The Research On The Regulation Of NGF To Rho/ROCK Signaling Pathway In Pathogenesis Of Asthma

Objective:Bronchial asthma(asthma)is one of the most common chronic disease affecting global public health.Asthma can be caused by a variety of factors and its pathogenesis is complex and has not yet been fully elucidated.Recent studies showed that the occurrence and the continuous state of asthma are closely related to immune-neural mechanisms.The nerve growth factor(NGF)acting as an intermediary substance contacting airway immune and neural mechanisms plays a very important role in the pathogenesis of asthma.The Rho GTP enzyme known as the small G protein,is one of the most clearly studied Ras-related monomeric GTP enzyme.It can activate its downstream target molecule-Rho associated coiled coil forming protein kinase(Rock)and regulate a variety of cell behaviors and functions,including cell shrinkage,migration and adhesion,growth and division,and as well as transformation and infiltration of tumor cells.Recent studies have found that Rho / Rock signaling pathway involved in the mechanism of the smooth muscle and non-smooth muscle dysfunction related diseases,which has attracted wide attention.But in bronchial asthma whether NGF caused tracheal smooth muscle contraction,increased airway resistance,and further caused the occurrence,development and continuous of asthma by Rho / Rock signal transduction pathway is unclear.The purpose of this project is to further explore the pathogenesis of asthma by analyzing the correlation between NGF and Rho / Rock signaling pathway,as well as to investigate the role of anti-NGF antibody and inhibitors of NGF in the treatment of asthma,to provide new ideas to research and will play a very important role in guiding asthma prevention.Methods:1.Forty healthy and clean female BALB / C mice,aged about 4 to 6 weeks with body weight of(17 ± 2)g,were fed in a clean grade environment,12 h lighting,room temperature 22-25 ?,humidity of 40% to 70% of the particles,and with free drinking of water.With random number table,the mice were divided into a control group(C group),asthma group(A group)and anti-NGF(N group),ROCK inhibitor Y-27632 intervention group(Y group),10 mice in each group.The mice of asthma group and intervention group were injected intraperitoneal with OVA suspension(OVA20?g + aluminum hydroxide 2mg gel)0.2 ml on day l and 14,and were sensitized on day 25 to 29 with 2% OVA in the spray tanknebulized,30 min once a day.The control group were given normal saline to sensitiz and stimulate.The intervention group were given nasal instillation of anti-NGF antibody(1:50 dilution liquid)50 ?l,or injected intraperitoneal with Y-27632(4mg/kg),30 minutes before the atomization each time,the control group and the asthma group were injected intraperitoneal with normal saline.2.Airway responsiveness in mice was measured 24 hours after the last stimulate using the non-invasive small animal spirometer.To determinate the basis of enhanced expiratory intermittent values(pulmonary enhanced pause,Penh values).3.Blood serum was collected byexcise the eye ball of mice,then take a drop of blood to make a blood smear.Ligate and resect of the right lung,upper lobes of it were embedded in paraffin,other lung tissues were rapidly placed in liquid nitrogen to storage.Left lung was lavaged with sterile saline through endotracheal intubation,and then recycling BALF.4.Blood smear and the BALF dregs smear were stained by Wright's stain after dry naturally.The white blood cells were counted and classified,and calculated the percentage of eosinophils by using the high magnification of microscope.5.Paraffin-embedded lung tissues were sliced into 4 ?m for HE staining.To observe inflammatory cell infiltration around the bronchial and vascularunder with an ordinary optical microscope.6.Double antibody sandwich enzyme-linked immunosorbent assay(Sandwich ELISA)was used to detect the concentration of IL-13,IFN-? in blood and BALF.The experimental process was refer to the reagent instructions.7.With SABC method,in accordance with the instructions of the kit.antibody against NGF,Rho A,ROCK and P-MLC were respectively at a dilution of 1:100,1:100,1:150 and 1:200.Each slice was selected randomly 5-10 bronchial view with high magnification(× 400),using a modified semiquantitative scoring system.the fraction of positive cells(F)was categorized into four groups,namely 0(0-1%),1(2-25%),2(26-75%)and 3(> 75%)and staining intensity(I)was scored as 0(negative),1(weak),2(moderate),3(strong).A combined score of Fx I,which had a range of 0 to 9,was then constructed.8.To determine and quantify of the total protein of lung tissue, protein sample was separated by SDS-PAGE gel electrophoresis,and then the gel separated proteins were transferred to nitrocellulose membranes.With 5% skim milk(PBST dissolved)blocked for 1 hour at room temperature,added an antibody and react overnight at 4° C,the membranes were washed with PBST and add second antibody,were incubated for 1 hour at room temperature,washed the membrane with PBST.Analyzed the result with infrared fluorescence scanning imaging system on the scan.9.To extract the total RNAaccording to the rapid extraction of high-purity total RNA