Primary Study Of The Expression And Function Of Slingshot In Peripheral Eosinophils Of Asthma Patients

BACKGROUNDAs a common chronic disease, the incidence of asthma increases year after year.It is fundamental that investigate the molecular mechanism of asthma whichincluding genetic factors, environment, immunity, inflammtion and reconstrution ofbronchus for controlling asthma. It has been approved that asthma is a polygenedisease influenced by genetic variants and environment. There is some controversy onthe mechanism of asthma, including the role of EOS in asthma.EOS is one of the effect cells in ashma. Over the last 20 years, a large body ofevidence has been generated to suggest that the eosinophil is a key proinflammatoryleukocyte in allergic asthma. But all drugs that aim directly at eosinophil are uselessat present, except the GCs which inhibit the imflamation of airway. The activation,migration, infiltration and enducing imflamation of eosinophil in asthma need futherexploration. We proceed with gene diffential expression in the perpheral blood eosinophilsand hoped to disclose the relationship between gene differential expression and thefunction of the eosinophil. In previous studies, 56 differential expressed cDNA cloneswere obtained by suppression subtractive hybridization (SSH). Because of theessence of method, these genes with differential expression must be confirmed. Inthese candidate genes, Slingshot gene was found up-regulation. Slingshot wasdiscovered several years ago, and the expression and function in eosinophil isn'tilluminated yet.Methods1. 30ml peripheral vein blood were drew respectively from 15 healthyvolunteers, 15 asthma patients and 15 patients with asthma by glucocorticoidstreatment.2. The eosinophils were isolated, purified and measured from each sample.Objected SSH-1L/β-Actin gene fragments were amplified simultaneously by RT-PCRfrom the total RNA.3. SSH-1L protein of the eosinophils was detected by Western blot method fromthe total protein of peripheral eosinophils. The expression of SSH-1L in differentgroups from the levels of gene and protein were compared.4. The location of SSH was detected with immunofluorescence in peripheraleosinophils of healthy volunteer and attack asthma patients.5. The expression of SSH-1L before and after glucocorticoids treatment inasthma patients was compared.6. Peripheral eosinophils of attack asthma patients were incubated and SSH-1Lgene fragments of them were amplified simultaneously by RT-PCR.7. All statistical tests were operated with the program SPSS 10.0(LSD/S-N-K orDunnett T₃) and P＜0.05 was considered statistically significant. Results1. The counts of peripheral eosinophils of attack asthma patients:(0.100×10⁹±0.045×10⁹)/L in control group, (0.120×10⁹±0.047×10⁹)/L in asthmapatients, (0.210×10⁹±0.125×10⁹)/L in asthma patients by glucocorticoids treatment.2. SSH-1L/β-Actin ratio significantly increased in untreated patients with asthmaattacks in comparison with healthy volunteers and patients treated withglucocorticoids(P=0.000, P=0.000). But there isn't difference between treatedpatients and healthy volunteers(P=0.821).3. The SSH-1L/β-Actin ratio of peripheral eosinophils protein samplesignificantly increased in untreated patients with asthma attacks in comparison withhealthy volunteers(P=0.024)but not in patients treated with glucocorticoids(P=0.761).4. SSH-1L phosphatases locate in kytoplasm of peripheral eosinophils andexpress well near cell wall by immunofluorescence.5. The compartion of SSH-1L expression before and after GCs treatment: TheSSH-1L/β-Actin ratio before treatment was 0.7403±0.1124, and significantlydecreased when treated with GCs(0.4101±0.0363, P=0.001). The SSH-1L/β-Actinratio of protein sample before treatment was 0.3410±0.1337, and significantlydecreased when treated with GCs(0.1543±0.0551, P=0.039).6. Dexamethasone incubation of peripheral eosinophils of attack asthma patientscan down regulate the expression of SSH-1L. The SSH-1L/β-Actin ratio of thecontrol group was 0.4574, 0.4269, 0.2294 at 0h, 24h, 48h respectively; TheSSH-1L/β-Actin ratio of the dexamethasone incubation group was 0.4469 at 0h(identical with the ratio in the same time of control group), 0.3213 at 24h (decreasedsignificantly in compassion with the control group in the same time), 0.1813 at 48h(decreased in compassion with the control group in the same time). Conclusion1. The significantly high expression of SSH-1L on both gene and protein level inperipheral eosinophils of asthma patients maybe play an important role in theactivation and migration of eosinophils.2. We found that SSH-1L phosphatases locate in kytopIasm of peripheraleosinophils and express well near cell wall, which suggested that the expression ofSSH-1L relative with the regulation of eosinophils migration.3. There isn't differential expression of SSH-1L on both gene and protein levelin peripheral eosinophils between GCs treatment patients with control group, butthere is differential expression of SSH-1L before and aider GCs treatment. Ourresearch give a convicing clinical evidence that glucocorticoid play an important rolein the treatment of asthma by inhibiting the expression of SSH-1L and then theactivation and migration of eosinophils.4. Dexamethasone incubation of peripheral eosinophils of attack asthma patientscan down regulate the expression of SSH-1L, which shows that glucocorticosteroidmay inhibit the activation and migration of eosinophils by inhibiting the expression ofSSH-1L.