Effect Of Methylprednisolone On The Expression Of CFTR Chloride Channel In The Lungs Of Mice Infected With Respiratory Syncytial Virus

Objective Respiratory syncytial virus(RSV)is the primary cause of hospitalization of viral respiratory tract infection in children in China.RSV mainly attacks the respiratory system,leading to airway obstruction,tracheal spasm and airway hyperresponsiveness through a variety of ways.Cystic fibrosis transmembrane transport regulator(CFTR)is a chloride channel mainly distributed in secretory epithelial cells of many organs throughout the body.In airway epithelial cells CFTR participates in the secretion of HCO3-and Cl-,and participates in the regulation of osmotic pressure,hydration and p H value of the Airway Surface Liquid(ASL)in the liquid layer of the airway surface,which helps to maintain the homeostasis of ASL.One of the important pathological features of RSV infection is airway mucus hypersecretion.CFTR can regulate airway mucus secretion by regulating respiratory epithelial ion transport.In our previous research work,we found that the expression levels of CFTR gene and protein changed after RSV infection in Calu-3 cells(human respiratory submucosal adenocarcinoma cells)and BALB/c mice lungs,suggesting the relationship between RSV and CFTR.Methylprednisolone(MP)is one of the commonly used glucocorticoid drugs in clinical treatment.Previous studies have reported that glucocorticoids can affect the gene and protein expression of CFTR channel in many ways.At the same time,in the clinical treatment of RSV infection,the application of glucocorticoids is still controversial.We want to explore whether methylprednisolone is helpful to the clinical treatment of RSV by studying the effect of methylprednisolone on the expression of CFTR gene and protein in the lungs of BALB/c mice infected with RSV.Research methods 1.Mice were randomly divided into control group(Control group),methylprednisolone group(MP group),infection group(RSV group),infection + methylprednisolone group(RSV + MP group).2.The mouse model of RSV infection was established: the mice were anaesthetized by intraperitoneal injection of 1% pentobarbital sodium.The mice in the infection group and the infection + methylprednisolone group were alternately instilled with RSV virus solution with a titer of 2.6x107 PFU into the nasal cavity of the RSV group and the RSV+MP group.The mice in the Control group and the MP group were given nasal drip with the same volume of Hep-2 cell culture medium in the same way.3.Drug intervention: the MP group and RSV + MP group was injected intraperitoneally with methylprednisolone 2 mg/kg on the 4th,5th and 6th day after infection,while the Control group and RSV group were injected with the same amount of normal saline.4.The successful establishment of the infection model was verified by RT-PCR: on the seventh day after RSV infection,the mice were killed and 20-30 mg lung tissue was taken to extract RNA,to detect the nucleic acid of RSV-G protein by ordinary RT-PCR to verify whether the RSV infection model was established successfully.5.The expression level of CFTR gene in each group was detected by q RT-PCR,and the relative RNA expression of each sample CFTR relative to internal reference GAPDH was calculated by 2-ΔΔCT method.6.The lung tissue protein of mice was extracted,and the expression level of CFTR protein in each group was detected by Western Blotting,and the gray value was semi-quantitatively analyzed by Image J software.7.Using IBM SPSS 19.0 statistical software,the mean ± standard error(Mean ± SEM)was used to represent the measurement data,and the t-test was used to analyze the differences among the measurement data groups,P ≤ 0.05 indicated that the difference was statistically significant.Research results.1.There was no significant difference in body weight before infection in each group,and there was no significant difference in body weight between Control group and MP group or RSV group and RSV + MP group on the 4th day after infection.After intraperitoneal injection of methylprednisolone,there was no significant difference in body weight between Control group and MP group on the 7th day after infection.The body weight of mice in RSV + MP group was lower than that in RSV group.2.There was no significant difference in the expression of CFTR gene between the Control group and MP group,the expression of CFTR gene in the RSV + MP group was lower than that in the RSV infection group. 3.There was no significant difference in the expression of CFTR protein between the Control group and MP group,the expression of CFTR protein in the RSV + MP group was lower than that in the RSV infection group.Research conclusion.1.Systemic glucocorticoid intervention in RSV-infected mice can lead to weight loss,which may be due to aggravation of infection or increased consumption caused by hormones.2.Systemic administration of glucocorticoid has no effect on the expression of CFTR gene and protein in the lungs of mice,but in the case of RSV infection,it can down-regulate the expression of CFTR gene and protein in the lungs of mice,which may not be conducive to the recovery of mice infected with RSV.