OBJECTIVE:To test whether acupuncture improves sleep by enhancing gamma-aminobutyric acid-ergic (GABA) neuronal activity in the thalamic reticular nucleus (TRN). METHODS:We established an animal model using p-chlorophenylalanine (PCPA). We assessed the impact of acupuncture on insomnia through wheel-running activity and sodium pentobarbital-induced sleep tests. Subsequently, the calcium indicator GCaMP6m was injected into the TRN of Vgat-Cre mice, and the activity of GABA neurons in the TRN was analyzed before and after acupuncture using fiber photometry recording. Additionally, cortical electroencephalography (EEG) recordings were compared between groups following chemogenetic inhibition of GABA neurons in the TRN. RESULTS:Following acupuncture, we observed a reduction in sleep latency and an increase in sleep duration in comparison to the PCPA-induced insomnia (P < 0.01). Furthermore, acupuncture positively influenced the diurnal phase of insomnia (P < 0.01). Ca2+ signals in GABA neurons within the TRN increased post-acupuncture (paired t-test, P< 0.01). Acupuncture also reduced the proportion of EEG power in the elevated Beta and Gamma frequencies associated with PCPA injection, while simultaneously increasing the Delta power ratio (P < 0.01). Chemogenetic inhibition of TRN GABA neurons blocked these acupuncture effects: the hM4Di + MA group showed no significant differences from the PCPA group in Beta and Gamma power, but exhibited elevated Beta and Theta activity compared to controls (P < 0.05). CONCLUSIONS:Acupuncture activates GABA neurons in the TRN, thereby regulating the sleep-wake cycle in the context of PCPA-induced insomnia.

Mitochondria are central to cellular metabolic reprogramming, and their energy metabolism pathways are indispensable for T‑cell activation, proliferation and differentiation. Mitochondrial metabolic reprogramming enhances T‑cell activity and antitumor function. Mitochondrial dynamics, including fusion, fission and transfer, regulate T‑cell tumor immune function by modulating the number, morphology and distribution of mitochondria, which is vital for the antitumor effects of T cells. The release of mitochondrial DNA can activate multiple innate immune signaling pathways, such as cyclic GMP‑AMP synthase‑stimulator of interferon genes, Toll‑like receptor 9, and NOD‑, LRR‑, and pyrin domain‑containing protein 3, serving a complex regulatory role in shaping the tumor immunosuppressive microenvironment and T‑cell antitumor immune responses. Notably, mitochondrial dysfunction is a major driver of tumor initiation and progression. T‑cell mitochondrial metabolic reprogramming, dynamic changes and mitochondrial DNA release all affect the antitumor immunity of tumor‑infiltrating T cells. The present review focuses on the relationship between mitochondria and T‑cell antitumor immune responses, exploring the core role of mitochondria in T‑cell tumor immunity from multiple aspects, including mitochondrial energy metabolism, mitochondrial dynamics and mitochondrial DNA. In addition, the present review examines state‑of‑the‑art research on antitumor therapies targeting mitochondria from multiple perspectives, with the aim of providing a reference for developing mitochondria‑targeted antitumor immunotherapy strategies.

