| ObjectiveCardiovascular disease is one of the main diseases that threaten human health and life,while myocardial ischemia is a major factor in cardiovascular disease.Acupuncture has been used for prevention and treatment of myocardial ischemia application for thousands of years,which has some features,such as,easy operation and obvious curative effect,but its mechanism of action,especially molecular biologicalmechanism is not entirely clear,to a certain extent affected the acupuncture on myocardial ischemia in clinical application and popularizationto a certain extent.Research shows that myocardial apoptosis widely exists in the process of myocardial ischemic andnecrosis,and apoptosis is tightly controlled by the genes,while Micro RNA(miRNA) has a key role in its regulation. Whethera acupuncture is interfered by miRNA gene regulation pathways for cardiac myocyte apoptosis? What miRNA was regulated by? Which pathway play does the main miRNA genes through?Based on it,the subject selected the model of myocardial ischemia in rats as the research object,Under the guidance of the theory "preventive treatment of disease" choosing "combined of Biao and ben acupoint" EA intervention in apoptosis as the starting point,using miRNA microarray screening, real-time quantitative PCR(q RT-PCR) and Western blotting(Western blot) techniques,we observed influence and control relationship onmyocardial cell apoptosis associated miRNAs and their target genes expression,in order to explore the regulation mechanisms of "specimen distribution points" acupuncture intervention on myocardial ischemia miRNA gene, with a view to providing theoretical support and scientific basis for acupuncture prevention of myocardial ischemia disease.Methods1.40 SPF male Wistar rats of weighing 180-220 g were randomly divided into the normal group, model group, Neiguan EA group(Neiguan group), Combined of Biao and ben acupointgroup(standard group), 10 rats in each group.The rats of the model group, Neiguan group and standard group were treated by ISO 2mg /(kg·d) subcutaneous injection for 14 days,the ECG changes by the BL-420 biology enginery system: the duration of QRS wave and QT wave extended as a successful model flag. The normal group rats were given the same amount of 0.9% saline injection subcutaneously for 14 consecutive days.The rats of the normal group and model group was fixed and no acupuncture.The rats of Neiguan would acupuncture bilateral(the distance is 5mm on the middle of wrist stripes and the depth of acupuncture is 0.5 cm), the standard group was treated bilateral "Neiguan", "Guan yuan"(the distance is 25 mm under umbilical of the rats and the depth of acupuncture is 0.5 cm)and bilateral "Zusanli"(the distance is 5mm under-outside knee rats fibular and the depth of acupuncture is 0.8 cm),Standard group ipsilateral points consisting of a pair of electrodes,the neiguan group and the standard group of single-hole beside the point at the start and another 0.5cm subcutaneous superficial stab a needle as an auxiliary electrode, after connecting the Hans LH202 H EA therapeuticapparatus,continuous wave,frequency of 2Hz,intensity of 1m A,electricity for 10 minutes.Daily for 1 times,a total of 21 days.At 22 days,rats were detected in the rat left ventricular diastolic end diastolic diameter(LVEDd),left ventricular systolic end diastolic diameter(LVESD), left ventricular ejection fraction(EF) and left ventricular short axis shortening(FS) by ultrasound echocardiography machine.The detection index of myocardial cell apoptosis(AI) in rats with TUNEL; The method of ELISA was detected serum creatine kinase isoenzyme(CK-MB),vascular endothelial cell adhesion molecule 1(VCAM-1), endothelin-1(ET- 1); The method of real-time PCR was detected miRNA-133-3p, miRNA-133-5p, miRNA-1-3p, miRNA-486 expression and target genes Nol3, Caspase- 3, Aifm2, Api5, RGD1564319, Aatk expression in myocardial tissue. Then the data for statistical was analyzed and the correlation of miRNA and its target gene was verified.2.Selection SPF male Wistar rats 12 and 3rats in each group, the method of treatment and drawing was the same as above. Expression with microarrays were detected in rat myocardial miRNA gene chip technology.3.The choice of SPF 70 male Wistar rats were randomly divided into the normal group, model group, Neiguan group, standard group, miRNA-133a-5p inhibitor group(inhibitor group), miRNA-133a-5p agonist group( agonist group), miRNA-133a-5p antagomir + Biao and Ben acupoints combination group(inhibitor + standard group), 10 rats in each group. The method of modeling with the model group, Neiguan, standard group, antagomir groups, agomir group and the antagomir + standard group were the same to(1).The rats of the antagomir