Molecular Mechanism Of The Dysfunction Of Muscarinic Receptors Inducing BHR

BACKGROUNDNowadays, researches suggest that asthma is a chronic inflammatory disease of the airway which is participated in mainly by Eosinophil, mast cell, T lymphocyte, and other inflammatory cells.It has been well documented that asthma is associated with both bronchial hyperresponsiveness(BHR) and airway inflammation. In the lung, the parasympathetic nerves provide the dominant autonomic control of airway smooth muscle. They release acetylcholine(ACh) onto muscarinic receptors, causing contraction and bronchoconstriction. In 1914 acetylcholine receptors were classified into nicotinic(N) and muscarinic(M) receptors by Sir Henry Dale. Muscarinic receptors were formally divided into five subtypes(M₁-M₅). Of the five cloned muscarinic receptor subtypes, human lung expresses only three receptor subtypes(M₁, M₂ and M₃) that are differentially distributed in the airways. M₁ receptors are localized in parasympathetic ganglia where they facilitate neurotransmission, and to the alveolar walls, where their function remains unclear. The selective agonist of M₁ receptors is McN-A-343, and its selective antagonist is pirenzipine. M₂ receptors are found on parasympathetic cholinergic nerve endings, where they serve to control neurotransmitter release,namely autoreceptor. Its selective agonist is pilocarpine and selective antagonists are gallamine, AF-DX116, methoctramine. And M₃ receptors are localized to the airway smooth muscle and glands, where they mediate smooth muscle contraction and secretion of glands. Its selective agonist has been not found yet and selective antagonists are 4—DAMP andHHD. Acetylcholine released from the parasympathetic nerves stimulates M3 muscarinic receptors on airway smooth muscle inducing contraction and bronchoconstriction. The amount of acetylcholine released from these nerves is limited locally by M2 muscarinic receptors. Studies showed that M2 muscarinic receptors are dysfunctional in asthma and in animal models of asthma. Decreased M2 muscarinic receptors function results in increased release of acetylcholine, bronchoconstriction, oversecretion of glands and bronchial hyperresponsiveness. Eosinophils are the main inflammation cell in asthma and in animal models of asthma. Eosinophils contain charged proteins such as major basic protein (MBP)> eosinophil cationic protein (ECP)? eosinophil peroxidase (EPO) and so on. and it is suggested that MBP is the key substance in inducing the dysfunction of M2 receptors and BHR. ObjectiveAfter making of the model of asthmatic guinea pig, we measured the total cell count and cell classification of BALF of guinea pigs, at the same time pulmonary histopathologic examination and immunohistochemistry of M2R and acetylcholinesterase(AChE) were performed. Finally, We investigated the expression of M2R mRNA> M3R mRNA and MBP mRNA in airway smooth muscle after dexamethasone therapy. In our experiment, we are bound to investigate the expression of muscarinic receptor (MR) mRNA and MBP mRNA in airway smooth muscle,localization of eosinophils to airway nerves and the infiltration of eosinophisl in the airway of asthmatic guinea pigs. Still we will study the more role of dexamethasone on therapy of asthma. Materials and MethodsMaking of the model of asthmatic guinea pig and grouping.Thirty healthy, parti-colored guinea pigs were randomly divided into three groups in which each group had ten guinea pigs: control group, asthma group and dexamethasone therapy group.Making of the model of asthmatic guinea pig: lml of 10% ovalbumin (OVA) saline suspension were injected into abdominal cavity of twenty guinea pigs of twogroups in the first day (The albumin flake was grand into fine powder before dissolution) . Then, aerosol of 1%OVA saline suspension was sprayed to challenge these sensitive guinea pigs by ultrasonic atomizing inhalation instrument in the fifteenth day. We stopped challenging until we found guinea pigs breathing quickly, nodding spasm, and rectus abdominis shrinking. From above two groups, one group was asthma group, the other group was treated by dexamethasone for two weeks. The last group was control group.Collection of BALF and measurement of the total cell number and cell classification.At the thirtieth day, we anesthetized them after the last challenge. Then, we poured 3ml Hanks into bronchus and lung, and retrieve BALF. We lavaged four times in all. BALF was centrifugated, and we took few of them to slide for smear and measured the total cell count and cell classification.Pulmonary histopathologic examination and Immunohistochemistry ofMzR and AChE.We cut out right lung after lavaging pulmonary alveoli. Then, we used 10% formalin to fix pulmonary tissue. Third, it was be paraffin embedding. The fourth step was to make it slices. Finally, some of the slices were stained by hematoxylin and Eosin(HE). All these steps were finished, we observed the change of pathology including eosinophil infiltration and epithelial cells abscission with light microscope which magnified 400 times.The other