Study On Mitochondrial Unfolded Protein Response Of Ventricular Myocytes In Rats With Spleen Deficiency

Objective:The effect of spleen qi deficiency on the pumping function of the heart has been studied,but whether the mechanism is related to mitochondrial injury,especially the reaction of mitochondrial unfolded protein is not yet clear.This research targeted left ventricular ejection,tail blood pressures,left ventricular myocyte mitochondria in the morphology,function and its unfolded protein response,atempting to explore the possible mechanism of spleen qi deficiency disease interferring the cardiac pumping blood.Material and method:1.Grouping After 1 week of adaptive feeding,sixteen SPF SD male rats were randomly divided into normal group and model group of spleen qi deficiency syndrome（model group）according to random number table method,with 8 rats in each group.2.Establishment and evaluation of spleen qi deficiency model2.1 Establishment Rats in model group were treated with a small amount of diet and fatigue stimulus:fasting for 48 hours after 24 hours of satiation,5 cycles for a total of 15 days,and drinking water freely throughout the whole process.At the same time,they swam till they were exhausted in 35～37℃water every day.2.2 Evaluation（1）Emaciation:Weight on an empty stomach of rats was measured,and it was up to standard when the model group was significantly lower than the normal group;（2）Anorexia inappetence:the daily food intake amount of rats was recorded in the metabolic cage for 24 hours,it was up to standard when the model group was significantly lower than the normal group;（3）Mental fatigue and tired:the grasping force of both forelimbs,the moving distance and standing times in the 5min open field experiment in the model group were significantly less than those in the normal group;（4）Withered and less fur:The three people evaluated independently and the results were consistent.The substandard rats were screened out.3.Blood pressure of caudal artery in rats Non-invasive arterial blood pressure device was used to measure tail artery blood pressures.The systolic pressure（SP）and diastolic pressure（SP）of the caudal artery were measured in 5 times by the same person at every other 3min for each rat during the same period of time,and the average values of 5 times were recorded.4.Cardiac ultrasound examination After Abdominal cavity anesthesia with 10%chloral hydrate,the left ventricular papillary muscle plane of the rats was detected,and the heart rate was recorded simultaneously.After Left ventricular end diastolic diameter,left ventricular end systolic diameter,left ventricular end diastolic volume,left ventricular end systolic volume were measured by image acquisition,left ventricle ejection fraction（LVEF）and left ventricle fraction shortening（LVFS）were calculated according to the formula.5.Specimen prepration After fasting for 24 hours,the rats were intraperitoneally anesthetized with 10%chloral hydrate.The abdominal hair was removed,the chest was cut open to get the heart,and the heart tissue was taked and frozened at-80℃for later.6.Ultrastructure of mitochondria in left ventricular myocytes The heart tissue was cut in1cm³at 4℃,fixed with 2.5%glutaraldehyde for 24h,rinsed with PBS buffer solution,fixed with 1%osmium acid,dehydrated with ethanol and acetone with the gradient,encased with epoxy resin,maded into slices,double staining with 3%uranium acetate and lead citrate,observed by transmission electron microscope.7.Evaluation of mitochondrial function of left ventricular myocytes Some ventricular muscle tissues were taken.Mitochondria were extracted according to the Mitochondria Extraction Kit instructions.The levels of adenosine triphosphatase（ATP）and malondialdehyde（MDA）were detected and calculated.8.Expression of protein related to the UPR^mtin left ventricular myocytes Western blotting was used to detect the expression of Lon protease（Lon P）and caseinolytic protease XP （ClpXP）complex,including Clp X and Clp P,in mitochondria of cardiomyocytes.9.Statistical analysis The data obtained in this experiment were expressed as mean±standard deviation（^—X±s）.The t-test of SPSS17.0 statistical software was used for analysis and comparison between groups.A P value of<0.05 was considered significantly.Results:1.Model results of spleen qi deficiency rats There was no death or loss of rats during the experiment.Compared with the normal group,the body weight,24h water intake,grip strength of both forelimbs,open field movement distance and standing times of rats in the model group decreased significantly.In addition,the three independently judged that the hair color of the model group was haggard,indicating that the model of spleen qi deficiency was copied successfully.2.Blood pressure and cardiac function of caudal artery in rats Compared with the normal group,the heart rate,systolic blood pressure,diastolic blood pressure,left ventricular ejection fraction and left ventricle fraction shortening in the model group were all decreased,with statistical significance（P<0.01）.3.Results of ultrastructural changes of mitochondria in left ventricular myocytes Most of the mitochondria in the two groups were oval,with clear edges,without swelling,degeneration and vacuolation.The cristae were lamellar with rich and clear layers.However,compared with the normal group,there were obvious dense particles in the mitochondria of the model group,and they were interwoven with each other.4.Evaluation of mitochondrial function of left ventricular myocytes Compared with the normal group,the ATP content in the model group decreased,while the MDA level increased,with statistical significance（P<0.01）.5.Expression of components related to the UPR^mtin left ventricular myocytes Compared with the normal group,the expression of Lon P and Clp X in the model group was up-regulated,with statistical significance（P<0.05 or P<0.01）,but the expression of Clp P was not significantly changed,with no statistical difference.Conclusion:1.The modern medical mechanism of how spleen qi deficiency disease disrupts the heart and affects cardiac ejection function might be related to mitochondrial protein de-homeostasis caused by wearker UPR^mtin myocardiac working cells.