Electroacupuncture interventions alleviates myocardial ischemia reperfusion injury through regulating gut microbiota in rats.

Electroacupuncture (EA) intervention has a remarkable cardioprotection against myocardial ischemia reperfusion injury (MIRI). Recently, it has been suggested that the gut microbiota plays an important role in regulating the progression and prognosis of MIRI. The purpose of this study was to illustrate the relationship between gut microbiota and cardioprotection of EA on MIRI. We conducted a MIRI model by ligating the left anterior descending coronary artery for 30 min followed by reperfusion in male Sprague Dawley rats, which then received 7 days of EA intervention. Echocardiography was employed to evaluate left ventricular function. Fecal samples were collected for microbial analysis by 16S rDNA high-throughput sequencing. Blood samples and myocardium were collected for inflammatory cytokine detection by enzyme linked immunosorbent assay (ELISA) and Western blot. Hematoxylin & eosin (HE) staining and immunofluorescence of ileum tissue were performed for intestinal damage evaluation. After 7 days of EA intervention, the left ventricular function was improved with significantly increased ejection fraction and fractional shortening. Furthermore, we found that EA intervention reversed the changed gut microbiota induced by MIRI, including Clostridiales, RF39, S24-7, Desulfovibrio, and Allobaculum, improved the impaired gut barrier, reduced the production and circulation of lipopolysaccharide (LPS), inhibited the level of interleukin 6 (IL-6) and interleukin 12 (IL-12) in periphery and decreased the expression of Toll like receptor 4 (TLR4) and IL-6 in myocardium. EA intervention could improve the impaired gut mucosal barrier and reduce the production and circulation of LPS after MIRI through regulating gut microbiota, thus inhibiting the circulation and myocardium inflammation and finally exerted the cardioprotective effect.

Effect of electroacupuncture on ventricular structure and function in rats with myocardial ischemia-reperfusion injury. / ç”µé’ˆå¯¹å¿ƒè‚Œç¼ºè¡€å†çŒæ³¨æŸä¼¤å¤§é¼ å¿ƒå®¤ç»“æž„å’ŒåŠŸèƒ½çš„å½±å“.

OBJECTIVES: To observe the effect of electroacupuncture (EA) on changes of ventricular structure and function in rats with myocardial ischemia-reperfusion injury (MIRI), so as to explore its potential mechanisms underlying improvement of ventricular remodeling after MIRI. METHODS: Forty male SD rats were randomly divided into 4 groups：sham operation group, model group, EA group and medication (sacubactril valsartan, LCZ696) group, with 10 rats in each group. The MIRI model was established by ligation of the left anterior descending coronary artery and reperfusion. EA (2 Hz/100 Hz, 2 mA) was applied to bilateral "Neiguan" (PC6) for 20 min, once every other day for 21 d. Rats of the medication group received gavage of LCZ696 (60 mg·kg-1·d-1). After the intervention, echocardiography was used to detect the ejection fraction (EF) and fractional shortening (FS) of the left ventricle, and the contents of serum tumor necrosis factor-α(TNF-α), vascular cell adhesion molecule-1(VCAM-1) and intercellular cell adhesion molecule-1(ICAM-1) were assayed by enzyme-linked immunosorbent assay. The pathological changes of myocardial tissue were observed after HE staining. The Masson staining was used to evaluate the myocardial collagen deposition and myocardial fibrosis. The mRNA expression levels of collagen Ⅰ and Ⅲ and connective tissue growth factor (CTGF) in the myocardial tissue were detected by quantitative real-time PCR, and the expression levels of IL-1ß and IL-18 were detected by Western blot. RESULTS: In contrast to the sham operation group, the EF and FS levels of the left ventricle were ob-viously decreased (P<0.001), while the contents of serum TNF-α, VCAM-1 and ICAM-1, the proportion of myocardial fibrosis area, the mRNA expression levels of myocardial collagen Ⅰ, collagen Ⅲ and CTGF, the expression levels of IL-1ß and IL-18 were significantly increased (P<0.001, P<0.000 1, P<0.05, P<0.01) in the model group. Compared with the model group, the EF and FS levels were remarkably increased (P<0.01), whereas the contents of serum TNF-α, VCAM-1 and ICAM-1, the proportion of myocardial fibrosis area, the mRNA expression levels of myocardial collagen Ⅰ, collagen Ⅲ and CTGF, and the expression levels of IL-1ß and IL-18 were significantly down-regulated (P<0.001, P<0.01, P<0.05) in both the medication and EA groups. No significant differences were found between the EA and medication groups in all the indexes mentioned above. CONCLUSIONS: EA can improve the left-ventricular fibrosis and function, delay or reverse ventricular remodeling in MIRI rats, which may be related to its functions in down-regulating myocardial inflammatory response and mRNA expression levels of myocardial collagen Ⅰ, collagen Ⅲ and CTGF.