Background Macrophage polarization plays an essential role in the anti-inflammation process. Moxibustion, a traditional Chinese medicine therapy, has been reported to have an anti-inflammatory effect via enhancing α-ketoglutarate (α-KG) and succinate levels. Succinate/α-KG ratio is a hallmark of M1 and M2 macrophage shift. Glutamate dehydrogenase 1 (GLUD1) is a vital enzyme for α-KG production and can be deacetylated by Sirtuin5 (SIRT5). Currently, the role of moxibustion in SIRT5-GLUD1-α-KG-related macrophage alteration in inflammatory diseases has not been discussed yet. Methods In this study, complete Freund's adjuvant (CFA)-induced adjuvant arthritis models were established. On day 4 post-CFA, moxibustion and acupoint MC3482 injection were administered. Foot volume was measured before and after the model was established, and after the moxibustion and acupoint injection interventions. ELISA assays were then performed to quantify inflammatory factors, including IL-1β, TNF-α, IL-4, TGF-β, succinate, and α-ketoglutarate (α-KG). Flow cytometry (FCM) and immunofluorescence were used to test M1- and M2-like macrophage expressions in the right arthrodial cartilages of mice. Furthermore, western blotting and immunoprecipitation (IP) were used to detect SIRT5, GLUD1, and GLUD1 succinylation expressions. Results Moxibustion and SIRT5 desuccinylation inhibitor MC3482 decreased inflammation by increasing M2 macrophage and reducing M1 macrophage levels in CFA model. The potential mechanism may relate to the effects of moxibustion and SIRT5 inhibition, which could invert succinate and α-KG levels in the CFA group, which displayed low succinate, high α-KG and increased GLUD1 succinylation modification after treatment. Conclusion This study supports that moxibustion's anti-inflammation effects are related to the consequences of macrophage conversion after SIRT5 post-translational modification.

IntroductionAlthough acupuncture is recommended by chronic obstructive pulmonary disease (COPD) treatment guidelines owing to its effects on dyspnea, the underlying neurobiological mechanisms of these effects remain unclear. This study aims to evaluate the efficacy of acupuncture in patients with stable COPD and explore the possible involvement of specific brain regions.MethodsThis is a prospective, multicenter, single-blind, randomized controlled trial. A total of 90 participants will be recruited from three centers and will be randomly assigned in a 1:1 ratio to undergo acupuncture at acupoints on the disease-affected meridian (DAM) or non-acupoints on the non-affected meridian (NAM), in addition to routine pharmacological treatments. All participants will undergo 30 min of acupuncture three times a week for 8 weeks and will be followed up for 12 months. The primary outcome will be the severity of dyspnea, as measured using the Borg Dyspnea Scale and a visual analog scale at rest and after exercise. The secondary outcomes will include the multidimensional profile of dyspnea using Dyspnea-12, the modified Medical Research Council Dyspnea Scale, and the COPD assessment test; quality of life assessments using St George's Respiratory Questionnaire and the Hospital Anxiety and Depression Scale; and additional measurements of exacerbation frequency, pulmonary function, and the 6-min walking distance. Magnetic resonance imaging (MRI) will be performed before and after exercise to explore the potential neurobiological mechanisms of exertional dyspnea. Anxiety and depression will be measured and analyzed for their correlation with the activation of specific brain areas involved in dyspnea.DiscussionThis randomized controlled trial aims to use a multidimensional evaluation of the efficacy of acupuncture in relieving dyspnea in patients with COPD in terms of emotion and quality of life and explore the neurobiological mechanisms underlying the effects of acupuncture on dyspnea from an imaging perspective. It is expected to provide strong evidence to support the use of acupuncture in relieving dyspnea in patients with COPD and those with aother diseases involving dyspnea. Additionally, it provides novel insights into the central mechanisms of acupuncture intervention and dyspnea.Trial registrationChinese Clinical Trial Registry (https://www.chictr.org.cn/): ChiCTR2300071725.

