| Part ?:Establishment of an Animal Model of Cough Hypersensitivity SyndromeBackgroundCough is a defensive nerve reflex of the body,which is conductive to the removal of respiratory secretions and harmful factors.However,frequent and severe cough will seriously affect patients' work,life and social activities,especially chronic cough.According to the epidemiological investigation,the prevalence of chronic cough is 10%,which varies with region or age.Most patients with acute and chronic cough can get a clear etiological diagnosis,and the symptoms can be relieved after targeted treatment.However,in 12%-42%of patients with chronic cough,after a comprehensive examination,the cause is still not clear and treatment is difficult,requiring further study.This type of chronic cough was called "refractory cough",and Moris proposed the concept of "cough hypersensitivity syndrome" in 2010.The study found that such patients had the following clinical characteristics:1)reduced cough threshold;2)increased TRPV1 expression in the airway;3)increased expression of neuropeptides in body fluids;4)histopathology has no diagnostic specificity.Currently,the preclinical research on cough is relatively chaotic.PM2.5,cigarette smoke and inflammatory mediators are the most commonly used cough induction methods,but there is no typical,stable and recognized cough model,let alone a mature model of cough hypersensitivity syndrome.Bradykinin is an inflammatory mediator,which is associated with chronic obstructive pulmonary disease,asthma and ACEI drug-related cough,and the latter is of high cough sensitivity,which belongs to the category of cough hypersensitivity syndrome.Therefore,this study attempted to establish an animal model of cough hypersensitivity syndrome by bradykinin inhalation.Methods In this study,male Hartly guinea pigs were given aerosol inhalation of bradykinin.In the first step,10 guinea pigs were randomly divided into two groups as follows:(1)normal saline group:normal saline was atomized for 10 minutes;(2)bradykinin group:inhaled bradykinin at 3mg/ml concentration for 10 minutes.Count the number of coughs within 10 minutes after atomization,and test whether bradykinin can induce cough reflex.The other 10 guinea pigs were randomly divided into two groups as follows:(3)the normal saline+capsaicin group:the normal saline was atomized for 10 minutes,and then the capsaicin was atomized at 60uM for 10 minutes after 30 minutes.(4)bradykinin group+capsaicin group:bradykinin was atomized at 3mg/ml for 10 minutes,and then capsaicin was atomized at 60uM for 10 minutes after 30 minutes.The number of coughs within the first 10 minutes of aerosol bradykinin was counted,and the effect of bradykinin pretreatment on the cough sensitivity of guinea pigs to capsaicin was tested.30 minuteslater,samples were collected and the expression levels of TRPV1 and neuropeptides such as NKA,CGRP,SP in serum,bronchoalveolar lavage fluid and lung tissue homogenate were detected by ELISA.The expression levels of TRPV1 and neuropeptides such as NKA,CGRP,SP in trachea and lung tissues were detected by immunohistochemistry.The pathological manifestations of trachea and lung tissues were observed by HE staining.The results were expressed as mean standard deviation(X±SD).SPSS 16.0 software was used to analyze all the data,and P<0.05 was used as the judgment standard of difference significance.Results1.Bradykinin-induced cough reflex1.1 Effect of bradykinin on cough frequency:Compared with normal saline group,the cough frequency of guinea pigs in bradykinin group increased(p<0.01),so the cough reflex was successfully induced.1.2 Effects of bradykinin on TRPV1 expression in cough reflex:(1)ELISA results:Compared with the normal saline group,the expression of TRPV1 in the lung tissues of guinea pigs in bradykinin group was increased(p<0.05)and that in bronchoalveolar lavage solution was increased(p<0.01),but there was no statistical difference between the two groups in the expression of blood.(2)Immunohistochemical results:Compared with the normal saline group,the expression of TRPV1 in the lung tissues of guinea pigs in the bradykinin group was increased(p<0.01),but there was no statistical difference in the expression of TRPV1 in the trachea tissues between the two groups.1.3 Effects of bradykinin on NKA expression during cough reflex:(1)ELISA results:Compared with the normal saline group,the expression of NKA in lung tissues and blood of guinea pigs in bradykinin group was increased(p<0.01),but there was no statistical difference in the expression of bronchoalveolar lavage solution between the two groups.