Electroacupuncture preconditioning protects against myocardial ischemia-reperfusion injury by modulating dynamic inflammatory response.

Background: The protective effects of electroacupuncture (EA) preconditioning against myocardial ischemia-reperfusion injury (MIRI) have been reported. However, the underlying mechanism remains unclear. Recent research has indicated that the dynamic inflammatory response following MIRI plays an essential role in the progression of myocardial injury. This study aimed to investigate the myocardial protective effects of EA preconditioning on MIRI in rats and to explore the relevant mechanism from the perspective of dynamic inflammatory response. Methods: A MIRI model was employed, and the rats were subjected to EA on Neiguan for four days prior to modeling. The myocardial protective effect of EA preconditioning was evaluated by echocardiography, Evans blue and triphenyltetrazolium chloride staining. Real-time polymerase chain reaction, Western blot, hematoxylin & eosin staining, and immunohistochemistry were utilized to detect the content of mitochondrial DNA, NOD receptor family protein 3 (NLRP3) inflammasome activation, neutrophil recruitment and macrophage infiltration in blood samples and myocardium below the ligation. Results: We found that EA preconditioning could accelerate the recovery of left ventricle function after MIRI and reduce the myocardial infarction area, thereby protecting the myocardium against MIRI. Furthermore, EA preconditioning was observed to ameliorate mitochondrial impairment, reduce the level of plasma mitochondrial DNA, modulate NLRP3 inflammasome activation, attenuate neutrophil infiltration, and promote the polarization of M1 macrophages towards M2 macrophages in the myocardium after MIRI. Conclusion: EA preconditioning could reduce plasma mtDNA, suppress overactivation of the NLRP3 inflammasome, facilitate the transition from the acute pro-inflammatory phase to the anti-inflammatory reparative phase after MIRI, and ultimately confer cardioprotective benefits.

[Effect of electroacupuncture preconditioning on expression of Gasdermin D, Caspase-1 and IL-1ß in rats with myocardial ischemia reperfusion injury].

OBJECTIVE: To observe the effect of electroacupuncture(EA) preconditioning on expression of Caspase-1, Gasdermin D(GSDMD) and interleukin-1ß(IL-1ß) in myocardial tissue of myocardial ischemia reperfusion injury (MIRI) rats in order to explore its underlying mechanisms in resisting MIRI. METHODS: Forty male rats were randomly divided into 4 groups: normal control (normal), sham operation (sham), MIRI model and EA groups. The MIRI model was established by ligation of the left anterior descending branch of the left coronary artery for 30 min and perfusion. EA (2 Hz/100 Hz, 1 mA) was applied to bilateral "Neiguan" (PC6) for 20 min, once a day for 3 consecutive days. The echocardiography was used to analyze the left ventricular end-diastolic dimension (LVEDD), left ventricular end-systolic dimension (LVESD) and left ventricular ejection fraction (LVEF, by using Teichholz formula) 4 h after modeling. The myocardial TTC staining was used to observe the proportion of the infarct area, and Western blot was used to detect the expression levels of GSDMD, Caspase-1, IL-1ß proteins in the myocardium. RESULTS: Compared with the normal group, the immunoactivity of GSDMD was increased in the sham group (P<0.05). Compared with the sham group, the LVEF was significantly decreased (P<0.000 1), while the myocardial infarction area, immunoactivity of GSDMD, and the expression levels of Caspase-1, GSDMD and IL-1ß proteins were considerably increased in the model group (P<0.000 1, P<0.001). In comparison with the model group, the decreased ejection fraction and the increased myocardial infarction area, and Caspase-1, GSDMD and IL-1ß expression were reversed in the EA group (P<0.001, P<0.000 1, P<0.01). CONCLUSION: EA preconditioning may ameliorate myocardial injury in MIRI rats which may be associated with its function in down-regulating the expression of myocardial Caspase-1 protein to reduce cardiomyocyte pyroptosis.

