Effects Of Ethanol Extract Of Usnea Longissima On Tracheal Smooth Muscle Contraction And Airway Inflammation In Asthmatic Mice

Asthma is a refractory respiratory system disease,which seriously affects people’s healthy and lives.Relieving tracheospasm and chronic inflammation of the airways is one of the main ways to treat asthma.At present,the main antispasmodic drugs in treating asthma are belong to beta-receptor agonists,and the long-time using these drugs is easy to lead to drug resistance and side effects.The traditional Chinese herbal medicines have the characteristics of mild effects,low toxicity,small side effects,low cost,and with comprehensive treating effect.It is an effective way to screen effective ingredients for treating asthma and other respiratory diseases from traditional Chinese herbal medicines that can be used to control asthma or inflammation.It is reported that Usnea longissima has the effectives of liver-clearing,phlegm-resolving,bleeding-stopping and disintoxication.Modern pharmacological studies also show that Usnea longissima is rich in usnic acid,which has a polychlorinated dibenzofurans structure.The pharmacological activities of usnic acid are include anti-inflammatory,anti-bacterial,anti-tumor,anti-virus and anti-tuberculosis.However,it has not been reported on the effects of Usnea longissima on respiratory diseases,especially on the antispasmodic and anti-inflammatory effects of asthma.Therefore,the relaxation effects of alcohol extracts of Usnea longissima（EUL）and usnic acid,one of its main components,on resting state and precontracted with 80mmol·L^-1KCl and 100μmol·L ACh respectivelyisolated from mouse airway,were studied by BL-420F bioassay system.By using the selective blockers Nifedipine,pyrazole 3（Pyr3）and Gadolinium of L-VDCCs,TRPC3 and STIM/Orai channels,the effects of EUL on respiratory resistance（Rrs）in vivo were detected by pulmonary function instrument to investigate the mechanism of inhibiting the contraction of tracheal smooth muscle.The effects of EUL and usnic acid on airway inflammation in asthmatic mice were studied by establishing asthma model in mice.The findings are as follows:1.EUL and usnic acid inhibited airway smooth muscle（ASM）contraction induced by 80 mmol·L^-1K⁺and 100μmol·L^-1ACh in a dose-dependent manner,it was inhibited completely at 0.032 mg·mL^-1of EUL and 0.003 mg·mL^-1of usnic acid,but had no effect on ASM contraction at rest.After Pyr3 and Gadolinium partially inhibited 100μmol·L^-1ACh-induced airway contraction,EUL and usnic acid could further relax the airway.The results showed that NSCCs also influenced the relaxation of precontracted tracheal smooth muscle induced by EUL and usnic acid.2.In the ACh-activated calcium-activated potassium channel,the addition of10m TEA inhibited the opening of KCa and induced the further contraction of trachea.0.032 mg·mL^-1EUL and 0.003 mg·mL^-1usnic acid still showed open action on ACh-induced.In calcium-activated BK_Ca,EUL also showed ability in relaxation of tracheal smooth muscle.0.032 mg·mL^-1EUL and 0.003 mg·mL^-1usnic acid significantly induced BK_Cachannel opening,and relaxed Paxiline-induced tracheal contractions.3.The results of lung pathological section showed that EUL and usnic acid could obviously attenuate inflammatory cell infiltration and airway remodeling in asthma mice.4.The results of eosinophil counts in bronchoalveolar lavage fluid and the detection of inflammatory factors showed that EUL and usnic acid could slow down the increase of eosinophil levels in bronchoalveolar lavage fluid of asthmatic mice and significantly decrease i NOS,TNF-α,proinflammatory factors（IL-4,IL-5,IL-13,NF-κB）and Muc5ac levels in lung tissue.5.At the living level,EUL and usnic acid could significantly reduce certain dose of ACh(6.25,12.5,25,50 mg·mL^-1)causing an increase in Rrs in normal mice and asthmatic mice.6.EUL and usnic acid had no significant tissue toxicity on tracheal smooth muscle tissue and no signal cytotoxicity on the proliferation of 16 HBE cells.The above results suggest that EUL and usnic acid may inhibit the extracellular calcium influx and intracellular cap ion releasation by inhibiting L-VDCCs,NSCCs and opening BK_Cachannels to inhibit the contracton of tracheal smooth muscle.Meanwhile,EUL and Usnic acid observably inhibited airway remodeling,inflammatory factor secretion and airway hyperresponsiveness in asthmatic mice.Therefore,Usnea longissima and usnic acid may be a potential new bronchodilator and airway inflammation suppressant in the treatment of asthma.