(2)Immunohistochemical results:Compared with the normal saline group,the expression of NKA in the lung tissues of guinea pigs in the bradykinin group was increased(p<0.01),but there was no statistical difference in the expression of NKA in the trachea tissues between the two groups.1.4 Effects of bradykinin on CGRP expression during cough reflex:(1)ELISA results:compared with the normal saline group,the expression of CGRP in bronchoalveolar lavage solution was increased in the bradykinin group(p<0.05),and no increased expression was found in lung tissue or blood.(2)Immunohistochemical results:Compared with the normal saline group,the expression of CGRP in trachea tissues of guinea pigs in bradykinin group was increased(p<0.01),but the expression in lung tissues was not increased.1.5 Effects of bradykinin on SP expression in cough reflex:(1)ELISA results:Compared with normal saline group,the expression of SP in lung tissue,blood and bronchoalveolar lavage fluid of guinea pigs in bradykinin group did not increase.(2)Immunohistochemical results:Compared with the normal saline group,the expression of SP in trachea and lung tissues of guinea pigs in bradykinin group did not increase.1.6 The trachea and lung tissue pathology observation:Pathological changes of both lung and tracheal tissue showed mild to moderate inflammatory changes with HE staining:The lining mucosal epithelium of trachea was slightly disordered.There was moderate infiltration of mixed inflammatory cells in the submucosal stroma,among which eosinophils were easy to be seen,submucosal edema and small blood vessels dilated and silted up.Alveolar dilatation and fusion,inflammatory secretions in alveolar cavity,loose alveolar septum and edema,partial capillary dilation and congestion were found in lung tissues.But there was no significant difference between the groups.2.Cough hypersensitivity syndrome induced by bradykinin2.1 Effect of bradykinin on the cough sensitivity to capsaicin:Compared with the normal saline+capsaicin group,the cough frequency of guinea pigs in bradykinin+capsaicin group increased(p<0.01),so the cough sensitivity to capsaicin increased,and the induction of cough hypersensitivity syndrome was successful.2.2 The expression of TRPV1 in cough hypersensitivity syndrome induced by bradykinin:(1)ELISA results:Compared with normal saline+capsaicin,the expression of TRPV1 in the lung tissues of guinea pigs in bradykinin+capsaicin group was increased(p<0.01),and the expression in bronchoalveolar lavage fluid and blood was not increased.(2)Immunohistochemical results:Compared with normal saline+capsaicin,the expression of TRPV1 in the lung tissues of guinea pigs in the bradykinin+capsaicin group was increased(p<0.01),while the expression in the tracheal tissues was not increased.2.3 The expression of NKA in cough hypersensitivity syndrome induced by bradykinin:(1)ELISA results:Compared with the normal saline+capsaicin group,the expression of NKA in lung tissues and blood of guinea pigs in bradykinin+capsaicin group increased(p<0.05),but there was no statistical difference in the expression of bronchoalveolar lavage solution between the two groups.(2)Immunohistochemical results:Compared with the normal saline+capsaicin group,the expression of NKA in the lung tissues of guinea pigs in the bradykinin+capsaicin group was increased(p<0.01),but the expression in the trachea tissues of the two groups showed no statistical difference.2.4 The expression of CGRP in cough hypersensitivity syndrome induced by bradykinin:(1)ELISA results:Compared with the normal saline+capsaicin group,the expression of CGRP in blood of guinea pigs in bradykinin+capsaicin group was increased(p<0.01),and no increased expression was found in lung tissue or bronchoalveolar lavage solution.Immunohistochemical results:CGRP expression in trachea and lung tissues of guinea pigs in bradykinin+capsaicin group was not increased compared with that in normal saline+capsaicin group.2.5 The expression of SP in cough hypersensitivity syndrome induced by bradykinin:(1)ELISA results:Compared with the normal saline+capsaicin group,the expression of SP in bronchoalveolar lavage solution of guinea pigs in bradykinin+capsaicin group was increased(P<0.05),and no increase was found in lung tissue or blood.Immunohistochemical results:Compared with normal saline+capsaicin group,the expression of SP in trachea of guinea pigs in bradykinin+capsaicin group was increased(p<0.01),but the expression in lung tissue was not increased.2.6 The trachea and lung tissue pathology observation:The trachea and lung tissue pathology observation:Pathological changes of both lung and tracheal tissue showed mild to moderate inflammatory changes with HE staining:The lining mucosal epithelium