slices were stained as following step: dealing with the slices with APES; slices dewaxed to water routinely; distilled water collocating 3%H2C>2 lately and destroying endogenesis enzyme for 5 to 10 minutes in room temperature; heated repairing antigen; dripping normal goat serum blocking reagent; dripping the first antibody diluted 150 times to the slices in 37 "C for one hour, then in 4°C for all night, finally 0.1 M PBS washing the slices three times and each times was two minutes; dripping biotining the second antibody in 20-27 °C for twenty minutes, the 0.1M PBS washing the slices three times, and each times was two minutes; dripping SABC to the slices in 20-27 °C for twenty minutes, the 0.1M PBS washing the slices fourtimes, and each times was five minutes; DAB displaying color; Hematoxylin lightly staining the slices again; observing the slices with microscope; taking photos.Synthesis of primers ofM2R mRNA, M3R mRNA,MBP mRNA and GAPDH.Primers of M2R mRNA, M3R receptor mRNA, MBP mRNA and GAPDH were synthesized by Shanghai BIOASIA Biology Technical Ltd. In this research, GAPDH was used an internal reference.Reverse transcription — polymerase chain reaction (RT-PCR) and semi-quantitative analysis of it.RNA Isolation and reverse transcription: the methods were according to the instruction of the RT-PCR kit. Sample products were taken from liquid nitrogen.Detection of the products after two PCR: Amplified fragments were separated in parallel with the coamplified actin reference fragments by electrophoresis in 2% agarose. Finally, we analyzed the comparison of integral calculus optics density (IOD) of target fragments and GAPDH( (IOD ) (taigets > /(IOD) ( gafdh > )to make semi-quantitative analysis.Statistics:Data of all groups were expressed with Mean±SD. All data were processed by SPPS 10.0 software. The statistical significance of differences between means of various groups was tested using analysis of variance with the One-Way ANOVA test and P<0.05 was considered significant. When equal variances assumed, we chose LSD(least significant difference) to analyze data. When equal variances not assumed, we chose Dunnetf s T3 to analyze data. ResultsThe total cell count and cell classification ofBALFCompared with the control and the asthmatic group, the number of EOS in the BALF of dexamethasone therapy group was significantly lower (p <0.01). After therapy of dexamethasone, the number of the total cell and EOS in the BALF was notability decreased, which prove that dexamethasone can restrain the infiltration of eosinophisl in the airway of asthmatic guinea pigs.Pulmonary histopathologic slice and Immunohistochemistry of M2R and AChb.In the pulmonary histopathologic slices of asthma group, we could find rather significant edema and hyperemia in the mucous membrane and tissue, and the number of eosinophil infiltration in them was more than that in pulmonary histopathologic slices of dexamethasone therapy group.In the slices of Immunohistochemistry of AchE of dexamethasone therapy group and asthma group, we found that nerve-associated EOS was significantly increased compared with control group. In the slices of Immunohistochemistry of M2R of asthma group, we could also find positive cells in the mucous membrane, underlayer mucous membrane and muscle of bronchiole. And the number of positive cells in the dexamethasone therapy group was less than that in asthma group, but still more than those in control group.The relative content of M2R mRNA, M3R mRNA and MBP mRNA of airway smooth muscle in three groups.The semi-quantitative analysis of RT-PCR of M2R mRNA, M3R mRNA and MBP mRNA in airway smooth muscle suggested that there were significant difference between asthma group and contol group, P<0.0\, and there were also significant difference between asthma group and dexamethasone therapy group, P<0.01.We found that the expression of M2R and MBP was increased in antigen-challenged guinea pigs, and that of M3R decreased, and dexamethasone can treat asthma by regulating the expression of M2R, M3R and MBP. Electrophoresis of RT-PCRThe results of sepharose electrophoresis of RT-PCR suggested that the four lanes of M2R, M3R, MBP and GAPDH were distinct to be seen. The results were in line with the request of RT-PCR. ConclusionThe number of EOS in the BALF of antigen-challenged guinea pigs was increased, EOS infiltration was evident in the lung tissues of antigen-challenged guinea pigs and eosinophils were associated with airway vagus in antigen-challenged guinea pigs.The expression of M2R in airway smooth muscle was increased in antigen-challenged guinea pigs, and that of M3R decreased.The expression of MBP in airway smooth muscle was increased in antigen-challenged guinea pigs.Dexamethasone could significantly inhibit inflammation cells effusion in the airway of asthmatic guinea pigs, especially EOS, and lighten tissue edema, reduce osmosis of capillary vessel and inhibit pathological immunity, inhibit the localization of eosinophils to airway nerves and regulate the expression of M2R, M3R and MBP to recover the function of M2 receptor.