Xiaoban Tongmai Mixture Of Traditional Chinese Medicine Serum On Rat Vascular Smooth Muscle Cell Proliferation And Apoptosis

Background: Restenosis after percutaneous transluminal Coronary Angiography (PTCA)is the main problem to influence the prognosis of intervention treatment to Coronary Heart Disease,and restricts Long-term effect of Percutaneous coro nary intervention seriously(PCI),it proves to be the hot point and difficulty in the field of cardiovascular. Abnormal proliferation of vascular smooth musc -le cells (VSMCs) is a critical component of atherosclerosis and arterial rest -enosis after angioplasty. Corresponding to the further study to the mechanism of RS molecule,finds that many factors has been related to a response to injury in which the growth factors such as basic fibroblast growth factor (FGF-2) and plateletderived growth factor (PDGF) are released, stimulating proliferation and migration of VSMCs, leading to the formation of a neointima. Binding of PDGF to its receptor leads to the activation of several cell-signaling pathways associated with both VSMC proliferation and VSMC migration which plays a important role in the proliferation and migration of VSMC, such as those related to mitogen-activated protein kinase (MAPK), extracellular signal regulated kinase(ERK)1/2, Phospha-Tidy linositol 3-kinase (PI3-kinase).The pathological base of restenosis is the formation of neointima, while the abnormal proliferation and insufficient apoptosis of vascular smooth muscle cells (VSMCs)are the main courses. Apoptosis of vascular smooth muscle cells (VSMCs) is a well established component of the remodeling that occurs during normal development of the circulatory system as well as during the course of neointimal formation after intervention for atherosclerosis. Because increased total cellularity is a prominent feature of an occluding neointima, the balance between proliferation and apoptosis during vessel healing appears central to this pathologic process. Indeed, accumulating evidence suggests that abnormal VSMC apoptosis leads toneointimal hyperplasia.However, despite the importance of VSMC apoptosis, the precise molecular mech anism underlying the regulation of apoptotic pathways in VSMC remains largely undetermined. Apoptosis is a multistage, genetically controlled process of selective cell deletion. Protein kinases regulate the early stages of apopt osis by phosphorylating key proteins, whereas akt, a family of signaling passway, are the main effectors whose activation results in the characteristic morpho- logical changes associated with programmed cell death such as membrane blebbing, chroma tin condensation, and DNA fragmentation.So vascular smooth muscle cell plays important role in restenosis, it is also the target cell in the research of restenosis prevention. The proliferation of vascular smooth muscle cells is a crucial pathophysiological process in the development of restenosis after coronary angioplasty. Corresponding to the further study to the mechanism of restenosis in the stage of molecular level, people find that there are many factors participate in the process of prolifer ation and apoptosis of vascular smooth muscle cell such as cell factor, growth factor et al, which affect cyclin and relating apoptosis gene of cell by active ting ERK, PI3K/Akt the passageways of signal transmission. So aiming at inter -vening the regulating proliferation and apoptosis of vascular smooth muscle cell is the point of penetration.The pathogenesis of restenosis asthenia in origin and asthenia in superf icia lity in the eyes of Traditional Chinese Medicine (TCM), the deficiency of viscera qi is root, blood stasis and Phlegm are the sign. The main therapies to prevent restenosis are invigorate the circulation of blood and remove stagnate, dispel Phlegm, regulating liver and spleen and so on. We consider that the main therapy to cure restenosis after PTCA operation is to nourish qi and assist yang, invigo -rate the circulation of blood and remove stagnate as complement in the angle of TCM. To play the role of regulating entirety and ameliorate local as the characteristic of Traditional Chinese Medicine fully. The aim of this study is to observe the influence of the blood serum of compound Xiao ban tong mai mixture to the proliferation and apoptosis of rat aortic vascular smooth muscle cell by cultivating cell in vitro, investigating the possible mechanism of Xiao ban tong mai mixture with the effect of nourishing qi and assisting yang, Clearing stasis to unimpeded vein, aiming at supplying feasible theory evidence to preven -tion of clinical restenosis by using Xiao ban tong mai mixture.PartⅠTo investigate the treatment to RS after PTCA operation