Expression Of Inositol-1,4,5-triphosphate And Ryanodine Receptor In Airway Smooth Muscle And Action On Airway Remodeling Of Asthma

Recently, there is a tendency that the mortality of asthma elevated year by year, especially in the developed countries. The asthma has become one of serious chronic diseases threatening public health around the world. Even though several specific drugs aimed to treat asthma have been developed, the attack of asthma is also not under control thoroughly till now. A number of pathological studies showed that not only inflammation appeared in airway, the airway remodeling (changes in airway) also occurred during asthma. The chief pathological changes of airway remodeling represent as thickening smooth muscle and basal membrane as well as hyalinization, which induce irreversible airway block and airway hyperresponsiveness(AHR). The airway remodeling is a characteristic of chronic asthma that is seen particularly in patients with severe asthma and a progressive decline in lung function. It is a feature of both fatal and nonfatal asthma. The proliferation of airway smooth muscle cells (ASMCs) is main reason of airway remodeling. Construction and proliferation of airway smooth muscle(ASM) is connected closely with [Ca2+]i. There are two kinds of calcium release channels in sarcoplasmic reticulum of most cells, that is ryanodine receptor (RyRs) and inositol-l,4,5-triphosphate receptor(EP3Rs). Numerous groups had investigated that RyRl-3 as well as IP3RI-3 expressed in vascular smooth muscle and changes in pulmonary artery hypertension or myocardial hypertrophy. But it is still unclear at present that which type of RyR and/or IP3R exists in airway smooth muscle. Thus our investigation mainly focused on the RyRs and /or IP3RS in the airway smooth muscle of rat. The mRNA expressions of RyR 1-3 and /or IP3R1-3 have been detected in both normal and chronic asthmatic rats with RT-PCR method. Antisense oligonucleotides technology has also been used to study the possible mechanism of RyRs and /or IP3RS in the development of asthma airway remodeling.Methods1. According the related sequences of rat RyRl-3 and IP3RI-3 registered in GenBank,6 pair primers have been designed as well as a pair of additional B -actin primer as an internal reference. mRNA expressions were detected by RT-PCR method with the template of total RNA extracted from airway smooth muscle. Connected with clone vector, PCR products were analysised by DNA sequencing.2. The chronic asthmatic rat model was established by using ovalbumin with aluminium hydroxide gel as adjuvant. Provocation was carried out 3 times every week with duration of 40 minutes for 10 weeks. The expressions of RyRl-3 and IP3RI-3 were detected every two weeks with RT-PCR method. The pathological sections of middle lobe of right lung were made and stain with HE. Inner and outer diameter, thickness of airway smooth muscle and mucosa coat of large medium and small airway were measured with image analysis software. The expression of ASM was detected with a-actin antibody by immunohistochemistry method.3. The rat airway smooth muscle cells (ASMCs) have been cultured primarily by using collagenase digestion and identified by immunocytochemistry methods.4. On the basis of related sequences of RyR1-3 and IP3R1-3, 2 pairs of sense and antisense oligonucleotide have been designed and introduced in ASMCs assisted with Lipofectamine2000. Before and after transfections, the mRNA expressions of RyR1-3 and IP3R1-3 were detected by using RT-PCR, the proliferation of ASMCs were measured with MTS/PES colorimetry method.5. The concentration changes of intracellular calcium were detected by using flow cytometry and laser confocal microscopy before and after transfections with sense and antisense RyR or IP3R oligonucleotide.Results1. 6 fragments with expected length have been amplified with RT-PCR method, which connected with clone vector to sequencing. The results indicated that the amplified fragments were in well accordance with that registered in GenBank.2. The image analysis of pathological sections of middle lobe of right lung showed lumens narrowing and various airw...