| Theoretical research Treatise on cold-induced diseases as established a theory system of six channels differentiation which has integrated the principle, method, formula and herbs into a new classified model, and always guided syndrome differentiation of clinical medicine in traditional Chinese medicine(TCM) effectively.“The coordination of five visceras” is the most real and important way of the therapeutic method. The internal essence of Harmonizing therapy is to dredge the qi and blood of viscera to make people recover. Zhang Zhongjing has enlarged the meaning of the Canon of Internal Medicine and has become the basic of theory and practice of“Harmonizing therapy”.It has been developed and it has been used the important principle of herbal prescription now. Nowadays, the CHD of PSMOS has become the familiar clinical symptom type,and rate of suffering from the disease is increased year after year in China.The secure and efficient,quality controllable new drug with explicit mechanism is still deficient,though TCM has been recognized by medical field.The research shows that the system of differentiation of syndromes of the six meridians in Treatiseon cold-induced diseases can play a guiding role in the occurrence,development, treatment of the CHD of PSMOS.The cause of CHD of PSMOS is stagnation of phlegm in heart vessel, the imbalance between vitality and heart blood and that between heart qi and heart blood. The imbalance between qi and blood and blockage of phlegm and blood stasis are the keys of treatment of the CHD of PSMOS. “The Methods of Harmonizing qi and blood” in Treatise on cold-induced diseases guides the prescription of the CHD of PSMOS as the main therapeutic principle to balance the qi and blood for the function of heart. Huoxue Huatan Fang is a empirical formula based on Qi-Blood Theory in Treatise on cold-induced diseases of “The Methods of Harmonizing qi and blood”.This formula can promote the clinlcal symptom of the CHD of PSMOS,and can also reduce blood lipid from pre-clinical research and experimental research. In addition, research shows that origin,shape,function,clinlcal symptom of qi and mitochondria have similar aspects,and mitochondrial DNA mutation is the pathogenic basis of cardiovasculardiseases.Feature of qi is its mitochondria.Therefore,this topic surrounded three targets “reducing blood fat”,“protecting critical organ”and“mitochondrial biogenesis”,and is aimed to exploring the influence of TCM on the prevention and treatment effects and mechanism of the CHD of PSMOS. The supporting theory and research results for the CHD of PSMOS would be offered and the reference data for exploring that the matter feature of qi is mitochondria would also be provided.Experimental researchThis topic has explored the influence of Huoxue Huatan Fang based on “The Methods of Harmonizing qi and blood ”of Treatise on cold-induced diseases on the prevention and treatment effects and mechanism of the CHD of PSMOS and studied the essence of Phlegm Stasis-Bizu of heart meridian.The supporting research results for exploring that the matter feature of qi is mitochondria would also be provided. ObjectiveMethod 1. Study on the influence of Huoxue Huatan Fang to the lipid metabolism of hyperlipidemia rats. Intragastric administration should be done for 8 weeks after feeding Wistar rats with high fat diet for 4 weeks,examing blood biochemical index,waiting for hyperlipidemia rats model building. Take the blood of inner canthus and make it centrifugation. Biochemical examination of blood was used to examine the content of Huoxue Huatan Fang in CHO, TG, HDL-C, LDL-C and FFA in the serum of hyperlipidemia rats after being treated at 4 weeks,6 weeks and 8 weeks. 2.Efficacy study on protective Effect of Huoxue Huatan Fang on hyperlipidemia rats subjected to the injury of myocardial ischemia and reperfusion.Hyperlipidemia rats subjected to the injury of myocardial ischemia and reperfusion model is used for ischemia for 40 minutes.After reperfusion for 2and 24 hours of the experiment,biochemical examination of blood was used to examine the content of CK-MB,LDH;Evans-TTC blue staining was applied to examine the myocardial infarct size; the change of cardiac muscle histomorphology after I-R was observed by Myocardium HE staining, oil red O staining;electron microscopy was used to observe theultrauctural Morphology of myocardial tissue after being treated for 8 weeks and reperfusion for 120 minutes. 3. Mechanism Study of Protective Effect of Huoxue Huatan Fang on hyperlipidemia rats subjected to the injury of myocardial ischemia and reperfusion.After administration for 8 weeks, hyperlipidemia rats subjected to myocardial ischemia and reperfusion subject ischemia for 40 minutes and reperfusion for 120 minutes.Biochemical examination and spectrophotometry were applied to examine the change of ROS、SDH、COX in myocardial tissue,and GSH-PX, CUZN-SOD,GSH-PX enzyme in serum after reperfusion for 2hutes,and analyze the effect of Huoxue Huatan Fang on myocardial mitochondrial function and oxidative stress. Analysis of FQ-PCR and Western blotting were used to study the transcription of m RNA and the protein expression level of the pathway of PGC-1α-NRF1-mt TFA about mitochondrial biogenesis and the pathway of PGC-1 α-PPAR α about myocardial fatty acids oxidation and the copy number of mitochondrial DNA(Mt DNA).Result 1.Effect of Huoxue Huatan Fang on lipid metabolism. Biochemical indicator of CHO、TG、LDL-C in the serum of High fat model group have increased significantly