Effects On Of Modified Taohe Chengqi Decoction And Its Composition And Different Extractions On Cell Proliferation And Apoptosis Of Diabetic Vascular Smooth Muscle Cell

Atherosclerosis can lead to serious complications mainly by affecting aorta, coronary artery and cerebral artery. Atherosclerotic cardiovascular disease (ACVD) is the number one killer in developed countries. It includes coronary heart disease, stroke, and acroteric artery disease. In the January 2003, IAS (international atherosclerosis society) issued Harmonized Clinical Guidelines on Prevention of Atherosclerotic Vascular Disease. This strengthened the prevention and cure of these diseases. Diabetes mellitus is one of the most risk factors for ACVD. As to the possible mechanism of atherosclerosis being caused by diabetes mellitus, it includes damaged function of endodermis, cell proliferation of vascular smooth muscle cell (VSMC) and apoptosis inhibition.This study made a review about the clinical and experimental application of prescriptionsof treatise on exogenous febrile diseases and Taohe Chengqi Decoction in recent 20 years. Reviewsalso included the investigations on cure and prevention of diabetic ACVD by western medicine and Chinese medicine and the relationship between diabetic ACVD and the cell proliferation and apoptosis inhibition of VSMC. We also made a summary about the studies on the affection of western and Chinese medicine for the cell proliferation and apoptosis inhibition of VSMC. We discussed some problems about serum pharmacology investigation in cell culture in vitro. At the same time, we based on previous investigation result of Taohe Chengqi Decoction for diabetic ACVD to observe effect of modified Taohe Chengqi Decoction and its composition and different extractions on cell proliferation and apoptosis of diabetic VSMC by the methods of methy thiazolyl tetrazolium (MTT), flow cytometer machine (FCM) and transmission electron microscope.1 Theoretical and literature studyPrescriptions of treatise on exogenous fibril diseases, another name is classical prescriptions, are famous for its less herbs, best composition and outstanding curative effects. It' s the best prescriptions for compound recipe investigation.Investigations for classical prescriptions in prevention and cure of diabetic complications were relatively limited. But they filled up blackness in this area, proved its effect and brought to light some active mechanism. For some classical prescriptions, the active components had been found for preventing and curing DM and its complications. Thus, to study the classical prescriptions is a possible breakthrough for the compound recipe research of traditional Chinese medicine (TCM). And pay attention to the prescription unpacking, pharmacokinetics and combination of prescription and syndrome is the future direction.As one of the classical prescription, Taohe Chengqi Decoction was famous for preventing and curing DM and its complications in both clinical and experimental researches. Some active mechanism and components had been found. As to the pathogenic factors of diabetic ACVD, it includes factors such as heredity, dysfunction of endothelial cell, metabolic error of lipid, dysfunction of SVMC, formation of immune complex and thrombosis. For DM, especially type 2 diabetes mellitus (T2DM), the long-term lightly inflammation of blood vessel endothelium accelerated the cell proliferation and inhibited apoptosis of VSMC. This led to the aorta complications of DM, such as atherosclerosis. So there was a close relationship between the diabetic ACVD and cell proliferation and apoptosis of VSMC. TCM studies showed that nearly 60-90 percents of patient with both DMand ACVD belonged to the syndrome of blood stasis.Researches showed that some western drugs could anti atherosclerosis by the mechanism of anti inflammation. These drugs included thiazoliddinediones (TZDs), regular insulin (RI), metformin, statins, angiotensin-converting-enyme inhibitor (ACEI), angiotensin receptor blockers (ARBs) and aspirin, etc. Thus these drugs were used to cure and prevent diabetic ACVD in clinical practice. At the same time, stem cell transplantation research was explored in practice. As to TCM researchers, we have improved symptoms and hemorheology by syndrome differentiation, unique prescription for special disease or syndrome. Researches showed that several Chinese medicines can prevent and cure diabetic ACVD and to affect the cell proliferation and apoptosis inhibition of diabetic VSMC might be its mechanism in anti arteriosclerosis.2 Experimental studies2.1 ObjectiveTo observe the effect of modified Taohe Chengqi Decoction and its composition and different extractions on cell proliferation and apoptosis of diabetic VSMC. Thus it helped to optimize the composition, elementally filter active ingredient and set forth the theoretical and clinical basis of Taohe Chengqi Decoction in prevent and cure diabetic ACVD.2.2 MethodsThis study was based on the former research result of Guangdong Natural Science Foundation. We used the 16th generation rabbit aorta VSMC strain of the Chinese preclinical medicine cell centre and cultured them from 16th to 21th generation. We used TZDs and metformin as control group and RI as model group to observe the effect of modified Taohe Chengqi Decoction and its composition and different extractions on cell proliferation and apoptosis of diabetic VSMC. All experiments divided into three stages. The first stage was to culture VSMC. The second stage was to use specific pathogen free (SPF) Sprague Dawley (SD) male rats and Chinese medicine to prepare blank serum and pastille serum. The third stage was to get diabetic VSMC model. The last stage was to compare each group with the methods of MTT, FCM and transmission electron microscope.2.3 ResultsOur study successfully mastered the techniques and situations related to VSMC culture, frozen and passage. We made sure that the dilution of rat blank serum was 5 times and the medium dosage was well enough. We successfully used the method of abdominal aorta blood sampling and separated enough rat serum for trial test and formal test. Results also showed that the RI dosage of 30mu/L and 300mu/L facilitated cell proliferate, but the eventually concentration dosage of 300mu/Lgot higher reproduction rate.Methods of