Sirtuine5 (SIRT5) is an important molecule involved in the pathology of inflammatory diseases. To investigate the impact of SIRT5 on the analgesic effectiveness of moxibustion, we established a complete Freund's adjuvant- (CFA-) induced inflammatory pain in mice model. Moxibustion was applied at the Zusanli (ST36) acupoint in mice with inflammatory pain. The analgesic effectiveness was evaluated by thermal hyperalgesia and mechanical allodynia tests in the right paws after CFA injection. The expression of inflammatory cytokines, including the pro-inflammatory factors IL-1β and TNF-α, and the anti-inflammatory factors IL-4 and TGF-β expressions, was evaluated using by ELISA. Furthermore, SIRT5 was evaluated by immunofluorescence and western blotting. The results showed that, compared with the CFA group, both thermal and mechanical pain thresholds increased with moxibustion and the SIRT5 inhibitor MC3482 intervention at ST36. Additionally, compared to the CFA-induced group, the inflammatory mediators, including IL-1β and TNF-α, decreased, while the anti-inflammatory cytokines IL-4 and TGF-β increased with moxibustion and MC3482 ST36 acupoint injection. Western blot results showed a decreased expression of SIRT5 at the ST36 site with moxibustion and MC3482 injection, compared to the CFA-induced group. SIRT5 expression in the right paw of mice injected with moxibustion and MC3482 was higher than that in the CFA-induced group. This study revealed that SIRT5 expression is involved in moxibustion analgesia and may be a potential mediator in the regulation of analgesia.

Objective: This study aimed to explore the efficacy and safety of qi-invigorating blood-activating tongmai decoction combined with rosuvastatin in the treatment of senile type 2 diabetes mellitus (T2DM) complicated with atherosclerosis (AS). Methods: The clinical data of 122 elderly patients with T2DM complicated with AS treated in Hospital of Chengdu University of Traditional Chinese Medicine from February 2020 to November 2021 were ret-rospectively analyzed. Among them, 57 patients treated with rosuvastatin alone were divided into a Monotherapy group, and 65 patients treated with qi-invigorating blood-activating tongmai decoction adjuvant combined with ro-suvastatin were divided into a combined group. The two groups were compared in terms of efficacy after treatment, incidence of adverse reactions after 8 weeks of treatment, and carotid plaque indexes, glucose metabolism indexes and lipid metabolism indexes before and after 8 weeks of treatment. Results: The Combined group showed a no-tably higher response rate than the Monotherapy group (P<0.05), but the two groups showed no significant differ-ence in the incidence of adverse reactions (P>0.05). After 8 weeks of treatment, the intima-media thickness (IMT), plaque area, fasting blood glucose, glycosylated hemoglobin (HbA1c), total cholesterol (TC), triacylglycerol (TG) and low-density lipoprotein-cholesterol (LDL-C) in the two groups decreased significantly, and high-density lipoprotein -cholesterol (HDL-C) in them increased significantly. Furthermore, the Combined group showed significantly higher levels of IMT, plaque area, fasting blood glucose, HbA1c, TC, TG and LDL-C, and a significantly lower HDL-C level than the Monotherapy group (P<0.05). Conclusion: Qi-invigorating blood-activating tongmai decoction can promote the therapeutic efficacy of rosuvastatin in elderly patients with T2DM complicated with AS.

Myocardial ischemia(Ml)is a common disease with high mortality and morbidity worldwide.Since the pathological process of Ml is very complicated,a comprehensive un-derstanding of its pathogenesis is the key to the treatment of MI.As chromatin plays a crucial role in regulating gene expression,and gene regulation is a fundamental process in developing and disease progression,combined analysis of the chromatin and gene can further reveal the pathological mechanism of MI.

The field of moxibustion research is expanding, with a rapid increase in publications in recent years. Moxibustion is a therapy that ignites moxa on the skin of humans, with an increase in peripheral skin temperature and localized redness. During this treatment, the recipient must remain still to prevent scalding and expose intervention sites for easy manipulation; however, maintaining a fixed posture during moxibustion is a big challenge for animals. Thus, manipulating moxibustion in small animals, such as mice, can lead to several difficulties for researchers. In addition, an uncomfortable posture for animals can lead to fear and resistance to moxibustion, increased risk of injury, diminished animal welfare, and less valid research data. An efficient, comfortable moxibustion method is needed to protect animal welfare and minimize the adverse effects on experimental results. However, moxibustion methods are highly variable and often have limited efficacy. More importantly, an uncomfortable moxibustion posture might cause a stress response, such as those observed with anxiety, fear, and anger, which could influence the research data. Therefore, strategies for animal moxibustion that inflict the least harm possible during the intervention are required. This protocol introduces a mouse tethering method for moxibustion intervention, minimizing mouse discomfort and improving study efficiency. Essential strategies for tethering mice and application of moxibustion are highlighted, and the structure of the tethering instrument is described.