kit and one-step reverse transcription-Real Time-PCR kit,applying Light Cycler480 fluorescent quantitative PCR instrument,c DNA was synthesized and completed the amplification.Then obtained a fluorescence amplification graph,and measured Ct values of the samples,compared using the 2-??CT.10.The data were analyzed with Graph Pad Prism statistical software,all of the results are presented as mean ± standard deviation(x± SD).The two groups were compared using T-test,pairwise comparisons among groups using ANOVA analysis with normal distribution and homogeneity of variance.For the non normal distribution,Non-parametric tests was used.P <0.05 was considered statistically significant.Results:1.The overall performance of mice in each group after excitation were difference.Mice of group A showed obvious shortness of breath,irritability security,scratching,salivation,stool incontinence,eyes chemosis,wheezing.Mice of group N and group Y showed some of these symptoms but to a less degree.No significant symptoms in mice of group C.2.Penh values in each group of mice increased with the Mch concentration.Penh values in the mice of group A were significantly higher(P <0.001)than those in group C under different concentrations of Mch inspire.N group and Ygroup were significant decreased compared with group A(P<0.001).It indicates that the airway resistance of asthma group was significantly increased,and the experimental models were successfully established.To the anti-NGF antibody group and Y-27632 group,airway resistance was significantly improved than asthma group.The differences were statistically significant.3.The percentage of eosinophils in blood and BALF smears was significantly increase in Group A than that in group C(P <0.001).There was significant decrease in Group N and Y compared to group A(P<0.001).The differences were statistically significant.4.HE staining of mouse lung tissues showed almost normal in bronchi and pulmonary structure of the group C mice.Mice of group A showed bronchial wall thickening,mucosal swelling,epithelial cells shedding,increased mucus secretion,bronchial stenosis,pulmonary vascular wall thickening,vascular stenosis.There were visible macrophages,eosinophils,lymphocytes and neutrophil infiltrated around the airway epithelial cells,bronchial and perivascular.The pathological changes of group N and group Y were significantly reduced than those in group A.The differences were statistically significant.5.The contentlevel of IL-13 was increased in serum of group A mice than that of group C(P <0.05),and was manifestly decreased in group N and group Y comparing to group A(P <0.01).The contentlevel of IL-13 was significantly increased in BALF of group A mice than that of group C(P <0.001),and was manifestly decreased in group Y comparing to group A(P <0.01),decreased in group N comparing to group A(P <0.05).IFN-? level in serum of group A was significantly decreased than that in group C(P <0.001),and were significantly increased in group N and group Y comparing to groups A(P<0.001).IFN-? level in BALF of group A was manifestly decreased than that in group C(P <0.01),and was increased in group N and group Y comparing to groups A(P<0.05).The differences were statistically significant.6.Of the two kinds of detection the expressions of NGF,Rho A,ROCK,P-MLC were significantly increased in lung tissue of group A than those in group C(each P <0.001).The expressions of Rho A,ROCK,P-MLC were significantly decreased in mice lung tissues of group N compared with those of group A(each P <0.001).The expressions of P-MLC detected by immunohistochemistry were significant declined in the mice lung tissue of group Y compared with those of group A(P <0.001),and were manifestly declined by Western-blot detection(P <0.01).The differences were statistically significant.7.Values of 2-??CT of NGF,Rho A,ROCK,P-MLC m RNA in lung tissues of group A mice were significantly increased compared with those of group C(each P <0.001).Values of 2-??CT of ROCK,P-MLC m RNA in lung tissue of group Y mice were significantly decreased than those in group A(each P <0.001).Values of 2-??CT of NGF,Rho A,ROCK,P-MLC m RNA in lung tissue of group N mice were significantly lower than those in group A(each P <0.001).The differences were statistically significant.Conclusions:1.NGF,Rho A,ROCK,P-MLC are all expressed in the lung tissue of mice,and were significantly higher in the asthma group than in the normal control group,suggesting that NGF,Rho A,ROCK,P-MLC may be involved in the pathogenesis of asthma.2.ROCK inhibitor Y-27632 can reduce the expression of ROCK?P-MLC m RNA and protein of asthmatic mice,decrease the content of IL-13 in serum and BALF of the mice,and increase the content of IFN-?.Prompted ROCK may be involved in the pathogenesis of asthma by enhancing the contraction of the bronchial smooth muscle and vascular smooth muscle,and increasing airway inflammation reaction.3.Anti-NGF antibody can inhibit the expression of Rho A,ROCK m RNA and protein in asthmatic mouse,thereby regulating the expression of P-MLC m RNA and protein in lung tissue of mice,suppressing asthma attack of mice.NGF involved in the pathogenesis of asthma by regulating Rho A / ROCK signal transduction pathways.