The Local Embedding Intervention Of Anti-nerve Growth Factor Microspheres In Asthmatic Rats And Its Mechanism

Background Bronchial asthma is a disease characterized by reversible airflow obstruction, airway inflammation and airway hyperresponsiveness. In recent years, as the development of molecular biology, molecular genetics and molecular immunology in the asthma research, it is confirmed that the exacerbation of asthma involves the nervous, endocrine, immune and many other mechanisms. Nerve growth factor (NGF) is a kind of neuropeptide. It plays an important role in neural development and differentiation. NGF is synthesized and secreted by the adrenal medullary cell. Whether asthma is a systemic disease or a separate airway disease? There is no conclusion. Our previous study found that the adrenaline was decreased due to the dysfunction of synthesis and release in asthmatic rats. NGF may initiate dysfunction of releasing adrenaline in asthmatic rats due to functional redundancy of adrenal medulla chromaffin cells to participate in the occurrence and development of asthma. After 14 days intervention with NGF antibody intraperitoneally injection in asthmatic rats we found that it could reduce the NGF expression in vivo while airway inflammation were also alleviated. However, there are still some difficulties in application of NGF antibody in patients with asthma. For example, the exogenous growth factor needs a multiple and large administered dose due to its short biological half life period which shows the short dilution and metabolism after use. In addition, NGF in vivo may also be neutralized by NGF antibody incuding the reduction of its normal physiological effect while the toxicity of NGF antibody after its systemic application is still unknown. Which can be a simple and effective method in inhibition of dysfunction of releasing adrenaline in order to relieve or suppress the asthma attack?In recent years, as drug delivery systems, microspheres and nanoparticles caused more and more people interested in them. Microsphere is a kind of polymer material suitable for wrapping or adsorption of drug carrier made of spherical or spherical particles, releases microspheres are based on biodegradable polymers as carrier material made of microspheres through the pores of the microspheres and the polymer material to control erosion degradation of the protein in vivo and in vitro release. The protein microsphere is commonly prepared by the (water-in-oil)-in-water (W1/O/W2) emulsion and solvent evaporation technique with some modification. In biodegradable polymer, polylactic acid (PLA) and Poly (D,L-lactic-co-glycollic acid) (PLGA) is widely used because of its non-toxic and slow-degradation characteristics in vivo.While it is already used by the U.S. FDA approved carrier material in the human body. In this research, the PLGA-protein microspheres with NGF and anti-NGF as coated drug are successfully prepared by the (water-in-oil)-in-water (W1/O/W2) emulsion and solvent evaporation technique. The particle diameter of microspheres is about 15μm, and the releasing is more than 15 days. Anti-NGF and NGF microspheres are respectively and directly injected into the adrenal gland of rat model of asthma as a local embedding way so as to evaluate the effect of lung function, lung histology structure, serological indicators and etc after intervention. We also discuss the effect and mechanism of the local intervention of anti-NGF microspheres of rat model of asthma in order to find a possible new method of prevention and treatment of asthma.Chapter One The preparation and evaluation of NGF and anti-NGF microspheresObjective To prepare NGF (anti-NGF) PLGA microspheres which possessed uniform size (15μm) and continuous releasing of NGF (anti-NGF) for 15 days with good effect in vitro to provide intervention agent to follow-up experiment.Method Using bovine serum albumin (BSA) as the carrier, NGF (anti-NGF) PLGA microspheres were prepared by the (water-in-oil)-in-water (W1/O/W2) emulsion and solvent evaporation technique. Then their appearance, size, Drug-loading rate, entrapment rate, in vitro release testing were evaluated.Result NGF microspheres(BSA/PLGA 15/100,NGF,anti-NGF/ BSA 1/2000) had smooth surface and spherical morphology with (15.5±5.2)μm particle size,8.45% drug loading,55.7%encapsulation efficiency. In vitro release of the first 3,6,9,12,15 days there were 32.14%, 44.5%,50.99%,63.51%,79.53%of