| Coronary heart disease (CHD) threatens the human health with high morbidity and mortality rate. In recent years, life rhythm is speeding up and social competition is fierce, it seem that CHD with depression, anxiety and insomnia are common and these mental disorders affect the development and prognosis of CHD, "psycho-cardiology" is gradually paid more attention. At present, western medicine combined the cardiovascular medicine with antidepressants and sedative hypnotics and temporarily solved some mental symptoms, but the side effects, low long-term effects, drug resistance and addiction have not been resolved effectively. Therefore, looking for a safe and effective complementary and alternative medicine on prevention and treatment of abnormal "psycho-cardiology" is a problem should to be mainly solved in the field of CHD.Currently traditional Chinese medicine plays an important role in the prevention and treatment of CHD with the good clinical effects in psycho-cardiological abnormality of patients with CHD, although there are a lot of research showing the effects of traditional Chinese medicine on CHD, while the drugs with single target used in clinical had no expected curative effects due to the lack of multiple factors’ composite models that are closely related with clinic. While there have no animal models and drug intervention studies on psycho-cardiological abnormality with CHD in the present stage. Therefore, established the animal model of psycho-cardiological abnormality with CHD is helpful for the further drug research. On the basis of existing study, sleep deprivation(SD) can cause anxiety, irritability, depression and other mental disorders, while myocardial ischemia and reperfusion (I/R) is the common pathophysiological process of the thrombolysis, percutaneous coronary intervention and coronary artery bypass surgery of CHD. So we established SD+I/R model to simulate psycho-cardiological abnormality with CHD and observed the pathological changes and drug intervention, so as to study the cardiovascular protective effects and mechanism of drug and provide basic support for drug research and development of CHD.TCM think that "Heart Governs the Blood" and "Heart Controls Consciousness", the qi, blood, Yin and Yang of the heart are impaired with the obstruction of phlegm and blood stasis when CHD occurs. The function of "Heart Governs the Blood" and "Heart Controls Consciousness" are abnormal and followed with the psycho-cardiological abnormality. Invigorating blood circulation and tranquillization method is the main method for psycho-cardiological abnormality with CHD. Huoxue Anshen Fang (HAF) based on the method of promoting blood circulation and tranquillization is formed by salvia miltiorrhiza, panax notoginseng, spina date seed and so on. It has good effects on the chest distress and pain as well as dysphoria and insomnia by invigorating the circulation of blood and tranquillization. Previous studies have found that HAF can relieve clinical symptoms effectively and improve the related parameters of myocardial injury, but there have no basic research on the CHD with anxiety, depression and insomnia. Therefore, invigorating the effect of HAF on the psycho-cardiological abnormality with CHD is significant. This experiment aimed at exploring the effect and mechanism of invigorating blood circulation and tranquillization method that regulating psycho-cardiological abnormality on SD+I/R injury, so as to provide the experimental basis for using the invigorating blood circulation and tranquillization method on psycho-cardiological abnormality with CHD, as well as to provide reference basis for the new drug development and research on prevention and treatment of psycho-cardiological abnormality with CHD.1. PurposeTo observe the pathological changes after different SD time and determine the time that makes the most obvious pathological changes. After that, the SD combined with myocardial I/R model was established to simulate clinical psycho-cardiological abnormality with CHD and observe the neuroendocrine changes, endothelial function, inflammation and myocardial injury degree to determine whether composite model is successful. On the basis of the successful model, we would explore the effect of HAF on SD+I/R injury by detecting cardiac function, myocardial enzyme, myocardial infarction area, myocardial tissue morphology and myocardial apoptosis to observe its’cardiovascular protective effect and the mechanism, so as to investigate the basic pathological changes of psycho-cardiological abnormality with CHD and the multiple-components, multiple-target and multiple-pathway effects of HAF, which provide the experimental basis for the invigorating blood circulation and tranquillization method using in the treatment of psycho-cardiological abnormality with CHD.2. MethodRats were randomly divided into control group and SD group, and the "improved multi-platform water environmental" method was used for SD intervene. Weight and corrected QT interval before SD and 2,5,7 days after SD were observed respectively, and the serum melatonin (MT), endothelin (ET-1), interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-a) were detected and then we determined the 7 days that pathological changes are most obvious for further SD intervene research.Then rats were randomly divided into groups:sham operation group, general model group, SD model group, HAF group.7 days for SD intervene and preventive drug administration. At the 7th day of SD, serum, plasma and heart of part of rats were taken, and other rats were anesthetized and left anterior descending coronary artery were ligated for 50 min and reperfused for 2 h,24 h