本文从多学科交叉技术在艾灸科研成果中转化的角度,以智能灸法机器人的研发为切入点,从实验设计、临床使用、文化传承等方面着重阐释智能灸法机器人的研发,以及研发过程中传统灸法的科研成果与人工智能、医学工程等学科技术的结合点,同时提出针灸与多学科交叉困难的问题,以及解决上述问题的方向,以期在"医工结合"的基础上,结合多学科交叉技术,为进一步融合科研成果,转化艾灸科研成果提供一定的参考.

Background. The pathogenesis of slow transit constipation (STC) is associated with exosomal miR-34c-5p. Electroacupuncture (EA) improves gastrointestinal motility in gastrointestinal disorders, especially STC. Our study aimed to explore the mechanism by which EA improves intestinal motility by modulating the release of exosomes and the transmission of exosomal miR-34c-5p. Methods. Fifty rats were randomly divided into five groups. STC model rats were induced, and GW4869, the exosome release inhibitor, was used to inhibit the release of exosome. The serum exosomes were authenticated under a transmission electron microscope and nanoparticle tracking analysis. RT-qPCR detected the expression of miR-34c-5p in serum exosomes and colonic tissues. The fecal number in 24 hours, Bristol scores, and intestinal transit rates were used to assess intestinal motility. Subsequently, hematoxylin and eosin (H&E) staining was used to examine the colonic mucosal histology. Finally, the expression of stem cell factor (SCF) and receptor tyrosine kinase (c-Kit) protein was measured using immunohistochemistry staining. Results. We found that EA upregulated exosomal miR-34c-5p in serum and downregulated miR-34c-5p in colonic tissues (P<0.01). EA improved fecal numbers in 24 hours, Bristol scores, and intestinal transit rates in STC rats (P<0.01). EA recovered the colonic histological structure and enhanced the expression of SCF and c-Kit protein (P<0.01). The therapeutic effect of EA was attenuated after inhibiting the release of the exosome. Conclusion. Our results indicated that EA improves intestinal motility in STC rats by transporting of exosomal miR-34c-5p targeting the SCF/c-Kit signaling pathway.

The paper reviewed the relevant studies on dyspnea treated with acupuncture over the past 20 years, as well as the underlying neuroendocrine mechanism from the perspective of central and peripheral vagus nerves, neurotransmitter, respiratory muscle function and anti-depression-anxiety function. It revealed that the central response area was regulated by acupuncture in treatment of dyspnea, which is similar to the area affected in acupuncture analgesia. Additionally, acupuncture generates its therapeutic effects on dyspnea through promoting the release of endogenous opioid peptides and the regulation of autonomic nerve, amygdale and hypothalamic-pituitary-adrenal axis.

IntroductionAlzheimer’s disease (AD) is the most common form of dementia worldwide. The biological mechanisms underlying the pathogenesis of AD aren’t completely clear. Studies have shown that the gut microbiota could be associated with AD pathogenesis; however, the pathways involved still need to be investigated.AimsTo explore the possible pathways of the involvement of gut microbiota in AD pathogenesis through metabolites and to identify new AD biomarkers.MethodsSeven-month-old APP/PS1 mice were used as AD models. The Morris water maze test was used to examine learning and memory ability. 16S rRNA gene sequencing and widely targeted metabolomics were used to identify the gut microbiota composition and fecal metabolic profile, respectively, followed by a combined analysis of microbiomics and metabolomics.ResultsImpaired learning abilities were observed in APP/PS1 mice. Statistically significant changes in the gut microbiota were detected, including a reduction in β-diversity, a higher ratio of Firmicutes/Bacteroidota, and multiple differential bacteria. Statistically significant changes in fecal metabolism were also detected, with 40 differential fecal metabolites and perturbations in the pyrimidine metabolism. Approximately 40% of the differential fecal metabolites were markedly associated with the gut microbiota, and the top two bacteria associated with the most differential metabolites were Bacillus firmus and Rikenella. Deoxycytidine, which causes changes in the pyrimidine metabolic pathway, was significantly correlated with Clostridium sp. Culture-27.ConclusionsGut microbiota may be involved in the pathological processes associated with cognitive impairment in AD by dysregulating pyrimidine metabolism. B. firmus, Rikenella, Clostridium sp. Culture-27, and deoxyuridine may be important biological markers for AD. Our findings provide new insights into the host-microbe crosstalk in AD pathology and contribute to the discovery of diagnostic markers and therapeutic targets for AD.