[Effect of electroacupuncture at "Neiguan" (PC 6) on cardiac function and inflammatory factors of acupoint area in rats with acute myocardial ischemia].

OBJECTIVE: To observe the effect of electroacupuncture (EA) at "Neiguan" (PC 6) on cardiac function and cardiomyocyte apoptosis in rats with acute myocardial ischemia (AMI), and to explore the correlation between myocardial protective effect of EA and inflammatory factors i.e. interleukin-1ß (IL-1ß) and interleukin-17 (IL-17) of "Neiguan" (PC 6) area. METHODS: A total of 40 male SD rats with normal ultrasonic cardiogram were randomized into a sham-operation group, a sham-operation plus EA group, a model group and an EA group, 10 rats in each group. The AMI model was established by ligating the left anterior descending (LAD) branch of the coronary artery in the model group and the EA group, while the threading without ligating was adopted in the sham-operation group and the sham-operation plus EA group. In the sham-operation plus EA group and the EA group, EA at bilateral "Neiguan" (PC 6) was applied, with disperse-dense wave, 2 Hz/100 Hz in frequency and 2 mA in density, once a day, 20 min a time for 3 days. The cardiac ejection fraction (EF) and fractional shortening (FS) were measured by ultrasonic cardiogram to evaluate the cardiac function, the cardiomyocyte apoptosis was detected by TUNEL staining, the infiltration of inflammatory factors of "Neiguan" (PC 6) area was observed by H.E. staining, the expression of inflammatory factors IL-1ß and IL-17 of "Neiguan" (PC 6) area was detected by immunofluorescence staining. RESULTS: Compared with the sham-operation group, EF and FS were decreased (P<0.001), the average optical density of cardiomyocyte apoptosis was increased (P<0.001), the infiltration of inflammatory factors was obvious in skin dermis of "Neiguan" (PC 6) area in the model group; the positive expression of IL-1ß and IL-17 of "Neiguan" (PC 6) area was increased in the model group and the sham-operation plus EA group (P<0.001, P<0.01). Compared with the model group, EF and FS were increased (P<0.01), the average optical density of cardiomyocyte apoptosis was decreased (P<0.01), the infiltration of inflammatory factors was aggravating in skin dermis of "Neiguan" (PC 6) area, and the positive expression of IL-1ß and IL-17 of "Neiguan" (PC 6) area was increased in the EA group (P<0.001, P<0.01). CONCLUSION: Electroacupuncture at "Neiguan" (PC 6) can improve the cardiac function and reduce the apoptosis of cardiomyocyte in rats with acute myocardial ischemia, its mechanism may be related to the regulation of the inflammatory factors of "Neiguan" (PC 6) area.