group and the antagomir+ standard group were injected subcutaneous 10 mg / kg were miRNA-133a-5p antagomir at the time of the 1st day、7th day and 14 th day;the agonist group was injected miRNA-133a-5p agomir agent which the dose and method was the same with the antagomir group. the method of normal group as the same of method 1.The rats of the normal group, model group, antagomir group and agonist group were only fixed and no acupuncture. The groups of Neiguan, standard and antagomir + standard were treated by EA and the method of treatment as the same with(1).The heart samples of rats were detected in 22 th day were detected miRNA-133-5p and its target gene Caspase-3, Aifm2 expression by real-time fluorescence quantitative PCR method; the expression of target gene Caspase-3, Aifm2 were detected by Western blot, and then analysis the datas.Results1.The value of LVEDd,LVESD with Model group rats were higher than those in the normal group,but EF and FS were lower than it(both P<0.01);Neiguan group and standard group were lower than those in the model group(both P<0.01) and the standard group was lower than those in Neiguan group(P<0.01,P<0.05);the value of EF and FS in Neiguan group and standard group were higher than those in the model group(P<0.01)and the standard group was higher than those in Neiguan group(P<0.05).2. The value of myocardial AI in the normal group was lower than those in the model group(P<0.01);Neiguan group and standard group were lower than those in model group(both P<0.01) and standard group was lower than those in Neiguan group(P<0.05).3. The serum levels of CK-MB, VCAM-1 and ET-1 in the model group were all higher than those in the normal group(P<0.01).The standard group, Neiguan group were lower than that those in the model group(P<0.01); the standard group was lower than that those in Neiguan group(P<0.05).4. The use of microarray technology were screened out 758 miRNA genes signals which 20 miRNA gene information has significant difference:12 were up-regulated and 8 down-regulated in the expression;the trend which miR-133a-5p and miR-133a-3p was upward, miR-1-3p and mir-486 was down obviously superior to other miRNA genes.Three kinds of biological information database by Target Scan, miRanda and Pic Tar were predicted the four miRNA genes associated with apoptosis target gene which the target genes of miR-133a-3p is Nol3, miR-133a-3p was Caspase- 3 and Aifm2, miR-1-3p was Api5, miR-486 was RGD1564319 and Aatk.5.(1)The expression of the model group rats myocardial miRNA-133-3p, miRNA-133-5p were lower than that in the normal group(both P<0.01), and miRNA-486 miRNA-1-3p were higher than those in the normal group(both P<0.01); The expression of miRNA-133-3p, miRNA-133-5p in Neiguan group and standard group were higher than those in the model group(respectively P<0.05,P<0.01,P<0.01,P<0.01), standard group were higher than those in Neiguan group(respecttively P<0.05, P<0.01).The expression of miRNA-1-3p, miRNA-486 in Neiguan group and standard group were lower than those in the model group(respectively P<0.05,P<0.05,P<0.01,P<0.01) and standard group were lower than those in Neiguan group(both P<0.05).(2) The expression of Nol3 in the model group was higher than that in the normal control group(P<0.01), there was no significant difference among Neiguan group, model group and standard group(both P>0.05); The expression of caspase-3 and Aifm2 were higher than those in the normal group(both P<0.01), Neiguan group and standard group were lower than those in the model group(both P<0.01) and standard group were lower than those in Neiguan group(both P<0.01);The expression of Api5 in the model group was lower than those in the normal group(P<0.01), but there was no significant difference between Neiguan group and model group(P>0.05), the standard group was lower than that in the model group(P<0.05). The expression of RGD1564319 in the normal group was no significant difference with the model group(P>0.05);The expression of Aatk in the model group was higher than those in the normal control group(P < 0.01), there was no significant difference between Neiguan group and model group(P>0.05), but the standard group was higher than those in the model group(P<0.05).(3) The rusult of correlation between the groups as a whole and intensity correlation coefficient was analyzed that there was moderate negative correlation between miRNA-133a-3p and Nol3, miRNA-1-3p and Api5; there was positive correlation between miRNA-486 and Aatk; but was mild negative correlation between miRNA-486 and RGD1564319; There were highly negatively correlated among miRNA-133a-5p and Cas-pase-3, Aifm2.6.