to pattern differentiationObjective: To elaborate on the feasibility of Xiaobantongmai mixture which had nourishing qi and assisting yang, invigorating the circulation of blood and removing stagnate to prevent artery from restenosis after PTCA operation in the eyes of Traditional Chinese Medicine.Methods: According to the features of age, symptom, sign that restenosis patients had, taking the theory of yin-yang which the core of TCM as starting point, combining related classical literature that record in Hang Di Nei Jing and Jin Kui Yao Lue,applying the basic theory of yin- yang, qi and blood in TCM and ideology of treatment according to pattern differentiation, and associating research findings of modern pharmacology seek theoretical evidence of Xiao ban tong mai mixture which had nourishing qi and assisting yang, invigorating the circulation of blood and removing stagnate to prevent artery from restenosis after PTCA operation.Results: Xiao ban tong mai mixture could be applied to prevent restenosis after PTCA operation.PartⅡTo observe the influence of Xiaobantongmai mixture to the proliferation of the rat aortic vascular smooth muscle cellObjective: According different proportion serum of Xiaobantongmai mixture to affect proliferation of the rat aortic vascular smooth muscle cell cultivated in vitro, understanded whether had the effect of Xiaobantongmai mixture to affect proliferation of the rat aortic vascular smooth muscle cell cultivated in vitro or not, and made sure the best giving concentration.Methods: The preparation of herbal serum. Choosing thirty male SD rats, weight between 180 and 220 gram, age between 6 and 8 weeks, rats were randomly assigned into two groups with 15 in each group (the normal serum group, the herbal serum group),the herbal serum group:Taking in Xiao ban tongmai mixture apozemwhose dose of intragastric administration was forty gram every kilogram everyday, Xiaobantongmai mixture was boiled in routine, the frequency of Xiaobantongmai mixture was twice a day, 12 hours interval, while giving the same dose normal saline to the normal serum group, continuing 7 days. At last time, rats were gave the herb two times as original dose,the interval of the two times was one hour ,after 1 hour at the last time, then took blood in a sterile operation manner 1 hour before gave herb. Rats were anesthesed by chloral hydrate.The sample of blood was laid up in a four centigrade degree refrigerator till the next day after placed in normal temperature 4 hours. 2000 rpm in the centrifuge for 15 minutes, then filtrated though 0.22um filter membrane, and packed the blood serum respectively in EP pipe, conserved in negative 80 centigrade degree in refrigerator, inactivated it in 56 centigrade degree water for 30 minutes before herb serum being used.Cells culture. The A10 rat aortic vascular smooth muscle cell line was obtained from the American Type Culture Collection (ATCC). The A10 rat VSMC derived from thoracic aorta of fetal rats were prepared as previously described. The cells were maintained in a humidified incubator with 5% CO2 and grown on 100-mm culture dishes in Dulbecco's modified Eagle's medium(DMEM) with high glucose, that was supplemented with 10% fetal bovine serum 100 units/ml penicillin, and 100 ug/ml streptomycin. The cells were split 1:10 when approaching confluence. Aliquots in DMEM/fetal bovine serum/DMSO (4:5:1) were prepared for storage in liquid nitrogen. The medium was replaced every 2 days until the time of all experiments.the medium was changed to serum-free DMEM 24h before experiments. The influence of serum of Xiaobantongmai mixture to proliferation of rat aortic vascular smooth muscle cell were detected using MTT color metric assay. There are four groups in all according to different proportion serum of Xiao ban tong mai mixture,the concentration of herbal blood serum was five percentage, ten percentage,twenty percentage, and the normal serum.There were ten repeated holes in each group, the five percentage serum and ten percentage serum were consisited of twenty percentage serum and the normal serum,every hole kept concentration as twenty percentage serum .cells were cultivated in 37 centigrade, a humidified incubator with 5%CO2 and grown on 100-mm culture dishes in Dulbecco's modified Eagle's medium (DMEM) with high glucose, that was supplemented with 10% fetal bovine serum 100 units/ml penicillin, and 100 ug/ml streptomycin for 24 hours,48 hours,72 hours respectively, then exchanged Dulbecco's modified Eagle's medium with no fetal bovine serum at different time, then added MTT (5mg/ml )20ul,continued cultured for 4 hours,throwed the nutrient solution away ,detected the each time point value of OD by Microplate