Background The pathological process of myocardial ischemia (MI) is very complicated. Acupuncture at PC6 has been proved to be effective against MI injury, but the mechanism remains unclear. This study investigated the mechanism that underlies the effect of acupuncture on MI through full-length transcriptome. Methods Adult male C57/BL6 mice were randomly divided into control, MI, and PC6 groups. Mice in MI and PC6 group generated MI model by ligating the left anterior descending (LAD) coronary artery. The samples were collected 5 days after acupuncture treatment. Results The results showed that treatment by acupuncture improved cardiac function, decreased myocardial infraction area, and reduced the levels of cTnT and cTnI. Based on full-length transcriptome sequencing, 5083 differential expression genes (DEGs) and 324 DEGs were identified in the MI group and PC6 group, respectively. These genes regulated by acupuncture were mainly enriched in the inflammatory response pathway. Alternative splicing (AS) is a post-transcriptional action that contributes to the diversity of protein. In all samples, 8237 AS events associated with 1994 genes were found. Some differential AS-involved genes were enriched in the pathway related to heart disease. We also identified 602 new genes, 4 of which may the novel targets of acupuncture in MI. Conclusions Our findings suggest that the effect of acupuncture on MI may be based on the multi-level regulation of the transcriptome.

Aldosterone induces cardiac electrical and structural remodeling, which leads to the development of heart failure and/or atrial fibrillation (AF). However, it remains unknown whether aldosterone-induced remodeling may modulate the efficacy of anti-AF drugs. In this study, we aimed to jeopardize the structural and functional remodeling by aldosterone in rats with aorto-venocaval shunts (AVS rats) and evaluate the effect of acehytisine in this model. An AVS operation was performed on rats (n = 6, male) and it was accompanied by the intraperitoneal infusion of aldosterone (AVS + Ald) at 2.0 µg/h for 28 d. The cardiopathy was characterized by echocardiography, electrophysiologic and hemodynamic testing, and morphometric examination in comparison with sham-operated rats (n = 3), sham + Ald (n = 6), and AVS (n = 5). Aldosterone accelerated the progression from asymptomatic heart failure to overt heart failure and induced sustained AF resistant to electrical fibrillation in one out of six rats. In addition, it prolonged PR, QT interval and Wenckebach cycle length. Acehytisine failed to suppress AF in the AVS + Ald rats. In conclusion, aldosterone jeopardized electrical remodeling and blunted the electrophysiological response to acehytisine on AF.

目的:为改进目前小鼠艾灸实验效率低下的不足,设计一种新型的小鼠穴位批量艾灸器.方法:新型小鼠艾灸器在传统穴位艾灸方式的基础上,采用木头及磁铁材料,制作小鼠固定床、层置架及艾灸架;运用层置架,以安全且批量固定小鼠;艾灸架可吸附多个磁铁,以精确调节艾条与穴位距离.结果:新型小鼠批量艾灸器精准调控灸距,在艾灸多只小鼠的同时,有效降低烫伤率,缩短实验进程.结论:新型小鼠批量艾灸器简便效廉,节省空间,有助于规范艾灸实验,可在小鼠艾灸实验中推广使用,为研究人员提供一种新的选择方案.

