse W locus encoding kit gene）were used to investigate the effect of EA on blood glucose as well as the mechanism; The blood glucose and insulin resistance level and the intestinal flora were evaluated. The level of intestinal junction protein, inflammatory cytokines in the serum, interstitial cells of Cajal content, and colonic motility were detected. Lastly, the IKKß/NF-κB-JNK-IRS-1-AKT pathway was explored. RESULTS: EA lowered the blood glucose level, altered the gut microbiota, and promoted colonic motility in T2D mice. EA-altered microbiota decreased the blood glucose level and insulin resistance in the antibiotics-treated diabetic mice. EA increased tight junction protein, lowered inflammatory factors, and regulated the IKKß/NF-κB-JNK-IRS-1-AKT pathway in the liver and muscles. EA could not reduce the blood glucose and regulated gut microbiota in the KitW/Wv mice model. CONCLUSIONS: EA promoted intestinal motility to regulate the intestinal flora, thereby reducing the level of systemic inflammation, and ultimately lowering the blood glucose by the IKKß/NF-κB-JNK-IRS-1-AKT signal pathway.

Electroacupuncture accelerates the delayed intestinal transit in POI by suppressing M1 like muscularis macrophages and IL6 secretion.

BACKGROUND: Electroacupuncture (EA) at ST-36 could accelerate the delayed gastrointestinal (GI) motility in many GI motility dysfunction models, but the definite effect and mechanisms are unclear. In this study, we intended to investigate the effects of EA on intestinal manipulation (IM) mice model and involved mechanisms. METHODS: Male C57BL/6 mice were randomized into five groups: normal control, intestinal manipulation (IM), IM with sham EA (SEA), IM with high-frequency EA (HEA), and IM with low-frequency EA (LEA). The GI transit was evaluated. The infiltration of muscularis macrophages (MMφ) and its phenotype were analyzed with flow cytometry. Magnetic-activated cell sorting was applied to isolate MMφ, and the relationship between the MMφ and interstitial cells of Cajal (ICCs) was further investigated. RESULTS: (1) Compared with the IM group, HEA and LEA attenuated the delayed intestinal transit. (2) Both the HEA and LEA obviously reduced the MMφ and suppressed the M1 activation of the MMφ in the ileum. (3) EA restored the disrupted ICC networks through inhibiting the release of IL6 by the MMφ. CONCLUSION: (1) Electroacupuncture at acupoint ST-36 could accelerate the delayed intestinal transit in the IM murine model by restoring the ICC networks. (2) EA protected the ICCs through reducing the MMφ, inhibiting its M1 polarization and its IL6 secretion.

Electroacupuncture preserves intestinal barrier integrity through modulating the gut microbiota in DSS-induced chronic colitis.

AIMS: Electroacupuncture (EA) at ST36 has been verified to ameliorate experimental acute colitis. However, the effect of EA on chronic colitis and its mechanism has not yet been explored. This study aimed to assess the protective effect of EA against chronic colitis and the related mechanisms. MAIN METHODS: Chronic colitis was induced by dextran sulfate sodium (DSS) in C57BL/6 mice, and EA was applied throughout the entire experiment. Colonic inflammation and intestinal barrier integrity were evaluated. Alterations in the gut microbiota were analyzed by 16S rRNA gene sequencing. The fecal microbiota transplantation (FMT) experiment was used to further confirm the effect of the gut microbiota on the barrier protective effect of EA. The potential molecular mechanisms were explored by western blotting. KEY FINDINGS: (1) EA lowered the disease activity index (DAI) and histological scores, decreased the levels of TNFα, IL1ß, IL6 and iNOS, and increased the IL10 level in DSS-induced chronic colitis. (2) EA upregulated the protein expression of ZO-1, Occludin, E-Cadherin and mucin2 (MUC2), reduced the apoptosis and proliferation of intestinal epithelial cells (IECs) and intestinal permeability. (3) EA enhanced the gut microbiota diversity and restored the community structure. (4) Both the low-frequency EA (LEA) FMT and high-frequency EA (HEA) FMT maintained the intestinal barrier integrity. (5) EA promoted activation of the mitogen activated protein kinase (MAPK) signaling pathway. SIGNIFICANCE: EA can relieve chronic experimental colitis, and this effect may depend on activation of the MAPK signaling pathway through modulation of the gut microbiota to preserve the intestinal barrier.

Electroacupuncture at ST-36 ameliorates DSS-induced acute colitis via regulating macrophage polarization induced by suppressing NLRP3/IL-1ß and promoting Nrf2/HO-1.