of trachea was slightly disordered.There was moderate infiltration of mixed inflammatory cells in the submucosal stroma,among which eosinophils were easy to be seen,submucosal edema and small blood vessels dilated and silted up.Alveolar dilatation and fusion,inflammatory secretions in alveolar cavity,loose alveolar septum and edema,partial capillary dilation and congestion were found in lung tissues.But there was no significant difference between the groups.ConclusionsBradykinin can induce not only cough reflex but also cough hypersensitivity syndrome,both of which are dependent on the TRPV1-neuropeptidesmechanism.The model of cough hypersensitivity syndrome induced by bradykinin showed increased expression of TRPV1 and neuropeptides,which had the pathophysiological characteristics of patients with cough hypersensitivity syndrome,and the model of cough hypersensitivity syndrome was successfully established.Part?: Evaluation of the Therapeutic Effects of Compounds that Regulate TRPV1 Sensitivity in Patients with Cough Hypersensitivity SyndromeBackgroundClinical studies have found that in patients with chronic cough,the expression of TRPV1 and coughsensitivity to the stimulation of capsaicin increased,and there was a correlation between them.Capsaicin induces cough by specifically activating TRPV1.TRPV1,known as transient receptor potential vanniloid receptor 1(TRPV1),is the most important cough receptor discovered in recent years.Currently,many researchers have begun to develop TRPV1 antagonists for the treatment of chronic cough,some of which have been proved effective in animal experiments,and some have entered clinical trials.The main disadvantages of these drugs are hyperthermia,impaired thermal sensation,increased risk of skin cancer,and a lack of stable pharmacokinetics and pharmacodynamics.Therefore,the treatment of chronic cough needs to find a new breakthrough.This study attempts to explore whether B2 receptor antagonists,PKC antagonists,intracellular calcium ion chelating agents,menthol that can regulate the sensitivity of TRPV1 have the effect of relieving cough.MethodsThe study was conducted in two steps with male Hartly guinea pigs.In the first step,30 guinea pigs were randomly divided into 6 groups as follows:(1)bradykinin group;(2)B2 receptor antagonists+bradykinin group;(3)PKC antagonists+bradykinin group;(4)calcium ion chelating agents+bradykinin group;(5)TRPV1 receptor antagonists+bradykinin group;(6)menthol+bradykinin group.In addition to the blank control group,each group was given different antagonist intervention in advance,and bradykinin atomization was given 1 hour later,and the number of coughs within 10 minutes after bradykinin atomization was counted.The other 30 guinea pigs were randomly divided into 6 groups as follows:(1)bradykinin+capsaicin group;(2)B2 receptor antagonists+bradykinin+capsaicin group;(3)PKC antagonists+bradykinin+capsaicin group;(4)calcium ion chelating agents+bradykinin+capsaicin group;(5)TRPV1 receptor antagonists+bradykinin+capsaicin group;(6)menthol+bradykinin+capsaicin group.In addition to the blank control group,each group was given different antagonists pre-intervention,nextwas given bradykinin atomization for 10 minutesl hour later,and then was given capsaicin atomization half an hour later,cough times within 10 minutes was counted.The results were expressed as mean standard deviation(X-SD).SPSS 16.0 software was used to analyze all the data,and P<0.05 was used as the judgment standard of difference significance.Results1.The inhibiting effect of TRPV1 sensitivity regulating drugs on cough reflexCough frequency of each intervention group was lower than that of bradykinin group(p<0.01).2.The inhibiting effect of TRPV1 sensitive drugs on increased coughsensitivity.Compared with the bradykinin+capsaicin group,the cough frequency of B2 receptor antagonists+bradykinin+capsaicin group,menthol+bradykinin+capsaicin groupwas lower with p<0.05.And that of PKC antagonists+bradykinin+capsaicin group,calcium ion chelator+bradykinin+capsaicin group and TRPV1 receptor antagonists+bradykinin+capsaicin group was lower with p<0.01.ConclusionsAs TRPV1 antagonists,B2 receptor antagonists,PKC antagonists,intracellular calcium ion chelating agents and menthol that can regulate the sensitivity of TRPV1 all have the effect of inhibiting cough reflex and increased cough sensitivity,so they have therapeutic effect on cough hypersenstivity syndrome.Part?:the Treatment Mechanism of TRPV1 Sensitivity Regulating Drugs for Cough Hypersensitivity SyndromeBackgroundClinical studies have found that TRPV1 expression in airway of patients with chronic cough was increased,and neuropeptides expression