基于NF-κB对Nav1.7的调控探讨祛风宣肺方治疗难治性慢性咳嗽的机制OA

Objective To investigate the therapeutic effects and mechanisms of Qufeng Xuanfei Formula in guinea pigs with cough hypersensitivity induced by neurogenic inflammation.Methods According to the random number table method,30 guinea pigs were randomly divided into the blank group,model group,Qufeng Xuanfei Formula group,pregabalin group,and voltage-gated sodium channel(Nav)1.7 inhibitor group,with 6 animals per group.Except the blank group,the guinea pigs of the other groups were immunocompromised by intraperitoneal injection of cyclophosphamide(3×10-2 g/kg);2 days later,they were sensitized by intraperitoneal injection of 1 mL normal saline containing 2 mg ovalbumin and 100 mg aluminum hydroxide;3 weeks later,they received booster sensitization by intraperitoneal injection of 1 mL normal saline containing 0.01 mg ovalbumin and 100 mg aluminum hydroxide.The blank group was injected intraperitoneally with the same volume of normal saline.Three weeks after booster sensitization,1%ovalbumin solution was administered by aerosol inhalation for challenge.Successful modeling was defined as cough frequency≥10 times within 3 minutes after challenge and was accompanied by typical coughing behaviors.After successful modeling,the Qufeng Xuanfei Formula group was given Qufeng Xuanfei Formula(5 g/kg)by gavage;the blank group and model group were given an equal volume of normal saline by gavage;the pregabalin group was given pregabalin solution(3×10-2 g/kg)by gavage;the Nav1.7 inhibitor group received aerosol inhalation of Nav1.7 inhibitor PF-05089771 solution(100 μmol/L)for 20 minutes each time;all interventions were administered once daily for 7 days.The general condition of guinea pigs was observed.Cough frequency was measured by capsaicin aerosol stimulation.Serum levels of interleukin(IL)-4,IL-6,IL-1β,and tumor necrosis factor-α(TNF-α)were detected by enzyme-linked immunosorbent assay.The mRNA expressions of Nav1.7,substance P(SP),and nuclear factor-κB(NF-κB)p65 in the dorsal root ganglia were detected by real-time PCR.The protein expressions of Nav1.7,SP,NF-κB p65,and phosphorylated NF-κB p65(p-NF-κB p65)in the dorsal root ganglia were detected by western blotting.Results Compared with the blank group,guinea pigs in the model group exhibited typical behavioral characteristics including increased respiratory rate,frequent coughing,obvious abdominal muscle contraction,forelimb extension,neck extension,and open-mouth breathing.Their general condition was poor,manifested as reduced food and water intake,decreased activity,preference for curling up,listless mental state,and fluffy coat.After capsaicin stimulation,cough frequency increased,serum levels of IL-4,IL-6,IL-1β,and TNF-α increased,and the mRNA expressions of Nav1.7,SP,and NF-κB p65 in dorsal root ganglia,as well as the protein expressions of Nav1.7,SP,p-NF-κB p65,and p-NF-κB p65/NF-κB p65 were all elevated(P<0.05).Compared with the model group,guinea pigs in each treatment group showed decreased spontaneous cough frequency over time,gradual recovery of food and water intake,increased activity,improved mental state,and gradually lustrous coat.Cough frequency induced by capsaicin decreased,serum levels of IL-4,IL-6,IL-1β,and TNF-α decreased,and in the Qufeng Xuanfei Formula group and Nav1.7 inhibitor group,the mRNA expressions of Nav1.7,SP,and NF-κB p65 in the dorsal root ganglia,as well as the protein expressions of Nav1.7,SP,p-NF-κB p65,and p-NF-κB p65/NF-κB p65 were all decreased(P<0.05).Conclusion Qufeng Xuanfei Formula can effectively alleviate cough symptoms in guinea pigs with cough hypersensitivity.Its mechanism may involve inhibiting the activity of the NF-κB signaling pathway in the dorsal root ganglia,thereby down-regulating Nav1.7 expression and alleviating neurogenic inflammation.