(1) The expression of miRNA-133a-5p in the model group rats was lower than that of the normal group(P<0.01); Neiguan group and standard group, agomir group, antagomir + standard group were higher than those in the model group(both P<0.01), but the antagomir group was lower than those in the model group(P<0.01); the standard group, agomir group, antagomir + standard group were higher than those in Neiguan group(respectively P<0.01,P<0.01, P<0.05), the antagomir group was lower than those in Neiguan group(P<0.01); the excited agent group was higher than those in the standard group(P<0.01), the antagomir group is lower than those in the standard group(P < 0.01),but there had no significant difference between the antagomir+standard group and standard group(P>0.05).(2) The expression of Caspase-3, Aifm2 in the model group(q RT-PCR) were higher than those in the normal group(both P<0.01); Neiguan group and standard group, agomir group, antagomir + standard group were lower than those in the model group(both P<0.01), the antagomir group was higher than those in the model group(respectively P < 0.01,P < 0.05);the standard group and excited agent group were lower than those in Neiguan group(both P<0.01),the antagomir group was higher than those in Neiguan group(both P<0.01), but there had no significant difference between the antagomir+standard group and Neiguan group(P>0.05);The antagomir group was higher than those in the standard group(both P<0.01), there had no significant difference among agomir group, antagomir + group and standard group(P>0.05).(3)The expression of Caspase-3 and Aifm2(Western-blot) in the model group were higher than those in the normal group(P<0.01);the standard group, Neiguan group, agomir group, antagomir + group were lower than those in the model group(P<0.01), the antagomir group was higher than those in the model group(P<0.05); the standard group and agonist group were lower than that in Neiguan group(P<0.01), the antagomir group was higher than that in Neiguan group(P< 0.01), the antagomir + standard group(Caspase-3) had no significant difference with Neiguan group(P>0.05), the standard + standard group(Aifm2) was lower that in Neiguan group(P<0.01); the antagomir group was higher than that in the standard group(both P<0.01), but there had no significant difference among the agomir group, antagomir + standard group and the standard group(P>0.05).(4) The rusult of correlation between the groups as a whole and intensity correlation coefficient was analyzed that its was a strong negative correlation between miRNA-133a-5p and Caspase-3, Aifm2(q RT-PCR, Western-blot detection).Conclusion1. EA could reduce the values of LVEDd and LVESD and improve the values of EF and FS.It aslo could effectively improve myocardial ischemia which was caused by apoptosis compensatory myocardial thickening, cardiac dysfunction and reduced degree of myocardial cell apoptosis,and the method of the combined of Biao and ben acupoint EA has better efficacy than that in Neiguan EA.2. The combined of Biao and ben acupoint and Neiguan EA could effectively reduce the value of CK-MB, VCAM-1 and ET-1 in order to confronted the anti-ischemic for enzymes and other indicators of change after myocardial ischemia-induced apoptosis, and The EA methods of the combined of Biao and ben acupoint has better effect than single acupuncture.3.The number of miRNA genes were involved in isoproterenol induced myocardial ischemia in the process of apoptosis which was showed mainly that the expression of miRNA-133-3p and miRNA-133-5p were down, but miRNA-486 and miRNA-1-3p were rise, the combined of Biao and ben acupoint EA could effectively improve the expression of miRNA-133-3p and miRNA-133-5p,but reduce its expression of miRNA-1-3p and miRNA-486, the protection mechanism was fulfilled by the way of regulation these four ways miRNA genes in order to inhibition apoptosis myocardium.4.The miRNA genes and its target genes were the main pathways for affecting apoptosis of myocardial ischemia; in combined of Biao and ben acupoint EA in treating myocardial ischemia of miRNA gene signal transduction pathway, gene regulation and control signals was the main way between the miRNA-133-5p and the expression of Caspase-3,Aifm2 in which the miRNA gene signal transduction pathway of the combined of Biao and ben acupoint EA prevention and treatment myocardial ischemia.5. Caspase-3 and Aifm2 were the main factors in activating cell apoptosis pathway;The effection of the combined of Biao and ben acupoint EA inhibition cell apoptosis and protective myocardial ischemia was completed with double channels regulatory by lifting the expression of miRNA-133-5p and decreasing the expression of caspase-3, Aifm2 expression of double channel regulatory pathways. |
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