reader device, Data are presented as mean±S.E.M. of the number of independent experiments shown in each Figure,then obtain the optimal experimental concentration.Results: The result of MTT color metric assay revealed that there was no significant difference between the five percentage herbal serum at different time with 24 hours, 48 hours, 72 hours and the normal serum. There was no significant difference between normal serum and 5 percentage herbal serum, while on the percentage of 10 and 20 herbal serum, there was significant difference about the value of OD comparing to normal serum, illuminated that 10 and 20 herbal serum had effect on the proliferation of rat vascular smooth muscle cell, and the most evident effect was between 24 hours and 48 hours, in addition, the inhibition ratio of 20 percentage was obviously higher than 10 percentage. The herbal blood serum inhibited the proliferation of rat aortic vascular smooth muscle cell in a concentration-dependent and time-dependent manner.PartⅢObjective: According to observe the influence of Xiaobantongmai mixture to the cell cycle of the rats'aortic vascular smooth muscle cell and regulated the protein that related, understanded the active mechanism of blood serum of Xiaobantongmai mixture to inhibite proliferation of the rat aortic vascular smooth muscle cell.Methods: The rat vascular smooth muscle cells were plated at culture dish. According to the result of MTT, choosed the optimal concentration of herbal serum, cells was divided into normal serum and herbal serum. The media were then removed and fresh growth media with drugs were added, acted on the cell for 12 hours and 24 hours respectively. The medium was carefully removed and combined with a 1ml phosphate-buffered saline (PBS) rinse to constitute the"detachedsample". The adherent cells were trypsinized and suspended in growth medium. Samples of the adherent and detached suspensions were counted and assessed for viability by exclusion of trypan blue. The remaining cell suspensions were washed 1×with PBS and then suspended in PBS before being fixed and processed for flow cytometric analyses of DNA content as described previously. Percentages of proliferation cells and cells in the G1, S, and G2/M stages of the cell cycle were determined with a DNA histogram-fitting program.Attempts were made to collect a minimum of 104events/sample for subse quent analyses.Choosed the optimal concentration of herbal serum, cell was divided to normal serum and herbal serum, collected cell after serum acted on the cell for 6 hours,12 hours,18 hours,24 hours respectively .Then VSMC were starved as described above for 24 hours and then treated with the various agents indicated in the text. They were then washed twice with phosphate-buffered saline (PBS) and lysed in ice-cold lysis buffer. Cells were detected the expression of cyclin Dl,cyclin E,p27 protein of cells were detected by Western blot. Then carried out statistical analysis on the result. Data are presented as mean±S.E.M. of the number of independent experiments shown in each Figure. Differences from control were assessed by analysis of variance with ANOVA tests when multiple comparisons were required.Results: The analytical result of cell cycle detected by Flow cytometry show that serum of Xiaobantongmai mixture inhibited the transformation from G0/G1 phase to S phase in a definite range. comparing to the control group , herbal serum delayed the process of cell cycle obviously,the proportion that on G0/G1 phase was more than normal serum, while on the S phase the proportion went down sharply, cell blocked on the G1 phase. The analytical result of Western blot revealed that 20 percentage of Xiaobantongmai mixture serum was in a time- dependent manner, postponed the expression of cyclin Dl,cyclin E obviously, there was significant difference between the control group and the herb group, p27 was not affected by herbal serum, but comparing to normal serum, there was no distinction on the 6 hours , on the 12 hours, 18 hours, 24 hours, there was significant difference.PartⅣObjective: According to observe the influence of Xiaobantongmai mixture to the expression of phosphorylatal ERK1/2, discussed the signal pathway of Xiao bantongmai mixture serum to inhabited the proliferation of rat vascular smooth muscle cell.Methods: Choosing the optimal experimental concentration, divided into the normal serum and the herbal serum, acted on the rat aortic vascular smooth muscle cell for 24 hours repectively. VSMC were starved as described above for 24 hours and then treated with the various agents indicated in the text.then went on cultured for 4 hours, while collected cell. They were then washed twice with phosphate-buffered