[Effect of electroacupuncture on expression of IL-23/IL-17 axis and TLR4 in infarcted tissue in rats with myocardial infarction].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on expression of interleukin (IL) -23/IL-17 axis and Toll-like receptor 4 (TLR4) in the infarcted tissue in rats with myocardial infarction (MI), and to explore the mechanism of EA on alleviating MI injury. METHODS: Forty male SD rats were randomly divided into a sham-operation group, a sham-operation plus EA group, a model group and an EA group, 10 rats in each group. The MI models were established by ligation of left anterior descending coronary artery in the model group and EA group, while only threading was performed in the sham-operation group and sham-operation plus EA group. The rats in the sham-operation plus EA group and EA group were treated with EA at "Neiguan" (PC 6), disperse-dense wave, 2 Hz/100 Hz, 2 mA, once a day, 20 min each time, for 3 days. After the intervention, the ejection fraction (EF) was measured by echocardiography to evaluate the cardiac function; the infarct area was measured by TTC staining; the HE staining was used to observe the morphological changes of myocardial tissue; the levels of IL-23 and IL-17 in infarcted tissue were detected by ELISA; the protein expression of TLR4 in infarcted tissue was detected by Western blot. RESULTS: Compared with the sham-operation group, the EF was decreased (P<0.01), the infarct area was increased (P<0.01), the myocardial fiber injury was obvious, accompanied by inflammatory cell infiltration, and the contents of IL-23, IL-17 and the expression of TLR4 in infarcted tissue were increased in the model group (P<0.01). Compared with the model group, the EF was increased (P<0.05), the infarct area was reduced (P<0.05), the myocardial fiber injury was significantly improved, the inflammatory cell infiltration was reduced, and the contents of IL-23, IL-17 and TLR4 expression in infarcted tissue were decreased in the EA group (P<0.05). CONCLUSION: EA may alleviate the excessive inflammatory response after MI by inhibiting the expression of IL-23/IL-17 axis in MI rats, and TLR4 may be involved during the process.

[Effect of electroacupuncture pretreatment on transient receptor potential vanilloid 1(TRPV1)/calcitonin gene-related peptide(CGRP) signal and NF-κB p65 protein expression in rats with acute myocardial ischemia].

OBJECTIVE: To observe the effect of electroacupuncture(EA)pretreatment on transient receptor potential vanilloid 1(TRPV1)/calcitonin gene-related peptide(CGRP)signal and nuclear factor-κB p65 (NF-κB p65) protein expression in myocardial tissue of acute myocardial ischemic injury (AMI) rats, and to investigate the possible mechanism of electroacupuncture pretreatment against AMI. METHODS: A total of 60 adult male SD rats were randomly divided into blank control, sham operation, model and EA pretreatment groups, 15 rats in each group. The acute myocardial ischemia model was established by ligating the left anterior descending (LAD)branch of the coronary artery in the model group and EA pretreatment group, while threading but no ligating at left anterior descending branch of the coronary artery was applied in the sham operation group. In the EA pretreatment group, bilateral "Neiguan"(PC6) acupoints were selected, with intensity of 2 mA and frequency of 2 Hz/100 Hz, for 20 min, once daily for 7 days before modeling. Electrocardiogram (ECG) was recorded by physiological signal acquisition system, and the ST segment potential offset values of standard Ⅱ lead were analyzed before surgery，30 min and 24 h after operation. The TTC staining was used to observe the percentage of myocardial infarction area. The HE staining was used to observe the pathological changes of myocardial tissue and the degree of inflammatory cell infiltration. And Western blot was used to detect TRPV1/CGRP signal and NF-κB p65 protein expression levels in myocardial tissue. RESULTS: Compared with the sham operation group, the ECG-J point potential in the model group was significantly increased at 30 min and decreased at 24 h after operation (P<0.05), myocardial infarction area increased significantly (P<0.05), the myocardial fibers were obviously disordered, inflammatory cell infiltration was obvious, and the expressions of TRPV1，CGRP and NF-κB p65 proteins were all increased (P<0.05). Compared with the model group, the EA pretreatment group was decreased in the ECG-J point potential at 30 min after operation(P<0.05), significantly reduced in myocardial infarction area (P<0.05), improved in the morphology of myocardial fibers, reduced ininflammatory cell infiltration, and increased in the protein expressions of TRPV1 and CGRP in myocardium (P<0.05), significantly decreased in the protein expression of NF-κB p65 (P<0.05). CONCLUSION: EA pretreatment may enhance TRPV1/CGRP signaling, down-regulate NF-κB p65 protein expression, reduce myocardial inflammatory response status, improve AMI injury, and reduce myocardial infarction area.

Proteomic and metabolomic characterization of cardiac tissue in acute myocardial ischemia injury rats.

