小青龙汤对β_(2)肾上腺素能受体减敏哮喘小鼠RhoGDI_(2)/GRK_(2)/β-arrestin信号传导的影响

Effect of Xiaoqinglong Decoction(小青龙汤)on RhoGDI_(2)/GRK_(2)/β-arrestin Signal Transduction in β_(2)-AR Desensitized Asthmatic Mice

目的探讨小青龙汤对β_(2)肾上腺素能受体(β_(2)-AR)减敏哮喘小鼠的可能作用机制。方法30只SPF级雌性BALB/c小鼠随机分为空白组、模型组、小青龙汤组(中药组)、地塞米松组及小青龙汤加地塞米松组(中药加地塞米松组),每组6只。除空白对照组外,其余各组小鼠通过用卵蛋白(OVA)致敏激发及沙丁胺醇反复刺激来进行造模。造模后自激发第1天起,小青龙汤组每天灌服小青龙汤0.76 g/100 g,地塞米松组每天以腹腔注射地塞米松0.07 mg/100 g,小青龙汤加地塞米松组每天灌服小青龙汤及腹腔注射地塞米松,剂量同前,连续7 d。末次给予OVA激发后24 h,采用EMK动物肺功能测量系统监测各组小鼠的气道阻力,苏木素-伊红(HE)染色法观察小鼠肺组织病理情况,逆转录PCR(RT-PCR)分别检测肺组织中β_(2)-AR、Rho鸟苷酸解离抑制因子2(RhoGDI_(2))、β-AR激酶(GRK_(2))、β-抑制蛋白(β-arrestin)的mRNA表达,Western blot测定肺组织中β_(2)-AR、RhoGDI_(2)、GRK_(2)、β-arrestin含量。结果病理组织学观察发现β_(2)-AR减敏哮喘小鼠气道炎症浸润,各级支气管管壁显著增厚,管道狭窄,且较空白组的病理表现明显加重,经给药后均有不同减轻,以小青龙汤加地塞米松组最优;小鼠气道阻力测定显示随着乙酰甲胆碱(Mch)给药浓度的增加,模型组气道阻力较空白组逐渐增加,给药后各组均有下降趋势,以中药加地塞米松组下降最为明显(P<0.05);肺组织中β_(2)-ARmRNA及β_(2)-AR的表达明显下降,经药物干预后两者均有不同程度的上升,其中肺组织中β_(2)-ARmRNA的表达以小青龙汤加地塞米松组最优,而小青龙汤与地塞米松组之间差异无统计学意义;经造模后与空白组相比,小鼠肺组织中RhoGDI_(2)、GRK_(2)、β-arrestin及它们的mRNA的表达均有不同程度的增强(P<0.05),经药物干预后与模型组相比,小青龙汤组、地塞米松组及小青龙汤加地塞米松组中RhoGDI_(2)、GRK_(2)、β-arrestin及它们的mRNA的表达均下降(P<0.05),且小青龙汤组与地塞米松组之间无明显差异(P>0.05)。结论小青龙汤对β_(2)-AR减敏哮喘小鼠的作用机制可能通过影响肺组织β_(2)-AR的表达及RhoGDI_(2)/GRK_(2)/β-arrestin信号传导来实现,且效果与地塞米松相当。

Objective To explore the possible mechanism of Xiaoqinglong Decoction(小青龙汤)on β_(2)adrenergic receptor(β_(2)-AR)sensitized asthmatic mice.Methods Thirty SPF female BALB/c mice were randomly divided into blank group,model group,Xiaoqinglong Decoction group,dexamethasone group and Xiaoqinglong Decoction plus dexamethasone group,with 6 mice in each group.In addition to the blank control group,mice in other groups were sensitized with ovalbumin(OVA)and repeatedly stimulated with salbutamol.From the first day after stimulation,Xiaoqinglong Decoction group was given Xiaoqinglong Decoction 0.76 g/(100 g·d)every day,dexamethasone group was given dexamethasone 0.07 mg/(100 g·d)intraperitoneally,and Xiaoqinglong Decoction plus dexamethasone group was given Xiaoqinglong Decoction and dexamethasone intraperitoneally every day for 7 consecutive days.24 hours after the last OVA stimulate,the airway resistance of mice in each group was monitored by EMK animal lung function measurement system,the pathological injury of lung tissue was observed by Hematoxylin and Eosin(HE)staining,the mRNA expressions of β_(2)-AR,Rho guanosine acid dissociation inhibitor 2(RhoGDI_(2)),β-AR kinase(GRK_(2))and β-inhibitory protein(β-arrestin)in lung tissue were detected by Reverse transcription PCR(RT-PCR),and the contents of β_(2)-AR,RhoGDI_(2),GRK_(2)and β-arrestin content in lung tissue were detected by Western blot.Results Histopathological observation showed that airway inflammation infiltration,bronchial wall thickening and tube narrowing were significantly increased in β_(2)-AR desensitized asthmatic mice,and the pathological manifestations were significantly worse than those in the blank group,which were alleviated in different ways after administration,and Xiaoqinglong Decoction plus dexamethasone group was the best.The measurement of airway resistance in mice showed that with the increase of Methacholine(Mch)concentration,the airway resistance in the model group gradually increased compared with that in the blank group,and decreased in all groups after administration,especially in the Xiaoqinglong Decoction plus dexamethasone group(P<0.05).The expressions of β_(2)-ARmRNA and β_(2)-AR in lung tissue decreased significantly,and both increased in varying degrees after drug intervention.The expression of β_(2)-ARmRNA in lung tissue was the best in Xiaoqinglong Decoction plus dexamethasone group,but there was no significant difference between Xiaoqinglong Decoction and dexamethasone group.After modeling,compared with those of the blank group,the expressions of RhoGDI_(2),GRK_(2),β-arrestin and their mRNA in lung tissue of the mice were increased in varying degrees(P<0.05).After drug intervention,compared with those of the model group,the expressions of RhoGDI_(2),GRK_(2),β-arrestin and their mRNA in Xiaoqinglong Decoction group,dexamethasone group and Xiaoqinglong Decoction plus dexamethasone group decreased(P<0.05),and there was no significant difference between Xiaoqinglong Decoction group and dexamethasone group(P>0.05).Conclusion The mechanism of Xiaoqinglong Decoction on β_(2)-AR desensitized asthmatic mice may be realized by affecting the expressions of lung β_(2)-AR and RhoGDI_(2)/GRK_(2)/β-arrestin signal transduction,and the effect is equivalent to dexamethasone.