| Objective: The previous research found that transgelin-2 is an acupuncture anti-asthmatic target protein.CAT12 can bind to transgelin-2 and can relax airway smooth muscle to some extent.This study will further demonstrate the relaxation function of airway smooth muscle of CAT12 in vivo and in vitro,in order to provide a new approach for target discovery and innovative drug development based on the material base of acupuncture effects.Methods: 1.Effects of CAT12 on tension of rat isolated tracheal spirals in vitro The rat isolated tracheal spirals were fixed in the tension detection system and equilibrated with 1.5 g loads for 1 h.The final concentration of 6.6×10-5 g/m L ACh stimulated the rat isolated tracheal spirals by 73%.When the tension value was stable(1.5 min),NS,ISO(800 ng/m L)and CAT12(800 ng/m L)were added at a final concentration of 800 ng/m L to observe the effect of CAT12 on the tension of rat isolated tracheal spirals.2.Effects of CAT12 on airway resistance of Mch-induced airway hyperresponsiveness mouse model The normal mice were anesthetized and intubated with a small animal pulmonary function monitor.Ns,ISO(10 ?g/kg),CAT12(100 ng/kg)were inhaled directlyinto normal mice.The volume of nebulization was 25 ?L,and the time was 2 min.And then inhaled Mch(3,6,12,24,48 mg/m L).The changes of airway resistance of mice were recorded by a small animal pulmonary function apparatus.3.Effects of CAT12 on airway resistance of OVA-induced asthmatic mouse model Normal mice were intraperitoneally injected with 0.2 m L of OVA and Al(OH)3 mixture.14 days later,25 ?L of 0.4% OVA challenge solution was inhaled to stimulate asthma,and OVA-induced mouse asthma model was established.Inhalation of NS,positive control TB(10 ?g/kg),CAT12(1,10,100 ng/kg)10 min before the challenge,and record changes of mice airway resistance within 50 min after administration.The volume of nebulization was 25 ?L,and the time was 2 mins.4.Effects of CAT12 on airway resistance of HDM-induced asthmatic mouse model Normal mice were intraperitoneally injected with 0.2 m L of HDM and Al(OH)3 mixture.14 days later,the mice were placed in a 40 cm× 30 cm× 30 cm closed atomization box,and HDM was inhaled to stimulate asthma at a concentration of 3 ?g/m L.The asthma model induced by HDM was established with 15 m L volume,2.5 m L/min atomization rate,30 min atomization time and 7 days continuous atomization.On the 21 st day,airway resistance was detected.Nebulized NS,positive control ISO(10 ?g/kg),CAT12(1?10?100 ng/kg),followed by aerosol inhalation 1.5,3,6,12,24 mg/m L Mch challenge.The volume of nebulization was 25 ?L,and the time was 2 min.The changes of airway resistance were recorded by a small animal pulmonary function apparatus.Results: 1.Effects of CAT12 on tension of rat isolated tracheal spirals in vitro The final concentration of 6.6×10-5 g/m L ACh stimulated the rat isolated tracheal spirals by 73%.Compared with the model group,the changes of the tension of the spiral strips in the ISO group was statistically significant within 1-10 minutes(P<0.05),and the sum of the tension of the spiral strips in the ISO group was statistically significant(P<0.05)The results showed that ISO could be used as a positive control drug to relax the tracheal spirals.Compared with the model group,CAT12 Group had significant difference in the tension of the tracheal spiral in the 2-10 min(P<0.05),CAT12 group had significant difference in the sum of the tension of the tracheal spiral in the 1-10 min(P<0.05).The results suggested that CAT12 could relax rat isolated tracheal spirals.2.Effects of CAT12 on airway resistance of Mch-induced mouse airway hyperresponsiveness model The airway resistance of the model group increased with the increase of Mch concentration,and the airway resistance of the model group was significantly higher than that of the NS group(P<0.05).Compared with the model group,the changes of airway resistance in ISO group were statistically significant under the stimulation of 48 mg/m L Mch(P<0.05).Compared with the model group,the changes of airway resistance in CAT12 group were statistically significant under the stimulation of 12,24,48 mg/m L Mch(P<0.05).In conclusion,CAT12 has the effects of reducing airway resistance in Mch-induced mouse airway hyperresponsiveness model.3.Effects of CAT12 on airway resistance of OVA-induced mouse asthma model After OVA stimulation,the Airway Resistance of the model group was significantly increased,which was 229% higher than that of the NS group(P<0.05),suggesting that the asthma model was successfully induced by OVA.The airway resistance of TB group decreased by 31% compared with that in model group(P<0.05).Compared with the model group,1,10 and 100 ng/kg CAT12 decreased airway resistance by 58%,64% and 77% respectively(P<0.05).CAT12 of 100 ng / kg had the strongest effect on Reducing Airway Resistance,2.4 times of that of TB.In conclusion,CAT12 has the effect of reducing airway resistance in OVA-induced mouse asthma model.4.Effects of CAT12 on airway resistance of HDM-induced mouse asthma model After 7-days HDM stimulation,the resistance of mice in the model group increased significantly.Under the stimulation of 24 mg/m L Mch,the airway resistance of the model group was 1267±68%,which increased 2.3 times more than the control group,indicating that the asthma model induced by HDM was successfully established.Compared with the model group,the changes of airway resistance in CAT12 group were statistically significant(P<0.05)under the stimulation of 12 and 24 mg/m L Mch,and those in ISO group were statistically significant(P<0.05)under the stimulation of 24 mg/m L Mch.CAT12 was 20% more effective than ISO in reducing airway resistance in mice.In conclusion,CAT12 has the effect of reducing airway resistance in HDM-induced mouse asthma model.Conclusion: CAT12 has a good effect of relaxing rat isolated tracheal spiral in vitro and can decrease airway resistance in vivo,providing a good experimental basis for target drug discovery based on acupuncture anti-asthmatic effects. |
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