the NGF released from PLGA microspheres.Conclusion (water-in-oil)-in-water (W1/O/W2) emulsion and solvent evaporation technique could be successfully and reproducibly prepared package containing nerve growth factor microspheres which possess best spherical morphology and uniform particle size (15μm or so). The results showed that the higher the protein/polymer ratio, the higher the drug loading into the microspheres, and the lower the efficiency of protein encapsulation in the microspheres. NGF(anti-NGF) microspheres maintained a sustained release of NGF(anti-NGF) for at least 15 days in vitro. Chapter Two The local embedding intervention of anti-nerve growth factor microspheres in asthmatic rats and its mechanismObjective Using anti-NGF and NGF microspheres injected directly into the asthmatic rat adrenal gland, to achieve partial intervention of anti-NGF microsphere treatment of asthma, identify potential new method for the treatment of asthma.Method 32 male SD rats were randomly divided into normal control group, asthma group, NGF microspheres group and anti-NGF microspheres group before the rats were intervented. To investigate the behavior of rats, lung function testing, light microscopy of lung biopsy, electron microscopy of adrenal medulla cell ultrastructure changes,NGF and phenylethanolamine N-methyltransferase (PNMT) expression in the adrenal gland by immunohistochemistry method and serumal NGF, cortisol, corticosterone, epinephrine and norepinephrine concentration changes by ELISA assay.Results Behavior in asthma rats showed varying degrees of sneezing, runny nose, wheezing, scratching the head and face, irritability holes, incontinence, increased aggression and other acts, while in the rats from anti-NGF group showed relatively slighter symptoms and the rats turned to be more quiet. Pulmonary function testing prompted asthma, anti-NGF group, NGF rats showed significant airway hyperresp- onsiveness, while anti-NGF group compared with asthma, RL value is reduced, Cdyn value increased. HE staining of lung tissue sections: asthma group were obvious signs of bronchoconstriction and inflammatory cell infiltration around small vessels and alveolar spaces, interstitial lung inflammatory cell infiltration, bronchial epithelial cells can be seen off, while anti-NGF group were not as much bronchoconstriction and inflammatory cell infiltration as asthma group. And also they have more complete tracheal epithelium with no significant inflammatory exudation cavity phenomenon. Electron microscopy: asthma, NGF group adrenal medulla cells in all groups appeared vacuolated changes, uneven distribution of chromaffin granules, the quantity and concentration were significantly lower than normal; NGF group seemed on the adrenal medulla cell membrane and villous clubbing processes; anti-NGF group had no significant vacuolar changes in chromaffin granules and the concentration close to the normal group. Image analysis revealed that anti-NGF group PNMT and NGF immunohistochemistry results mean gray values were (193.39±0.95), (173.78±3.23), and the asthma group was significantly different (P< 0.05,0.01); The ELISA results showed that:(1) The average concentration levels of epinephrine in each group were the control group > anti-NGF group> asthma> NGF group.Anti-NGF group compared with asthmatic group, as well as asthma group and control group, NGF group and asthma group, were significant different (all P<0.01). (2) The average concentration levels of norepinephrine in each group were the NGF group> asthma group>anti-NGF group> control group. Anti-NGF group compared with asthmatic group, as well as asthma group and control group, NGF group and asthma group, were significant different (all P<0.01). (3) There was no significant difference among the groups of the average concentration levels of cortisol (all P> 0.05). (4) The average concentration levels of norepinephrine in each group were control group>anti-NGF group>asthma group>NGF group. Anti-NGF group compared with asthma group, as well as asthma group and control group, NGF group and asthma group, were significant different.(P＜0.01, 0.01,0.05).Conclusion Local embedding of anti-NGF microspheres can alleviate inflammatory infiltration in lung tissue and improve lung function of rat model of asthma. The mechanism may be the anti-NGF reverse NGF's priming of the adrenal medulla cell transdifferentiation process after the antagonism of it.