MTT, FCM and transmission electron microscope showed as follows. Method of MTT showed that there was no significant difference between fore and after adding serums for both the blank group and promoting blood circulation and purging heat and stool group. As to the comparison after adding serums, the model group showed significant difference from the blank group(P＜0.01) . It showed that the method to induce the VSMC proliferate with RI was successful. Studies showed significant inhibitory action of diabetic VSMC proliferate in the promoting blood circulation and purging heat and stool group, modified Taohe Chengqi Decoction group and TZDs group. And the promoting blood circulation and purging heat and stool group got the highest inhibition rate that was 39.8%. As to different method of extraction groups, all three groups showed significant difference in inhibitory action (P＜0.01) . But the normal butyl alcohol group got the highest inhibition rate.Method of FCM showed that regular insulin could promote cell proliferation and inhibit apoptosis of diabetic VSMC. But there was no significant difference between the blank group and the model group in the phases of GO/G1, S and G2M. Compared to the model group, the TZDs group, the promoting blood circulation group, the promoting blood circulation and purging heat and stool group, the ethyl acetate group and normal butyl alcohol group showed significant difference in the promotion of apoptosis (P＜0.01). The active ingredients might be found in these 5 groups. As to the mechanism, the TZDs group and the ethyl acetate group might be related to inhibition of GO/G1 phase and G2M phase. The promoting blood circulation group and the promoting blood circulation and purging heat and stool group might be related to the inhibition of G2M phase. As to the normal butyl alcohol group, its mechanism might be related to the inhibition of S phase and G2M phase.With the transmission electron microscope, we found out that the normal VSMC was with even chromatin, rich in cytochondriome, endoplasmic reticulum and lysosome. The cell membrane was integrated and the microvillus was abundant. As to the proliferous VSMC, we could see the concentrated chromatin. As to the VSMC at the stage of apoptosis, we could the strongly concentrated chromatin turned into half moon shape, the cytomembrane was vacuolar and apoptosis corpus could be found. Results showed that there was no apoptosis corpus in the blank group, the model group and the metformin group. There was different degree of apoptosis in the TZDs group, the modified Taohe Chengqi Decoction group, the promoting blood circulation group, the supplementing qi and nourishing yin group, the purging heat and stool group, the promoting blood circulation and purging heat and stool group, the water extracting alcohol precipitating group, the ethyl acetate group andnormal butyl alcohol group. Some of them could be seen apoptosis corpus.As to the changes of mitochondrion, there could be seen normal shape, amount and ridges in the group of blank group, the metformin group and the supplementing qi and nourishing yin group. The ridge was distinct in the model group. There could be seen scarce of mitochondrion and blur ridges in the TZDs group, the modified raohe Chengqi Decoction group, the promoting blood circulation group, the purging heat and stool group, the promoting blood circulation and purging heat and stool group, the water extracting alcohol precipitating group, the ethyl acetate group and normal butyl alcohol group.2.4 ConclusionThe modified Taohe Chengqi Decoction might anti diabetic ACVD through inhibition of proliferate and promotion of apoptosis for diabetic VSMC. Its main ingredients for prevent and cure diabetic ACVD might exist in the promoting blood circulation group and the promoting blood circulation and purging heat and stool group. The TZDs group also had this active ingredient.For different methods of extraction, there might be more ingredients for preventing and curing diabetic ACVD in the ethyl acetate group and normal butyl alcohol group. As to the mechanism, the TZDs group and the ethylacetate group might be related to inhibition of GO/GI phase and G2M phase. The promoting blood circulation group and the promoting blood circulation and purging heat and stool group might be related to the inhibition of G2M phase. As to the normal butyl alcohol group, its mechanism might be related to the inhibition of S phase and G2M phase.3 Innovative points3.1 It was a positive explore and innovation for the development of the treatise on exogenous febrile diseases.This study based on the theory of TCM. It was the combination of either prescription and syndrome and prescription and disease. We integrally studied the classical prescriptions in the treatise on exogenous febrile diseases. It based on the close relationship among the apoptosis and proliferation of VSMC, atheroma and diabetic ACVD. It used unpacking prescription theory and different methods of extraction to investigate compound of classical prescription. It was a positive explore and innovation for the development of the treatise on exogenous febrile diseases and gave new idea for the research and development of classical prescription modernization. 3.2 It elementally found out the substantial foundation and active mechanism for the modified Taohe Chengqi Decoction in affecting the apoptosis and proliferation of diabetic VSMC.After the compared investigation about the different composition and exaction, we elementally found out the substantial foundation and active mechanism for the modified Taohe Chengqi Decoction in affecting the apoptosis and proliferation of diabetic VSMC. It provided scientific basis for the further ingredients qualification and separation. It was a positive explore for TCM reformation of the dosage forms. It was also an attempt for analyzing the ingredients groups of this prescription. 3.3 To explore the active mechanism of TCM compound for preventing and curing diabetic ACVD.This study used cell biology techniques to investigate the possible active mechanism the prevention and cure of diabetic ACVD by modified Taohe Chengqi Decoction and its composition and different extractions. And we conferred that one of the active mechanism was affect the cell proliferation and apoptosis of diabetic VSMC.