BACKGROUND: Electroacupuncture (EA) at ST-36 can attenuate acute experimental colitis, but the mechanisms are unclear. We investigated the role of macrophages in the anti-inflammatory effects of EA and its molecular mechanisms. METHODS: Male C57BL/6 mice were randomized into five groups: normal control, dextran sulfate sodium (DSS)-induced acute colitis (DSS), DSS with sham EA (SEA), DSS with high-frequency EA (HEA) and DSS with low-frequency EA (LEA). Body weight, colon length, DAI score and histological score were evaluated during colitis progression. Serum and colonic levels of pro- and anti-inflammatory cytokines were detected with ELISA, cytometric beads array, RT-PCR and western blotting analysis. Colonic macrophage subsets were determined using flow cytometry. Magnetic-activated cell sorting was applied to isolate colonic macrophages, and molecular mechanisms were explored with western blotting, RT-PCR and immunofluorescence. RESULTS: (1) Compared with the DSS group, HEA and LEA attenuated body weight loss and decreased DAI and histological scores. (2) Serum levels and colonic protein and mRNA levels of IL-1ß, TNFα, IL-6, IL-12 and IL17 were markedly decreased with HEA and LEA. IL-10 level was increased with HEA. (3) M1 macrophage percentage increased, while M2 macrophage percentage decreased in the DSS group; HEA and LEA reversed these proportions. (4) NLRP3/IL-1ß protein and mRNA levels in isolated macrophages decreased with HEA and LEA compared with the DSS treatment group; (5) HEA increased Nrf2/HO-1 levels compared with levels in DSS mice. CONCLUSION: The anti-inflammatory effects of EA on DSS-induced acute colitis may rely on regulating macrophage polarization, NLRP3/IL-1ß suppression and Nrf2/HO-1 promotion.

Electroacupuncture promotes the gastrointestinal motility of diabetic mice by CNP/NPR-B-cGMP and PDE3A-cGMP signaling.

BACKGROUND: Electroacupuncture (EA) can promote gastrointestinal (GI) motility of diabetic mice, but the mechanism is not clearly elucidated. Natriuretic peptides (NPs) were related to the diabetes-induced gut dysfunction of mice, which may be associated with ICC (interstitial cells of cajal). Besides, EA could increase the ICC of diabetic mice. Our aim was to explore whether EA can promote the gut motility by CNP/NPR-B-cGMP and PDE3A-cGMP signaling in diabetic mice, and the relationship between NPs and ICC. METHODS: Wild C57BL/6 male mice were divided into five groups: control group, diabetic mellitus (DM group), diabetic mellitus plus sham EA group (SEA), diabetic mellitus plus low-frequency EA group (LEA), and diabetic mellitus plus high-frequency group (HEA). Gastrointestinal motility was assessed by gastric emptying and GI transit test. Immunofluorescence staining was applied to assess the expression level of CNP, NPR-B, and c-Kit. Western blot, PCR, and ELISA were used to detect the level of CNP, NPR-B, PDE2A, PDE3A, c-Kit, mSCF, and cGMP content. The correlativity between NPR-B and mSCF was evaluated by Pearson's correlation and linear regression analyses. KEY RESULTS: (a) EA could improve the GI dysfunction of diabetic mice. (b) CNP, NPR-B, and cGMP contents were decreased, but the level of PDE3A, c-Kit, and mSCF was increased in the EA groups. (c) There was a negative correlation between NPR-B and mSCF among the groups. CONCLUSIONS AND INFERENCES: Electroacupuncture promotes the GI function by CNP/NPR-B-cGMP and PDE3A-cGMP signaling in diabetic mice; up-regulated mSCF/c-Kit signaling by EA may be mediated partially via down-regulation of CNP/NPR-B signaling.

Electroacupuncture at ST-36 Protects Interstitial Cells of Cajal via Sustaining Heme Oxygenase-1 Positive M2 Macrophages in the Stomach of Diabetic Mice.

BACKGROUND: Electroacupuncture (EA) at ST-36 has been reported to improve delayed gastric emptying and protect the networks of ICC in diabetic models. However, the mechanisms of the effects of EA are still unclear. The purpose of this study was to investigate whether the HO-1 positive M2 macrophages participate in the protective effects of EA for the ICC networks. METHODS: Male C57BL/6 mice were randomized into five groups: the normal control group, diabetic group (DM), diabetic mice with sham EA group (SEA), diabetic mice with low frequency EA group (LEA), and diabetic mice with high frequency EA group (HEA). ICC network changes were detected by Ano1 immunostaining. F4/80 and HO-1 costaining was used to measure HO-1 positive macrophage expression. Western blot and PCR methods were applied to monitor HO-1, IL-10, and macrophage markers, respectively. The serum MDA levels were detected by a commercial kit. RESULTS: This study presents the following results: (1) Compared with the control group, ICC networks were severely disrupted in the DM group, but no obvious changes were found in the LEA and HEA groups. (2) Many HO-1 positive macrophages could be observed in the LEA and HEA groups, and the expression of HO-1 was also markedly upregulated. (3) The IL-10 expression was obviously upregulated in the LEA and HEA groups. (4) The serum MDA levels were decreased in the real EA group. (5) When compared to the DM group, the expression of CD163 and Arg-1 was increased in the LEA and HEA groups, but the iNOS expression was decreased. CONCLUSION: The protective effects of EA on the networks of ICC may rely on the HO-1 positive macrophages to mediate anti-inflammatory and antioxidative stress effects.