Changes Of Cardiac Function In Rats With Spleen - Qi Deficiency Syndrome And Its Mechanism

Purpose: Traditional Chinese medicine basic theory is that the spleen is "the foundation of the day after tomorrow", "the source of qi and blood biochemical" is to provide the required material life activities the day after tomorrow is the most important organs. While "spleen blood system in operation, the Lord" is the core function of the spleen. Modern medicine has clearly, to maintain life activities is the most basic material represented by adenosine triphosphate(ATP) of energy. And the source and formation of ATP is including the digestion and absorption of matter, blood transport, transmembrane transport and platform for mitochondrial oxidative phosphorylation and other biological processes. Therefore, blood "the spleen and the main operation, the function and energy metabolism of modern medicine biological processes has the close inner link.But traditional Chinese medicine theory is that "the spleen and the main operation and the blood" is like two different core function of the spleen, the spleen governs the blood "contains the" life blood, blood, blood taken ". We according to the theory of TCM and modern medicine biological process and mechanism of energy metabolism, blood believed that spleen should be associated with the generation of blood component; Spleen ejection is related to stop bleeding and blood coagulation mechanism. Line for the "blood" suggests the following scientific hypothesis: "the spleen governs the blood" function may be part of "the spleen and the main operation" function, namely material after digestion and absorption, to the whole body tissue cells through blood circulation system conveying part function. The mechanism may be temper led to the change of the BNP, thus affecting the c AMP- PKA pathway, lead to cardiac function has changed.The purpose of this work, the use of electrophysiology and molecular biology techniques, observation spleen deficiency model rat cardiac function changes, and discuss the possible mechanism of molecular biology, to clarify TCM "spleen blood system in operation, the Lord", especially "transport" and function of the relationship between scientific connotation at the same time, for the clinical cardiovascular disease "from the spleen differentiation" to provide experimental basis.Methods:1 In experimental animals and group By experimental animal center of benxi(liaoning immortality biological technology co., LTD.) to buy only SPF 30 male SD rats, weight 200 ±10 g, license SCXK(liao- 2010-0001), after a week adaptive breeding, were randomly divided into normal group and the two groups of spleen-deficiency model group, each group 15 only.2 Spleen deficiency syndrome model replication and evaluation:Temper deficiency syndrome in rats model replication method According to the basic theory of TCM etiology, with reference to the Chinese medicine new medicine clinical research guiding principles [1] and the experimental animal model of TCM methodology [2] in the preparation of pixu(spleen deficient) model, the method of using diet not festival add weary internal injuries composite factor replication temper deficiency syndrome model in rats. 24 hours free diet water, fast water for 48 hours; Swimming 25 minutes a day at the same time, the water temperature at 35 ℃~ 37 ℃, room temperature kept at 23 ± 1 ℃; For 2 weeks after the building model evaluation.Spleen deficiency syndrome model Cheng Mo evaluation method: based on the literature [3] macro characterization of the main symptoms of model rats respectively, god weakness, fatigue, less food stay, body weight, then has carried on the quantitative soft diarrhea, excluding wasn’t up to standard in rats.Normal group normal diet water, no stimulus.3 Methods of core index detection3.1 Left ventricular functionUsing small animal ultrasound imaging system is used to detect the rat cardiac function; The system by the Canadian Visua Sonics company buys, model VEVO2100 ultra-high resolution. In the model group after the success of the building, fasting for 24 hours, the normal group and model group rats was detected in the left heart function, testing parts choose the rat atrioventricular annulus, small animal echocardiography instrument internal Settings of electrocardiogram(ecg) model as instrumentation. After the completion of the connection, adjust the instrument parameters: Dynamic range(Dynamic range), 60 db. Display map Display(map), CS; Scan rate(Sweep speed), 1000 hz; Peer-to-peer(Contrast), 50; Luminance(Brightness) 50. Waiting for rats breathe smoothly, electrocardiogram(ecg), after can clearly show the start recording rats 10 seconds of echocardiography, instrument randomly selected from 10 seconds of footage 3 period of measurement, gives the average, to get each group rats left ventricular ejection fraction(LVEF), left ventricular end systolic volume(ESV), left ventricular stroke output(SV), left ventricular end-diastolic volume(EDV) average.3.2 Materials and molecular biology index detection:Based method: after the experiment, two groups of rats banned food 24 hours a day, 20% ethyl carbamate solution in the anesthesia in rats, the broken neck method is adopted to death, big abdomen of the skin, anatomy, 5 ml syringe to take big abdomen aortic blood; After then used medical scissors to cut open the chest, heart of rats, medical scalpel cut quickly take heart tissue, according to the detection index requires a blood or tissue samples preparation, respectively.The conventional HE staining to observe heart tissue morphology; Transmission electron microscopy(sem) to detect myocardial tissue cell morphological change.Using the Elisa method for determining the content of serum and myocardial tissue BNP, c AMP; Using the immunohistochemical method of myocardial cell BNP receptor protein expression.Using Western Blot method(Western Blot) determination of BNP in myocardialexpression; The Q-polymerase chain reaction(PCR) to detect b FGF, PKA m RNA expression. 