ABSTRACT
Objective:To observe the effect of electroacupuncture (EA) pretreatment on adenine nucleotides in the myocardial tissues of the myocardial ischemia-reperfusion injury (MIRI) rats, and to explore the mechanism of EA pretreatment on myocardial prevention and protection in MIRI rats. Methods:Forty SPF male Sprague-Dawley (SD) rats were randomly divided into 5 groups: a blank group, a sham operation group, a model group, an EA at Neiguan (PC 6) group and an EA at Hegu (LI 4) group, with 8 rats in each group. Rats in the blank group only received binding to the rat plate, 30 min/time, once a day for 7 d; on the 7th day, rats in the sham operation group were subjected to threading for 40 min at the left anterior descending coronary artery without ligation, and then the rats were allowed to stand for 60 min before collection of the specimens; on the 7th day, rats in the model group were subjected to threading at the left anterior descending coronary artery with ligation, for 40 min before the blood flow was restored, and then the rats were allowed to stand for 60 min before collection of the specimens; on the 7th day of pretreatment with EA at Neiguan (PC 6) or Hegu (LI 4) for 30 min per day (once a day for 7 d), rats in the EA at Neiguan (PC 6) group and EA at Hegu (LI 4) group were subjected to modeling and sample collection same as in the model group. The left ventricular myocardium of the lower left anterior descending coronary artery was collected from rats in all 5 groups. Hematoxylin-eosin (HE) staining and transmission electron microscope (TEM) were used to observe the changes in myocardial pathological morphology. The change in the adenine nucleotide level of myocardial tissue was measured by high performance liquid chromatography (HPLC). Results:The HE staining and ultrastructure showed that the myocardial injury was severer in the model group compared with the sham operation group. Compared with the model group, the myocardial injury in the EA at Neiguan (PC 6) and the EA at Hegu (LI 4) groups was mild or hardly any. The adenine nucleotide levels in the sham operation group and the model group were all decreased compared with the blank group (allP<0.05); compared with the sham operation group, the adenine nucleotide level of the model group was also decreased, but the difference was not statistically significant (P>0.05); compared with the model group, the adenine nucleotide level in the EA at Neiguan (PC 6) group was increased (P<0.05), and the adenine nucleotide level in the EA at Hegu (LI 4) group was significantly increased (P<0.01). The adenine nucleotide level in the EA at Hegu (LI 4) group was higher than that in the EA at Neiguan (PC 6) group, but the difference was not statistically significant (P>0.05). Compared with the EA at Neiguan (PC 6) group, the levels of adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) in the EA at Hegu (LI 4) group were significantly increased (allP<0.01). Conclusion:Both EA at Neiguan (PC 6) and Hegu (LI 4) can alleviate the pathological damage to myocardium in MIRI rats, and increase the adenine nucleotide level in myocardial tissues, and thus protect MIRI rats. EA at Hegu (LI 4) has a better protective effect than Neiguan (PC 6).
ABSTRACT
OBJECTIVE: To observe the effect of electroacupuncture (EA) and moxibustion (Moxi) pretreatment on myocardial pathological and structural changes and expression of autophagy related protein LC 3 Ⅰ/Ⅱ and Beclin 1 in rats with myocardial ischemia-reperfusion injury (MI/RI), so as to explore their mechanisms underlying improving MI/RI. METHODS: Forty SD rats were randomly divided into sham operation, model, ischemic preconditioning (IP), EA and Moxi groups (n＝8 in each group). EA (10 Hz/50 Hz，1 mA) or Moxi (ignited moxa stick) was respectively applied to bilateral "Neiguan"(PC 6) for 20 min, once daily for 7 days. The MI/RI model was established by occlusion of the anterior descending branch of the left coronary artery for 40 min, followed by reperfusion for 60 min. The left ventricular (LV) tissue samples were collected and analyzed for pathological (H.E. staining) and ultrastructural changes, for myocardial apoptosis (apoptotic index＝ number of apoptotic cells/total number of cardiomyocytes×100%) with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method, and for the expression of LC 3 and Beclin 1 in myocardial cells with Western blot. RESULTS: Following MI/RI, H．