saline(PBS) and lysed in ice-cold lysis buffer.To detected the expression of phosphorylatal ERK1/2 protein of rat vascular smooth muscle cell in the way of Western blot.Results: The result of Western blot revealed that Xiaobantongmai mixture serum could down-regulate the expression of phosphorylatal ERK1/2 of rat vascular smooth muscle cell, there was significant difference comparing to normal serum.PartⅤObjective: According to observe the influence of Xiaobantongmai mixture serum to the apoptosis of rat vascular smooth muscle cell, the expression of Akt protein which related the passageways of apoptosis, discussed signal transductive channel of apoptosis that induced by Xiaobantongmai mixture serum.Methods: Choosing the optimal experimental concentration, divided into the normal serum and the herbal serum, acted on the rat vascular smooth muscle cell for 48 hours repectively, then observed the ultrastructure through transmission electron microscope; Choosing the optimal experimental concentration, divided into the control group and the herbal group, acted on the rat aortic vascular smooth muscle cell for 24 hours and 48 hours. Apoptosis was determined by flow cytometry using the"Annexin V-FITC Apoptosis Detection Kit I", according to the manufacturers instructions. Choosing the optimal experimental concentration, divided into the control group and the herb group, acted on the rat aortic vascular smooth muscle cell for 24 hours repectively, VSMC were starved as described above for 24 hours and then treated with the various agents indicated in the text. Then went on cultured for 4 hours, while collected cell. They were then washed twice with phosphate buffered saline (PBS) and lysed in ice-cold lysis buffer. And analyzed the expression of phosphorylatal Akt of rat vascular smooth muscle cell in the way of Western blot.Results: The result of transmission electron microscope revealed that: the morphology of cell in normal serum was in a average state, the nucleolus was clear, while in the herbal serum the morphology of cell manifested typical apoptosis, for example, cell nucleus was irregular, concentratd cytoplasm, the nucleolus of the cell was disappear, the pyknosis of chromatin, gathered under the caryotheca aside, the swelling of endoplasmic reticulum etc. The result of Annexin V-FITC/PI double staining detected via Flow cytometry (FCM) showed that: Herbal blood serum acted on rat vascular smooth muscle cell for 24 hours and 48 hours, apoptotic rat vascular smooth muscle cell of rat could be increased obviously along with prolong acted time, there was significant difference comparing to normal blood serum on the time point of 24 hours and 48 hours,it also had significant difference between 24 hours and 48 hours. The result of Western blot revealed that Xiaobantongmai mixture serum could down-regulate the expression of phosphorylatal Akt of rat vascular smooth muscle cell in the herbal blood serum, and there was significant difference comparing to normal blood serum.Conclusions:1.The herbal serum of Xiaobantongmai mixture inhibited the proliferation of rat vascular smooth muscle cell in a concentration-dependent and time-dependent manner.2.The herbal serum of Xiaobantongmai mixture postponed the expression of Cyclin Dl,Cyclin E, promoted the expression of p27, as a result, the proliferation of rat vascular smooth muscle cell was inhabited.3.The herbal serum of Xiao ban tong mai mixture that influenced the trans for mation from G0/G1 phase to S phase and inhibited the proliferation of rat vascular smooth muscle cell was connected with the fall of the expression of phosphorylatal ERK1/2.4.The herbal serum of Xiaobantongmai mixture that influenced the transformation from G0/G1 phase to S phase and inhibited the proliferation of rat vascular smooth muscle cell was connected with the fall of the expression of phosphorylatal Akt, according to reduced the expression of phosphorylatal Akt to induce the apoptosis of rat vascular smooth muscle cell.5.According to influence the signal passageways of ERK1/2 and Akt, the proliferation of rat vascular smooth muscle cell was inhabited and the apoptosis of rat vascular smooth muscle cell was induced to some extent.6.The herbal serum of Xiaobantongmai mixture could influence the biological behavior of VSMC through angle and multi-point,could be used for the prevention from restenosis in clinical.7.To nourish qi and assist yang as the main therapy and invigorate the circulation of blood and remove stagnate as complement are feasible to cure restenosis, conform to the theory of Traditional Chinese Medicine that treatment according to pattern differentiation, and conform to research findings of modern molecular biology.