level in body fluid was increased.Vitro experiments have shown that TRPV1 is sensitive to inflammatory mediators,which makes it more likely to be activated by various stimuli and induce cough reflex.The activation of TRPV1 can cause the release of neuropeptides such as neuropeptide A(NKA),substance P(SP),calcitonin gene-related peptide(CGRP)from the c-fiber terminal,causing;neurogenic inflammation and constituting one of the mechanisms of cough.In this study,we attempted to investigate whether TRPV1 sensitivity regulating drug B2 receptor antagonists.PKC antagonists,intracellular calcium ion chelating agents and menthol caninhibit the expression of TRPV1 and neuropeptides,so as to clarify its cough mechanism.MethodsThe study was conducted in two steps with male Hartly guinea pigs.In the first step,30 guinea pigs were randomly divided into 6 groups as follows:(1)bradykinin group;(2)B2 receptor antagonists+bradykinin group;(3)PKC antagonists+bradykinin group;(4)calcium ion chelating agents+bradykinin group;(5)TRPV1 receptor antagonists+bradykinin group;(6)menthol+bradykinin group.In addition to the blank control group,each group was given different antagonists pre-intervention,1 hour later,the bradykinin was atomized for 10 minutes.The other 30 guinea pigs were randomly divided into 6 groups as follows:(1)bradykinin+capsaicin group;(2)B2 receptor antagonists+bradykinin+capsaicin group;(3)PKC antagonists+bradykinin+capsaicin group;(4)calcium ion chelating agents+bradykinin+capsaicin group;(5)TRPV1 receptor antagonists+bradykinin+capsaicin group;(6)menthol+bradykinin+capsaicin aroup.In addition to the blank control group,each group was given different antagonists pre-intervention,bradykinin atomization was given for 10 minutesl hour later,capsaicin atomization was given for 10 minutes half an hour later.30 minutes later,samples were collected and the expression levels of TRPV1,NKA,CGRP,SP in serum,bronchoalveolar lavage fluid and lung tissue homogenate were detected by ELISA.The expression levels of TRPV1,NKA,CGRP,SP in trachea and lung tissues were detected by immunohistochemistry.The results were expressed as mean standard deviation(X±SD).SPSS16.0 software was used to analyze all the data,and P<0.05 was used as the judgment standard of difference significance.Results1.Inhibition of TRPV1 sensitivity regulating drugs on the expression of TRPV1 and neuropeptides in cough reflex.1.1 Inhibition of TRPV1 and neuropeptide expression in cough reflex by B2 receptor antagonists.(1)Compared with bradykinin group,the expression of TRPV1 in bronchoalveolar lavage solution in guinea pigs(p<0.05)ofB2 receptor antagonist+bradykinin group was reduced,while the expression in lung tissue was not decreased.(2)Compared with the bradykinin group,the expression of NKA in the lung tissue of guinea pigs in the B2 receptor antagonists+bradykinin group was decreased:The results of ELISA(p<0.05)and immunohistochemistry(p<0.05)showed the same trend,and the expression in the blood was also decreased(p<0.05).1.2 PKC antagonists inhibited the expression of TRPV1 and neuropeptides in cough reflex.(1)Compared with the bradykinin group,the expression of TRPV1 in the lung tissue of the PKC antagonists+bradykinin group was decreased(p<0.05),and the results of ELISA(p<0.05)and immunohistochemistry(p<0.01)showed the same trend,but the expression was not decreased in the bronchoalveolar lavage fluid.(2)Compared with the bradykinin group,the expression of NKA in the lung tissues of guinea pigs in the PKC antagonists+bradykinin group was decreased,and the results of ELISA(p<0.01)and immunohistochemistry(p<0.01)showed the same trend,and the expression in the blood was also decreased(p<0.01).1.3 Inhibition of TRPV1 and neuropeptides expression in cough reflex by calcium ion chelator.(1)Compared with the bradykinin group,the expression of TRPV1 in bronchoalveolar lavage fluid in the calcium ion chelator+bradykinin group was decreased(p<0.05),while the expression in lung tissue was not decreased.(2)Compared with the bradykinin group,the expression of NKA in blood of guinea pigs in the calcium ion chelator+bradykinin group was decreased(p<0.01),and the expression in lung tissue was not decreased.(3)Compared with the bradykinin group,the expression of CGRP in tracheal tissue(p<0.05)and BALF(p<0.05)in the calcium ion chelator+bradykinin group was decreased.1.4 Inhibition of TRPV1 receptor antagonists on TRPV1 and neuropeptides expression in cough reflex.(1)Compared with the bradykinin group,the expression of TRPV1 in bronchoalveolar lavage fluid of guinea pigs(p<0.01)in TRPV1 receptor antagonists+bradykinin group was reduced,while no reduction was found in the expression of lung tissue.