| Research PurposeBy the comparative study of the different compatibility proportion of Chinese traditional medicine extracted from f istular onion stalk and rhubarb on the treatment of acute myocardial ischemia, the study aims to discuss the regulation effect and mechanism of blood stasis removing method on acute myocardial ischemia in rat MAPKs/IL-6signal pathway, thus preliminarily reveal the biological basis of the treatment of acute myocardial ischemia with the blood stasis removing method.MethodThis research selected64healthy pure SD male rats weighed for200-230g and aged3-4months, then they were randomly divided into normal control group, blank model group, fistular onion stalk extraction group, fistular onion stalk extraction and rhubarb extraction group of7:3,5:5,3:7, rhubarb extraction group and the nitroglycerin group, with a total8groups and8rats in each group. The acute myocardial ischemia models were prepared according to the acute myocardial ischemia model from Guo Yansong, rats were adaptively fed for1week, and were injected with3.5%sodium pentobarbital intraperitoneal (35mg/kg) for anesthesia, Rats were fixed supinely and were connected with the power lab/8s eight guided data analysis recorder, opened the chest to get records of a normal ECG, then the rats’pulses were cut to connect with the Y pulse interpolation, with the other end connected with the animal trace ventilator outlet, with a continuous inlet of10x60ml/min, and legated the left coronary artery to prepare the myocardial ischemia model, then delivered the drugs, then observed the indexes3hours later, each group was treated with the corresponding concentration of one drug, sham operation group and model group were given the same volume of saline, with a syringe5minutes after occlusion, blood collection and heart were taken4h later. Cut at the left of abdominal midline to locate the duodenum, covered the wound with saline gauze, took the blood and myocardial tissues4hours later, observed the relevant index, the drug-delivery volume was of2ml/kg, the normal control group and blank model group were given an equal volume of saline, the fistular onion stalk extraction group was delivered the drug at600mg kg-1, rhubarb extraction group at400mg kg-1, fistular onion stalk and rhubarb were mixed with the corresponding concentration of7:3,5:5,3:7in different groups, nitroglycerin group was delivered at80mg kg-1, observed for3h, and conducted ECG detection again, after observation, immediately clamped the rat thoracic wall temporarily by cl ipping the fast lines of rats, immediately took blood from the abdominal aorta and injected2ml blood into to the tube containing30UL of10%EDTA sodium and40ul of pancreatic peptide enzyme, mixed at4℃, centrifugal at3000rpm for10min, separated the plasma, and placed it in the refrigerator at-70℃for testing, the remaining blood of was centrifuged at3500rpm for20min, took the supernatant, packed and placed in the refrigerator at-70℃. Before the test, the sample was melted in cold water, mixed, and centrifuged at3500rpm for5min at4℃, then took the supernatant for testing. Recorded the electrocardiogram before and after administration, measured the average of ST segment shift and the changes of electrocardiogram of ST segment, with the standard Ⅱ electroca-rdiogram observation (standard voltage was0.1mv, paper shift speed was50mm/s). The pathological changes of rat myocardial tissues were analyzed through HE staining, the serum content of cTnl, CK-MB, LDH, IL-6were detected with ELISA assay. Chemical method was taken to detect myocardial H2S content. Western Blot method was applied for the detection of P-ERK, P-JNK, P-p38in acute myocardial ischemia in rat tissues, the real time fluorescent quantitative PCR technique was taken for the detection of IL-6mRNA expression in acute myocardial ischemia in myocardial tissues of rats; ultrasonic technology was used in the detection of cardiac function in acute myocardial ischemia rats;Results1. The influence on the ECG, left ventricular function and markers for myocardial infarction of acute myocardial ischemia rats:in comparison with the normal control group, the ST segment increased significantly in the blank model group (P<0.01); and the blank model group got no significant difference from the rhubarb extraction group (P>0.05), all the other treatment tissue groups could reduce rat ST segments (P<0.05, P<0.01), the effect in fistular onion stalk extraction group was the most remarkable (P<0.01), the groups of blood stasis removing method got no signi ficant difference (P>0.05).Compared with the normal group, the serum cTnl of rats in blank model group was significantly increased (P<0.01); and serum cTnI of rats in all the treatment group were significantly decreased when compared with the blank model group (P<0.05, P<0.01), the decrease in fistular onion stalk extraction group was most obvious, and was better than any treatment group, the groups of blood stasis removing method got no significant difference (P>0.05).Compared with the normal group, the serum CK-MB of rats in blank model group was significantly increased (P<0.01); and serum CK-MB of rats in all the treatment group were significantly decreased when compared with the blank model group (P<0.05, P<0.01), the decrease in fistular onion stalk extraction group and the nitroglycerin group was most obvious, and with no significant difference between the groups (P>0.05), the effect in fistular onion stalk extraction group was better than that in the rhubarb extraction group and the group of blood stasis removing method.Compared with the normal group, the serum LDH of rats in blank model group was significantly increased (P<0.01); and serum LDH of rats in all the treatment group were significantly decreased when compared with the blank model group (P<0.05,P<0.01), there were no significant difference among the rhubarb extraction group, group of blood stasis removing method and the nitroglycerin group, and the effect was better than that in the fistular onion stalk extraction group, no significant differences were found in the groups of blood stasis removing method (P>0.05). Compared with the normal group, EF, FS, Em/Am values in the model group were significantly decreased (P<0.01); compared with the model group, the EF, FS, Em/Am values in each treatment group (except the rhubarb extraction group) were significantly increased (P<0.01); as for the influence on the EF value and FS value, no significant difference was found between the group of blood stasis removing method and the group of fistular