The Research Of Thrombin Involved In Airway Remodeling Through PERK1/2Signaling Pathway In Asthmatic Rat

Objective:Asthma is a chronic airway inflammatory disease characterized by airway hyperresponsiveness and reversible airflow obstruction. Airway remodeling is an important aspect of the disease, including subepithelial fibrosis, thickening of airway smooth muscle, hyperplasia of the glands and formation of new blood vessels. Airway remodeling may aggravate airway obstruction and airway hyperresponsiveness, resulting in lung function continues to work with progressive damage.Recent studies show that thrombin can promote airway remodeling and pERK1/2maybe an important signaling pathway.In our research, we use ELISA to detect the activity of thrombin in bronchoalveolar lavage fluid(BALF). we use HE staining, PAS staining and Masson staining to compare the severity of airway smooth muscle cell proliferation, goblet cell hyperplasia and collagen deposition of different each group of rats. We use immunohistochemical to detect TGF-β protein expression each group. We use western blot to detect pERK1/2expression in each group. We use our results to explore wether thrombin can promote airway remodeling through pERK1/2in asthmatic rats.Methods:Twenty four femal rats were randomly divided into normal group(n=6),asthma group(n=6),thrombin group(n=6), pERKl/2inhibitor PD98059group(n=6). The rats were sensitized with ovalbumin to establish the asthmatic mode. The asthma group received intraperitoneal injections of allergy causing solution on day1and8. From day15,give asthmatic mode the inhalation of2%OVA three times a week for6weeks,the same as thrombin group and PD98059group. thrombin group and PD98059group were treated with inhalation of thrombin before inhalation of OVA and PD98059group were treated with intraperitoneal injections of PD98059(3mg/kg) before inhalation from day15.The normal group received normal saline for sensibilization. At the end of the modeling, bronchoalveolar lavage fluid were collected and lung tissue were got from rat, and then divided into two groups, a group frozen in liquid nitrogen to prepare PCR and westernblot, another grpup were fixed in4%formaldehyde solution, to prepare for the line of paraffin sections. pERK1/2expression was observed in each group, and also the PAS staining, Masson staining and protein expression of TGF-beta.ELISA was used to detect the activity of thrombin.Result:1.General situation:the rats of group N have no dysphoria when excited, the respiratory smoothy, action sensitive and living habits normal, the coat color is shiny; Group A have the symptoms of varying degrees of irritability, shortness of breath, abdominal convulsions, incontinence when stimulated, the severe ones’breathing become slow or uneven rhythm, the rats fell down and not move. But after many times after excitation, the above-mentioned symptoms of asthma group was slightly reduced, the coat was dull; Group T have the same symptoms as Group A, but the degree was more serious. When the P group in the excited, the irritability phenomenon is lighter, faster breathing and occasional incontinence status come out, respiratory rate or rhythm have no change, coat was normal.2.The activity of thrombin:the activity of thrombin of group A was higher than group N. Group T and P were highest.3.Airway remodeling:smooth muscle of N group had no thickening, no the goblet cells hyperplasia and collagen deposition; group A had abnormal thickening of the smooth muscle, goblet cells were hyperplasia and collagen deposition is apparent; smooth muscle hyperplasia, goblet cell hyperplasia and collagen deposition of T group of was the most serious.smooth muscle proliferation to a lesser extent in group P goblet cells, and collagen fiber deposition was significantly reduced.4.pERK1/2expression:expression level of group N is very low, group A had higher expression levels and statistically significant compaired with the N group.(P< 0.05). The highest expression levels of T group was statistically significant compared with group A.The expression level of the P group was significantly lower, compared with group A (P<0.05).5. TGF-β1expression:expression level of group N is very low, group A had higher expression levels and statistically significant compaired with the N group.(P<0.05). The highest expression levels of T group was statistically significant compared with group A.The expression level of the P group was significantly lower, compared with group A (P<0.05).Conclusions:1. The activity of thrombin is increased in asthmatic rats.2. Thrombin can improve phosphorylation levels of ERK1/2in asthmatic rats.3. Thrombin can regulate the expression of TGF-β1through pERK1/2signaling pathway, which participate in airway remodeling.