The Effect Of Total Alkaloids Of Sophora Flavescens On Rat Airway Smooth Muscle Cell Proliferation

Asthma is a common disease worldwide, difficult diseases. It is non-specific chronic airway inflammatory disorders. The World Health Organization (WHO) reports that the health and economic burden which brings by Asthma is more than the sum of AIDS and TB . Over the past 10 years, asthma morbidity and mortality is increasing due to the level of dust and air pollution increased year by year. The asthma prevalence rate of Asthmatic children in our country is form 0.52% to 3.34% in 2000, which is increase of 64.8% than 10 years ago, and it shows a rising trend. Therefore, it is a brook no delay task to research and development of effective drug treatment of asthmat.Airway remodeling is an important pathological feature of asthma. It can stimulate airway inflammation to sustain and develop. It also can cause persistent airway hyperreactivity and irreversible airflow obstruction. Airway remodeling happens not only in Advanced asthma, but also in early asthma. It is the pathology foundation of refractory recurrent asthma. Airway remodeling is hot in Academic research. It is conducive to study it for early prevention and treatment of asthma and prevent further deterioration. Extracellular matrix deposition and smooth muscle cell proliferation are two main performances of Airway remodeling. Extracellular matrix deposition includes fibroblast proliferation, collagen deposition an so on. Smooth muscle cell proliferation includes smooth muscle cell proliferation and hypertrophy. Airway smooth muscle cell plays a decisive role in the process of airway remodeling as the main effector cells in airway contraction. Smooth muscle hyperplasia and hypertrophy are the basic of of airway remodeling structure. It can cause airway wall thickening , deformation and airway stenosis. it can lead the contractile response and contractility to non-specific stimulus of all levels of bronchuses to be multiplied enhanced. This subject take the airway smooth muscle cell proliferation in vitro as the breakthrough point and study the effect of total alkaloids of Sophora Flavescens on rat airway smooth muscle cell proliferation.This subject is divided into three parts. The first part is to set up and optimize the ways of extracted total alkaloids from Sophora. The second part is to set up the way of culture air way smooth muscle cells of rat. The last part is that observe the effect of total alkaloids extracted from Sophora flavescens on the ASMCs proliferation model which is sensitized by endothelin -1 (ET-1).The experiment of the first part use the acidic water immersion and ultrasonic extraction to extract total alkaloids from Sophora Flavescens. And it use 732-Strongly Acidic Cation Exchange Resin to purified total alkaloids. In order to optimize the extraction of total alkaloids process from Sophora, the experiment makes concentration of hydrochloric acid, volume of solid and liquid ratio, ultrasonic extraction time and ammonia concentration of ethanol as the influencing factorsIn the design of the orthogonal experiment of L9 (3~4) And deal with the results by SPSS 16.0. The optimum operation condition was confirmed through the orthogonal experiment: A2B2C2D1, maintaining the following conditions : 10g coarse Matrine soaked in 100mL of 0.2% hydrochloric acid solution for 4h, ultrasonic extraction 40min, 732-Strongly Acidic Cation Exchange Resin absorb total alkaloids fully, use of 0.5% ammonia ethanol extract them. The process is less energy-consuming and reagent. The extraction time is short. So is an Energy-saving and efficient extraction method. The total alkaloids of Sophora Flavescens have a variety of pharmacological effects which may be of mutual synergies and common fight against the disease at a certain extent. So the optimization of Extraction Process of total alkaloids in Sophora flavescens lay the foundation for further development and application of Matrine. In addition, it can provide a rich source of total alkaloids of Sophora flavescens for the further extraction of alkaloid compounds monomers.The experiment of the second part take cell survival rate and cell proliferation activity as biological index, study on which one is better to culture ASMCs between tissue explant method. and enzyme digestion method. According to the experiment, enzyme digestion method is very cumbersome, the time that it costs is so long which can increase infected bacteria opportunities. Besides that many conditions are much more difficult to control in enzyme digestion method. The tissue explant method is the better one. Te time it costs to primary culture cells is shorter than enzyme digestion method. The cell survival rate and cell proliferation activity is higher than enzyme digestion method, too. So this method is not only simple, low-cost, but also keep the cells have good status for further cells experiments. To sum up, tissue explant method have the promotion and practical value to culture ASMCs. And it can widely used in in asthma and other airway diseases on the cellular level.The third part is the innovation of the subject. We observed the effect of serum contained total alkaloids matrine on ASMCs proliferation model which is sensitized by Endothelin -1(ET-1) for the first time. We used ET-1 to sensitize ASMCs because of the Endothelin Receptor exist in the lung tissue widely and airway smooth muscle has the highest density of them. The most important thing is that ET-1 can significantly enhance the effect of epidermal growth factor (EGF) on airway smooth muscle cell proliferation by binding with its receptor. ET-1 is one of the important factors of airway remodeling. Thus, it may show that total alkaloids matrine can effect on airway remodeling and may also indirectly show they role in the treatment of asthma to observe their effect on airway smooth muscle cell proliferation sensitized by ET-1. Dexamethasone used in this experiment as a positive control drug because of it can decrease smooth muscle hyperplasia and airway wall collagen deposition without damage the airway. Total alkaloids of Sophora flavescens have a certain degree of anti-role on rats. We gained high, medium and low dose after we have inspected the half lethal dose in mice and had a series of conversion. The test proved that the three-dose does not lead rats to deadth. Eventually the part of the experimental results show that high doses of total alkaloids matrine can inhibit the proliferation of ASMCs, medium and low doses of their role were not obvious, but they also played an inhibitory effect to a certain extent. Therefore, total alkaloids of Sophora flavescens can inhibit ASMCs proliferation which sensitized by ET-1, and can inhibit airway remodeling. Total alkaloids of Sophora flavescens have many pharmacological effects, such as anti-inflammatory effects, regulate the immune system, inhibit fibroblast proliferation , inhibit ASMCs proliferation, and so on. These effects can be coordinated and jointly control airway remodeling, thus they can treat asthma effectually. So total alkaloids of Sophora flavescens have has the advantages of treating asthma. This study provides a theory for total alkaloids of Sophora flavescens role in treatment of asthma, and laid a foundation for study the inhibition mechanism of the proliferation of ASMCs further.To sum up, this subject takes the airway smooth muscle cells proliferation in airway remodeling as the breakthrough point, confirms the new pharmacological effect of Sophora Flavescens. The new pharmacological effect is that total alkaloids of Sophora flavescens can inhibit ASMC proliferation. This result enhance the medicinal value of Sophora flavescens. At present, there is no effect treatment of airway remodelings. Since the total alkaloids of Sophora flavescens have multiple pharmacological effects, they have potential to become an effective drug of preventing and treating the airway remodeling and asthma. They are expected to become new drugs for clinical treatment of asthma.