E. staining revealed a disorder of arrangement of cardiomyocytes with vague border, inflammatory cell infiltration, intracellular swelling with bleeding, necrosis and dissolution of partial striated muscles of the left ventricle under light microscope, and dual staining of Uranyl acetate and leadnitrate showed atrophy, arrangement disorder, dissolution, necrosis, and interstitial edema of partial myocardial fibers, mitochondrial structural disorder, vacolation, and large body of autophagosomes with bilayers, etc. in ultrastructure, which was relatively lighter in both EA and Moxi groups. The apoptosis index, expression levels of myocardial LC 3 Ⅱ and Beclin 1 and the ratio of LC 3 Ⅱ/LC 3 Ⅰ were significantly higher in the model group than those in the sham operation group (P<0.01), but the expression level of LC 3 Ⅰ was considerably down-regulated in the model group relevant to the sham operation group (P<0.01). Following the intervention and MI preconditioning, the increased apoptosis index and expression levels of LC 3Ⅱ and Beclin 1 proteins and the ratio of LC 3Ⅱ/LC 3 Ⅰ were obviously down-regulated in the IP, EA and Moxi groups relevant to the model group (P<0.01), and the decreased expression of LC 3 Ⅰ protein was up-regulated obviously in the 3 treatment groups (P<0.05，P<0.01). The effects of EA were significantly superior to those of IP and Moxi groups in down-regulating apoptosis index and expression of LC 3 Ⅱ and Beclin 1 and the ratio of LC 3 Ⅱ/LC 3 Ⅰ and in up-regulating expression of LC 3 Ⅰ (P<0.05, P<0.01). CONCLUSION: Both EA and Moxi preconditioning of PC 6 have a protective effect on ischemic myocardium in MI/RI rats, which is probably related to their effects in regulating expression of myocardial autophagy proteins as LC 3 Ⅰ/Ⅱ and Beclin 1.
ABSTRACT
Objective:To observe the effect of point-moxibustion on gastrointestinal motility,mRNA and protein expressions of ghrelin and growth hormone secretagogue receptor 1a (GHSR-1a) in lateral septal nucleus of rats with diabetic gastroparesis (DGP),and to investigate the central regulatory mechanism of DGP treatment with point-moxibustion.Methods:Forty SPF-grade Sprague-Dawley (SD) rats were randomly divided into a blank group,a model group,an electroacupuncture (EA) group and a point-moxibustion group,with 10 rats in each group.A DGP rat model was established by intraperitoneal injection of 2％ streptozotocin (STZ) with 8-week irregular high-sugar and high-fat diet in the model group,the EA group and the point-moxibustion group;and rats in the blank group were injected intraperitoneally with 0.1 mmoL/L (pH 4.5) citric acid-sodium citrate buffer with 8-week normal diet.Eight weeks later,rats in the EA group were treated by EA at Zusanli (ST 36),Liangmen (ST 21) and Sanyinjiao (SP 6);while rats in the point-moxibustion group were treated by point-moxibustion at Zusanli (ST 36),Liangmen (ST 21) and Sanyinjiao (SP 6) for successive 15 d.Rats in the blank group and the model group were fixed as the control without intervention.After treatment,intestinal propulsion rate and gastric emptying rate were measured.The mRNA and protein expressions of ghrelin and GHSR-1a in the lateral septal nucleus were detected by real-time polymerase chain reaction (RT-PCR) and Western blot (WB).Results:Compared with the blank group,the intestinal propulsion rate and gastric emptying rate of the model group were significantly lower (both P＜0.01);compared with the model group,the intestinal propulsion rate and gastric emptying rate of the EA group and the point-moxibustion group increased significantly (all P＜0.05).The mRNA and protein expressions of ghrelin and GHSR-1a were lower in the model group than those in the blank group (all P＜0.01).The mRNA and protein expressions of ghrelin and GHSR-1a were significantly higher in the EA group and the point-moxibustion group than those in the model group (all P＜0.05).There were no statistically significant differences between the EA group and the point-moxibustion group (all P＞0.05).Conclusion:Point-moxibustion at Zusanli (ST 36),Liangmen (ST 21) and Sanyinjiao (SP 6) can increase the intestinal propulsion rate and gastric emptying rate of DGP rats,and promote the mRNA and protein expressions of ghrelin and GHSR-1a in the central nervous system.The mechanism may be related to the activation of ghrelin pathway in hypothalamic arcuate nucleus-lateral septal nucleus.