Additional file 2: Table S2. The differentially expressed genes in PC6 group compared to MI group.

A newly-developed "Mouse Forelimb Fixator" and two types of "Batch Mice Moxibustion Device" on the basis of our "Mouse Safe and Fast Fixation Board" (developed in 2012) were introduced in the present paper. The Forelimb Fixator inserted into the base part of the apparatus in tenon and mortise style is used to control the mouse's posture with the forelimbs' acupoints fully exposed, and can realize simultaneous fixation of several mice at the same time. By using the mobility of the base of the single-hole moxibustion frame and the magnet, the distance between the acupoint surface and the tip of the ignited moxa stick can be accurately controlled, and several acupoints of different meridians can be simultaneously stimulated at the same time. Utilizing the porous transparent moxibustion board, the Batch Mice Moxibustion Device can meet the requirement of moxibustion at multiple acupoints at the same time. In addition, these devices are convenient in operation, innovative in creativity, save manpower and material resources, and help improve experimental efficiency and research on moxibustion.

Phosphorylation is one of the most important posttranslational modifications and regulates the physiological process. While recent studies highlight a major role of phosphorylation in the regulation of sleep–wake cycles to a lesser extent, the phosphoproteome in the suprachiasmatic nucleus (SCN) is not well-understood. Herein, we reported that the EA treatment elicits partial reparation of circadian rhythmicity when mice were exposure to constant darkness for long time. We investigated the effects of EA on circadian rhythms in constant darkness between EA stimulation and free-running control. Next, mass spectrometry–based phosphoproteome was utilized to explore the molecular characteristics of EA-induced phosphorylation modification in the SCN. A total of 6,192 distinct phosphosites on 2,488 proteins were quantified. Functional annotation analysis and protein–protein interaction networks demonstrated the most significant enriched phosphor-proteins and phosphosites involved in postsynapse and glutamatergic synapse. The current data indicated that most of the altered molecules are structural proteins. The target proteins, NMDAR and CAMK2, were selected for verification, consistent with the results of LC–MS/MS. These findings revealed a complete profile of phosphorylation modification in response to EA.

Background: Sympathetic and parasympathetic nerve remodeling play an important role in cardiac function after myocardial ischemia (MI) injury. Increasing evidence indicates that electroacupuncture (EA) can regulate cardiac function by modulating the autonomic nervous system (ANS), but little is known about its effectiveness on neural remodeling post-MI. Objectives: To investigate the role of EA in ANS remodeling post-MI. Methods: Adult male C57/BL6 mice were equally divided into the Control (Ctrl), MI and EA groups after generating the MI model by ligating the left anterior descending (LAD) coronary artery. Echocardiography and 2,3,5-triphenyltetrazolium (TTC) staining were employed to evaluate cardiac function and infarct size after EA treatment for five consecutive days. Serum norepinephrine (NE) levels were measured by ELISA to quantify sympathetic activation. Then, ANS remodeling was detected by immunohistochemistry (IHC), RT-qPCR, and Western blotting. Results: Our preliminary findings showed that EA increased ejection fraction and fractional shortening and reduced infarct area after MI injury. Serum NE levels in the EA group were significantly decreased compared with those in the MI group. IHC staining results demonstrated that the density of growth associated protein (GAP)43 and tyrosine hydroxylase (TH) positive nerve fibers in the EA group were decreased with increased choline acetyltransferase (CHAT) and vesicular acetylcholine transporter (VACHT). Meanwhile, the results verified that mRNA and protein expression of GAP43 and TH were significantly inhibited by EA treatment in the MI mice, accompanied by elevated CHAT and VACHT. Conclusions: EA treatment could improve cardiac function and reduce infarct size by modulating sympathetic and parasympathetic nerve remodeling post-MI, thus helping the cardiac ANS reach a new balance to try to protect the heart from further possible injury.