(2)Compared with the bradykinin group,the expression of NKA in the lung tissue of guinea pigs in the TRPV1 antagonists+bradykinin group was decreased,and the results of ELISA(p<0.01)and immunohistochemistry(p<0.01)showed the same trend,and the expression in the blood was also decreased(p<0.01).1.5 The inhibitory effect of menthol on TRPV1 and neuropeptides expression in cough reflex.(1)compared with the bradykinin group,TRPV1 receptor antagonists+bradykinin group reduced the expression of TRPV1 in bronchoalveolar lavage fluid of guinea pigs(p<0.05),while no reduction was found in the expression of lung tissue.(2)compared with the bradykinin group,the expression of NKA in the lung tissue of guinea pigs in the TRPV1 antagonists+bradykinin group was decreased,and the results of ELISA experiment(p<0.01)and immunohistochemical experiment(p<0.05)showed the same trend,and the expression in the blood was also decreased(p<0.01).2.The inhibitory effect of TRPV1 sensitivity regulating drugs on the expression ofTRPV1 and neuropeptides in cough hypersensitivity syndrome.Compared with bradykinin+capsaicin group,TRPV1 expression was not decreased in guinea pigs of each group.2.1 Inhibition of neuropeptides expression in cough hypersensitivity syndrome by B2 receptor antagonists.(1)Compared with the bradykinin+capsaicin group,the expression of NKA in the lung tissue of guinea pigs in the B2 receptor antagonists+bradykinin+capsaicin group was decreased,and the results of ELISA(p<0.05)and immunohistochemistry(p<0.05)showed the same trend.No decrease in NKA expression was found in the blood.(2)Compared with bradykinin+capsaicin group,the expression of CGRP in blood of guinea pigs(p<0.01)in B2 receptor antagonists+bradykinin+capsaicin group wasreduced.(3)Compared with bradykinin+capsaicin group,the expression of SP in trachea(p<0.05)and bronchoalveolar lavage fluid(p<0.05)in B2 receptor antagonists+bradykinin+capsaicin group wasreduced.2.2 The inhibitory effect of PKC antagonists on the expression of neuropeptides in cough hypersensitivity syndrome.(1)Compared with the bradykinin+capsaicin group,the expression of NKA in the lung tissue of the PKC antagonists+bradykinin+capsaicin group was decreased,and the results of ELISA experiment(p<0.05)and immunohistochemical experiment(p<0.01)showed the same trend,and the expression in the blood was also decreased(p<0.01).(2)Compared with bradykinin+capsaicin group,the expression of CGRP in blood of guinea pigs(p<0.01)in PKC antagonists+bradykinin+capsaicin group wasreduced.(3)Compared with the bradykinin+capsaicin group,the expression of SP in trachea(p<0.05)and bronchoalveolar lavage fluid(p<0.01)in the PKC antagonists+bradykinin+capsaicin group was decreased.2.3 Inhibitory effect of calcium ion chelating agents on the expression of neuropeptides in cough hypersensitivity syndrome.(1)Compared with the bradykinin+capsaicin group,the expression of NKA in blood of guinea pigs in the calcium ion chelator+bradykinin+capsaicin group was decreased(p<0.01),and the expression in lung tissue was not decreased.(2)Compared with the bradykinin+capsaicin group,the expression of CGRP in blood of guinea pigs in the calcium ion chelator+bradykinin+capsaicin group was decreased(p<0.01).2.4 Inhibition of neuropeptide expression in cough hypersensitivity syndrome by TRPV1 antagonists.(1)Compared with the bradykinin+capsaicin group,the expression of CGRP in the blood of guinea pigs(p<0.01)inthe TRPV1 antagonists+bradykinin+capsaicin group wasreduced.(2)Compared with the bradykinin+capsaicin group,the expression of SP in trachea(p<0.05)and bronchoalveolar lavage fluid(p<0.01)ofthe TRPV1 antagonists+bradykinin+capsaicin group wasreduced.2.5 Inhibitory effect of menthol on neuropeptides expression in cough hypersensitivity syndrome.(1)Compared with the bradykinin+capsaicin group,the expression of NKA in the lung tissue of guinea pigs in the menthol+bradykinin+capsaicin group was decreased.The results of ELISA experiment(p<0.05)and immunohistochemical experiment(p<0.05)showed the same trend,and the expression in the blood was also decreased(p<0.01).(2)Compared with the bradykinin+capsaicin group,the expression of CGRP in blood of guinea pigs in the menthol+bradykinin+capsaicin group was decreased(p<0.01).ConclusionsNot only TRPV1 antagonists,TRPV1 sensitivity regulating drugs such as BK2 receptor antagonists,PKC antagonists,intracellular calcium ion chelating agents and menthol all inhibited the expression of neuropeptides in cough reflex andcough hypersensitivity syndrome to varying degrees and in a diverse way,which is one of its therapeutic mechanisms. |
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