after being fed for 4 weeks,6 weeks, 8 weeks, 12 weeks compared with normal control group.Comparing with high fat model group,CHO 、 TG 、 LDL-C in the blood serum of hyperlipidemia rats has been reduced with dose effect and time effectiveness relationship by a high dose and a middle dose of Huoxue Huatan Fang in different extent.Blood fat has been reduced obviously with a high dose of Huoxue Huatan Fang after being taken medicine for 4 weeks, 6 weeks,8 weeks(P<0.05 or P<0.01).According to the calculation of AI number and non-HDL-C number,both thenumbers of all the dose groups of Huoxue Huatan Fang are lower than high fat model group,and high dose group and middle dose group are better than Group of positive control drug. 2.Drug efficacy study on protective effect of Huoxue Huatan Fang on hyperlipidemia rats subjected to the injury of myocardial ischemia and reperfusion.CK-MB, LDH of high fat model group do not have obvious differences after being feeding 8 weeks and myocardial ischemia and reperfusion for 120 minutes compared with normal control group.CK-MB, LDH of High fat model group also show a rising trend after be fed for 12 weeks and after reperfusion for 24 hours the myocardial infarct size is higher than normal control group on the 8 weeks, 12 weeks. After being treated for 4 weeks,LDH can be reduced after myocardial ischemia and reperfusion for 120 minutes(P<0.05)and the myocardial infarct size can be reduced obviously after reperfusion for 24 hours by the dose of Huoxue Huatan Fang.The dose reduces CK-MB after the operation for 120 minutes and can reduce the myocardial infarct size significantly(P<0.01)after being treated for 8 weeks.Comparing with normal control group,the result of light and electron microscopy shows that necrosis region can be found in myocardial cells of high fat model group,myocardial cells are tumid,inflammatory cytokines are invasive,lipid droplets are obvious,myocardial mitochondria are tumid,and mitochondrial cristae is in chaos after myocardial ischemia and reperfusion for 120 minutes.Huoxue Huatan Fang can protect myocardial cells and reduce swelling, inflammatory cell infiltration, reduce lipid droplets, protect mitochondria shapes.3.Mechanism Study of Protective Effect of Huoxue Huatan Fang on hyperlipidemia rats subjected to the injury of myocardial ischemia and reperfusion. MDA are increased significantly.T-SOD、Cu Zn–SOD、GSP-PX、SDH are decreased in different degrees.COX and ROS have no significant change after ischemia myocardial for 120 minutes in high fat model group comparing with normal control group.As for Huoxue Huatan Fang,T–SOD,GSP-PX,Cu Zn–SOD,SDH should be increased,MDA should be reduced,but SDH,COX should be increased slightly.Huoxue Huatan Fang can promote the expression of PGC-1á、PPAR、NRF、ITFA which are related to mitochondrial biogenesis after myocardial ischemia and reperfusion for 120 minutes for the rats in HLP model group.The transcription of m RNA and the protein expression level of the pathway of PGC-1 α-NRF1-mt TFA about mitochondrial biogenesis and the pathway of PGC-1α-PPARα about myocardial fatty acids oxidation of the rats of hyperlipidemia model group subjected to myocardial ischemia and reperfusion for 120 minutes have been increased remarkably, the copy number of mt DNA in Huoxue Huatan Fang group are increased obviously.Conclusion 1.Treaties of febrile diseases is a classic with six channels differentiation,and “He Method” can guide etiology and pathogenesis,progression of disease, clinical diagnosis and treatment and prescription-making of phlegm and blood stasis type of coronary heart disease. Huoxue Huatan Fang can have an important effect on the theory of the balance between the qi and blood for the function of heart based on this method. 2.The results of this study performed a synthetic judgment from lipid metabolism,myocardial enzymes,cardiac function,myocardial morphology and mitochondrial biogenesis.Andthe results shows that myocardial ischemia reperfusion injury of rats can be aggravated by hyperlipemia.It proved that the essence of existence of stagnation of phlegm in heart vessel. 3. Based on the “The Methods of Harmonizing qi and blood” of “Treatise on cold-induced diseases”, Huoxue Huatan Fang may have an influence on the pathway of fatty acid beta oxidation PGC-1α—PPARα and energy metabolism by reducing blood-fat.It promoted mitochondrial biogenesis of the pathway of PGC-1α- NRF1- mt TFA and the biogenesis of mt DNA by antioxidant stress.PGC-1αis used as the point of intersection to protect the the injury of myocardial ischemia and reperfusion in common. 4. Huoxue Huatan Fang has a certain influence on the protection of mitochondrial morphology,function of hyperlipidemia rats subjected to myocardial ischemia and reperfusion and promotes the expression of genes related to mitochondrial biogenesis by the effect of the balance between the qi and blood and promoting blood circulation and resolving phlegm based on the “The Methods of Harmonizing qi and blood” of “Treatise on cold-induced diseases”. And it offers the reference data to confirming that the matter feature of qi is mitochondria objectively. 5.The result shows that TCM theory has guide meaning to the treatment and prescription of the CHD of PSMOS. And it also shows that disease differentiation in western medicine should combine with syndrome differentiation in TCM on the clinical diagnosis treatment and the cell and molecular level to improve the development of TCM. |
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