[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.

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 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.