onion stalk extraction group (P>0.05), but the results were still better than that in the rhubarb extraction group and the nitroglycerin group (P<0.05); as for the influence on the Em/Am value, there were no significant differences among the groups of blood stasis removing method, or between the group of blood stasis removing method and the nitroglycerin group (P>0.05), and the effect was better than that in the fistular onion stalk extraction group.2. Morphological influences on the rat tissues of acute myocardial ischemia The syocardial tissue sections were HE stained, and the rat myocardial cells were observed with light microscopy to find out pathological changes, the results showed that, for the normal group:most of the myocardial fibers were of normal structure, and were arranged in bundles, cardiac muscle cells were in the oval-shaped or in the shape of short spindle, they were located in the cell center and the muscle fibers scattered in the capillaries. For the model group:myocardial fiber was swelling, part of the myocardial fiber fracture was found, small focal muscle fiber necrosis was noticed, and the muscle fiber structure was unclear; muscle fiber capillary was dilatated and congested, and focal infiltration of inflammatory cells was found. Most of the myocardial fiber structure of the fistular onion stalk extraction group was normal, and only a small number of muscle fiber swelling was found, which were arranged in bundles, the muscle fibers were scattered in the capillaries, and no focal necrosis was found. The groups of blood stasis removing method (7:3), in this group, part of the myocardial fiber structure was normal, a small number of muscle fiber swelling was detected, capillaries between the muscle fibers increased. In the rest of the treatment groups, focal myocardial swelling was found, with blurred structure, and part of the myocardial fiber was broken, and telangiectasia was found in a few capillaries of muscle fibers.3. Influence on the myocardial tissue of H2S of rats with acute myocardial ischemia Compared with the normal control group, the H2S decreased significantly in the blank model group (P<0.01); and the H2S level of each treatment group was significantly increase-ed when compared wi th the blank model group (P<0.01), the increase was most obvious in the group of blood stasis removing method (7:3), and the results were significantly better than that in the fistular onion stalk extraction group, rhubarb extraction group, the nitroglycerin group and other groups of blood stasis removing method.4. The influence on MAPKs of rats with acute myocardial ischemiaCompared with the normal control group, the P-ERK expression in model group was significantly increased (22.54%), suggesting a successful modeling. Compared with the model group, except for the group of blood stasis removing method (7:3)(P>0.05), P-ERK was significantly decreased in the other treatment groups, the inhibitory rate of each intervention group were:6.29%in the group of blood stasis removing method (5:5),8.94%in the nitroglycerin group,9.05%in the fistular onion stalk extraction group,11.17%in the rhubarb extraction group, and it was13.34%in the group of blood stasis removing method (3:7).Compared with the normal control group, the P-JNK protein expression in model group was significantly increased (44.58%), suggesting a successful modeling. Compared with the model group, the P-ERK was significantly decreased in each treatment group, and the inhibitory rate of each intervention group were:14.17%in the group of blood stasis removing method (3:7),24.83%in the group of blood stasis removing method (5:5),25.59%in the rhubarb extraction group,26.37%in the fistular onion stalk extraction group,26.56%in the nitroglycerin group, and31.73%in the group of blood stasis removing method (7:3).Compared with the normal control group, the P-p38protein expression in model group was significantly increased (88.0%), suggesting a successful modeling. Compared with the model group, the P-P38expression was significantly decreased in each treatment group, and the inhibitory rate of each intervention group were:18.91%in the rhubarb extraction group,24.31%in the group of blood stasis removing method (3:7),31.64%in the group of blood stasis removing method (5:5),44.20%in the fistular onion stalk extraction group,45.25%in the group of blood stasis removing method (7:3) and45.29%in the nitroglycerin group.5. The influence on myocardial tissue IL-6of rats with acute myocardial ischema Compared with the normal control group, the IL-6level of rats in model group was significantly increased (P<0.01), compared with the model group, except for the group of blood stasis removing method (3:7)(P>0.05), all the other treatment groups could decrease the level of serum IL-6, and the decrease of serum IL-6was most significant in the group of blood stasis removing method (7:3)(P<0.01), which was better than that in the group of blood stasis removing method(5:5), the rhubarb extraction group, the fistular onion stalk extraction group and the nitroglycerin group (P<0.05).Compared with the normal control group, the IL-6mRNA level of heart muscle tissue of rats in model group was significantly increased (P<0.05), with the amplification of1.00; compared with the blank model group, all the treatment groups can reduce the level of IL-6mRNA, and the inhibition rate of each intervention group was:16.49%in the fistular onion stalk extraction group,27.33%in the nitroglycerin group,30.5%in the rhubarb extraction group,41.32%in the group of blood stasis removing method (5:5),41.96%in the group of blood stasis removing method (3:7), and45.98%in the group of blood stasis removing method (7:3)Conclusion1. The immune mechanisms of the blood stasis removing method intervening acute myocardial ischemia may be related with the activation of MAPKs and IL-6mRNA expression and the influence on MAPKs/IL-6signal transduction pathway.2. The blood stasis removing method could inhibit myocardial enzyme leakage and reduce myocardial injury by improving ECG, and enhancing the cardiac function.3. The blood stasis removing method protects the ischemic myocardium through the regulation of gaseous molecules of H2S.4. Of the different proportions of blood stasis removing, the group of scallion stalk with rhubarb (7:3) got the best efficacy, but due to the differences of the action segments and targets, and the unclear drug ingredients, further studies on the evaluation of its overall advantage are still in need. |
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