The pathological process and mechanism of myocardial ischemia (MI) is very complicated, and remains unclear. An integrated proteomic-metabolomics analysis was applied to comprehensively understand the pathological changes and mechanism of MI. Male Sprague-Dawley rats were randomly divided into a mock surgery (MS) group and an MI group. The MI model was made by ligating the left anterior descending coronary artery, twenty-four hours after which, echocardiography was employed to assess left ventricular (LV) function variables. Blood samples and left ventricular tissues were collected for ELISA, metabolomics and proteomics analysis. The results showed that LV function, including ejection fraction (EF) and fractional shortening (FS), was significantly reduced and the level of cTnT in the serum increased after MI. iTRAQ proteomics showed that a total of 169 proteins were altered including 52 and 117 proteins with increased and decreased expression, respectively, which were mainly involved in the following activities: complement and coagulation cascades, tight junction, regulation of actin cytoskeleton, MAPK signaling pathway, endocytosis, NOD-like receptor signaling pathway, as well as phagosome coupled with vitamin digestion and absorption. Altered metabolomic profiling of this transition was mostly enriched in pathways including ABC transporters, glycerophospholipid metabolism, protein digestion and absorption and aminoacyl-tRNA biosynthesis. The integrated metabolomics and proteomics analysis indicated that myocardial injury after MI is closely related to several metabolic pathways, especially energy metabolism, amino acid metabolism, vascular smooth muscle contraction, gap junction and neuroactive ligand-receptor interaction. These findings may contribute to understanding the mechanism of MI and have implication for new therapeutic targets.

[Progress of Research on Mechanisms of Moxibustion Intervention Underlying Improvement of Blood Circulation].

OBJECTIVE: Moxibustion is one of the commonly used therapy of traditional Chinese medicine by applying burning dried mugwort on particular acupoints of the body surface. In the present paper，we reviewed progress of researches about the mechanisms of moxibustion treatment undering imporvement of blood circulation in recent 15 years. Research results displayed that moxibustion can dilate blood vessels to increase blood flow and improve microcirculation, not only in the local superficial vessels of body, but also in the deep tissues as the brain, stomach and mesentery, kidney, heart, etc., as well as in the distal blood vessels. The vasodilator action of moxibustion stimulation is related to nerve regulation, endothelium derived relaxing factors and vasodilator mediator, etc. through 1) interaction of acetylcholine (Ach)/muscarinic receptor (MR) and noradrenaline (NE)/α- or ß-receptor; 2) nitric oxide synthase (NOS)/NO/arachidonic acid/prostacyclin (PGI2)/endothelium-derived hyperpolarizing factor (EDHF) pathway; 3) EDHF/TRPV 4/KCa channel, cytochrome P 450 oxidase/epoxyeicosatrienoicacid (EET); 4) EET/TRPV 4/big conductance calcium-activated potassium channel (BKCa); 5) sulfuretted hydrogen (H2S)/ATP-sensitive potassium channel (KATP) or voltage-gated potassium channels (Kv 7); 6) NO/substance P (SP) or CGRP and adrenergic ß 2 receptor(R)/TRPV 1/adenosin A 1 R and A 2 R/NK 1 R pathway; 7) PGI2/adenylyl cyclase (cAMP)/PKA and vasoactive intestinal peptide (VIP), etc. in the vascular endothelium and smooth muscle. These research results may help us understand the effects and mechanisms of moxibustion in the treatment of different clinical conditions by improving microcirculation.

[From Treatment of Stress Disease by Acupuncture to Correlation between Acupuncture and Stress].

Stress disease is a body functional disorder caused by harmful stimuli overwhelming the nonspecific adaptive capabilities. Numerous evidence proved the feasibility of acupuncture in treating stress diseases. This paper aims at exploring new perspective in the study on the underlying mechanism of acupuncture in the treatment of stress diseases. The discussion is conducted by summarizing the pathogenesis of stress diseases and mechanisms of its treatment with acupuncture, in exploring the related factors of acupuncture and stress response, and the correlation between the two from the regulatory pathway, medium, characteristics and manifestations.