4 Statistical methods: this experiment all data statistical analysis by SPSS 13.0 statisticalsoftware, and the mean of ±standard deviation(`x±s) way to express the data as a result, thecorrection of t test(variance) when not neat or independent sample t-test variance(qi) moremeasurement data, with P < 0.05 for the difference is statistically significant.Results: 1 The rat cardiac function test results:Compared with normal group, spleen deficiency syndrome model group rats heart left ventricular end systolic volume(ESV) numerical increased significantly(P < 0.05); And the left ventricular end-diastolic volume(EDV), left ventricular ejection fraction(LVEF), left ventricular stroke volume(SV) is significantly lower than normal group(P < 0.05).2 Myocardial tissue HE staining results:Fibrous structure of normal rat myocardial cells show more clear, myocardial fibers arranged neatly, myocardial cell horizontal lines clear, there is no phenomenon of inflammatory cells infiltration, myocardial cell structure is normal; Spleen deficiency syndrome model group rats myocardial contraction, cell arranged disorder, cell necrosis, such as cell fatty degeneration and turbidity changes.3 Detection of myocardial tissue transmission electron microscopy(sem)Normal group arrange normal myocardial cells, muscle membrane structure is clear, myocardial fiber arrangement is rules, Z line is visible. Mitochondria is rich in the muscle fibers, the structure is in good condition, mitochondria swelling, degeneration and necrosis; Interstitial no fibrous tissue hyperplasia and inflammatory cell infiltration. Model group muscle fibers(edema), occasional leap plate separation, part of the muscle silk dissolved; A muscle silk belt, band structure is not clear, I Z line is still visible; Mitochondria is rich, but size is differ, mitochondrial matrix increased electron density.4 The BNP, c AMP content test resultsCompared with normal group, spleen deficiency syndrome model group rats the BNP, c AMP content in serum and myocardial tissue were significantly increased, with statistical significance(P < 0.05).5 The BNP receptor expression test resultsThe BNP receptor expression in rat myocardial tissue, see more at the cell membrane, cell cytoplasm has also expressed but less, a tan is positive expression. Group with the normal rat myocardial cell dyeing performance is weak positive, and spleen deficiency syndrome model group of positive expression cells was significantly more than the normal group; Compared with normal group, spleen deficiency syndrome model group the IOD value of BNP in the myocardial cells increased significantly, and the difference is statistically significant(P < 0.05).6 PKA and b FGF m RNA expression test resultsGet effective gene amplification, for the purpose gene amplification curves in accord with the s-shaped curve. Melting curve shows no impurity peak, is only one peak, and further illustrate the purpose gene can adopt the quantitative evaluation of the system. Contrast between the two groups of PKA and b FGF m RNA expression of spleen deficiency syndrome model group than the normal group significantly increased(P < 0.05).The experiment results show that the spleen deficiency model rats’ spleen to transport "function damage, the left ventricular end systolic volume increase, end-diastolic volume, stroke output changes, especially the left heart ejection fraction changes, shows that virtual temper" transport "function disorder occurs, cardiac ejection function also significantly affected, which show the" blood "function decline. This result supports the function of the spleen governs the blood " could be part of " the spleen and the main operation " function hypothesis.Summary of the results can be thought of, temper deficiency syndrome, spleen to transport "function of the" blood "function have also been affected, one of its possible mechanism is due to myocardial tissue the BNP and its receptor expression, exciting c AMP-PKA pathway and through b FGFm RNA excessive expression, resulting in myocardial tissue morphology change, eventually resulting in a decline in the left ventricular ejection function.Conclusion:1 Spleen deficiency syndrome model rats obviously decreased left ventricular ejection function. The left ventricular end systolic volume(ESV) rise; Left ventricular end-diastolic volume(EDV), left ventricular ejection fraction(LVEF), left ventricular stroke volume(SV) is significantly reduced.2 Myocardial tissue in rat model of spleen deficiency syndrome atrophy, inflammatory cells infiltration, the disordered arrangement of muscle fibers, muscle wire with nuclear dissolved; Uneven size of mitochondria.3 Myocardial tissue in rat model of spleen deficiency syndrome and serum BNP levels increase, the myocardial cell BNP receptor expression.4 Myocardial tissue in rat model of spleen deficiency syndrome c AMP levels rise, PKA and b FGFm RNA expression.