respectively to establish myocardial I/R model. General condition and weight of rats before and after SD, plasma eatecholamine (CA), neuropeptide Y (NPY), serum angiotensin II (AngⅡ), c-reactive protein (CRP), nitric oxide (NO), ET-1, MT, IL-6, TNF-a and myocardial tissue TNF-a after SD were observed; At 2 h after I/R, same detection indexes, creatine kinase isoenzyme (CK-MB) and lactate dehydrogenase (LDH) were tested, myocardial histopathologic change was observed with HE dyeing, myocardial apoptosis was detected by TUNEL method, positive expressions of myocardial NF-kB, Bcl2, Bax and Caspase-3 were analyzed by immune histochemical method, protein expressions of myocardial NF-κBp65, Bcl2, Bax and Caspase-3 were detected by western blot; At 24 h after I/R, the percentage of the infarct area was observed calculated by TTC-Evans blue staining method; Before I/R,2 h and 24 h after I/R, the heart ejection fraction (EF) and heart rate (HR) were detected by ultrasounds.3. Results3.1 Sleep deprivation time:previous studies have found that QTc interval was lengthen, MT level was reduced, ET-1, IL-6 and TNF-a levels were increased gradually with the more days of SD. After 7 days of SD, all detection indexes’differences have statistical significance when compared with the control group (P<0.05). So we chose the 7 days of SD for further research.3.2 Effect of HAF on neuroendocrine:Rats were manic and irritability with significantly reduced weights and serum MT and increased plasma CA, NPY, serum AngⅡ, ET-1, NO, IL-6, TNF-α and myocardial TNF-α after 7 days of SD, all detection indexes’differences have statistical significance when compared with the general model group (P<0.05). While HAF could reduce NPY, AngⅡ, ET-1, IL6 and TNF-a levels and inhibit the reduced MT level after SD, all detection indexes’differences have statistical significance when compared with SD model group (P<0.05); After I/R, the concentration of AngⅡ, ET-1, IL-6, TNF-a and CRP in SD model group were higher than general model group, while HAF could reduce all detection indexes levels after SD+I/R, the differences were statistically significant (P<0.05).3.3 SD combined with myocardial I/R model:Compare with the sham operation group and general model group (I/R), SD model group (SD+I/R) had the significantly higher level of serum AngⅡ, CRP, ET-1, IL-6, TNF-a, CK-MB, LDH and myocardial TNF-a, significantly lower level of EF, obviously larger infarction area, obviously severe myocardial tissue pathological damage, significantly higher apoptosis index, significantly higher expressions of NF-kB, Caspase-3 and lower ratio of Bcb/Bax, all detection indexes’differences have statistical significance when compared with the general model group (P<0.05), which have suggested that myocardial injury of SD+I/R is more than severe than simple I/R. It has been proved that SD combined with myocardial I/R model is established successfully.3.4 Effect of HAF on cardiac function and myocardial enzyme:Compare with the SD model group, HAF could obviously improve the EF of 2 h and 24 h after I/R, decreased serum LDH and CK-MB of 2 h after I/R, the differences were statistically significant (P<0.05).3.5 Effect of HAF on myocardial infarction area:Compare with the SD model group, HAF could obviously reduce the percentage of infarct size in ischemia area 24 h after I/R, the differences were statistically significant (P<0.05).3.6 Effect of HAF on myocardial tissue pathology:HAF group rats’myocardial tissue injury degree is lighter than the SD model group rats, and arrangement of the myocardial cell are rule with less inflammatory cells infiltration.3.7 Effect of HAF on myocardial apoptosis and related pathways:Compare with the SD model group, HAF could reduce the apoptosis index and expressions of NF-kB and Caspase-3, at the same time increased Bcb/Bax ratio, the differences were statistically significant (P<0.05)4. Conclusion(1) Different SD days had different effects on the cardiovascular pathogenic factors.7 days of SD had the most obvious pathological changes and significantly increased the risk of cardiovascular disease. (2) SD could reduce the weight and lead to poor mental state and neuroendocrine disorder, further caused the endothelial dysfunction and inflammation, increase the risk of cardiovascular pathogenic factors; HAF could improve SD-induced neuroendocrine disorder, further alleviate vascular endothelial dysfunction and inflammation, reduce cardiovascular pathogenic factors. (3) After SD+I/R, EF was reduced, myocardial enzyme was increased, myocardial tissue pathological injury was severe, myocardial infarction area and myocardial apoptosis were increased, mycardial injury is more severe than single I/R injury, which indicated that SD can aggravate the I/R injury. It is proved that SD combined with myocardial I/R model is established successfully, which clarify the pathological changes of psycho-cardiological abnormality with CHD and provide basic support for the treatment with traditional Chinese medicine. (4) HAF not only improved neuroendocrine disorder caused by SD, reduced cardiovascular pathological factors and risk, but also improved SD+I/R injury by improving cardiac function, decreasing infarction area and myocardial apoptosis, alleviating pathological tissue injury, which may be related to inhibiting the release of NPY and reducing inflammation factors TNF-a after SD, further adjusting the inflammation and apoptosis NF-kB pathway, reducing the activation of NF-κB and expression of Caspase-3 and increasing the Bcb/Bax ratio, protecting the myocardial injury with multiple targets and multiple pathways. |
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