| BackgroundInfluenza,COVID-19 and other respiratory infectious diseases are outbreaks frequently and seriously endanger human health,becoming a major threat and challenge in the field of public health in China and the world.Influenza prevention is mainly based on vaccines,but the prevention and control effect is seriously affected by faster virus mutation,lower vaccination rate and more side effects of oral drug prevention.Therefore,research and development of various safe and effective preventive measures,enriching the means of prevention and control,and improving the ability of influenza prevention and control will become the focus of future influenza prevention.Chinese medicine has a long history of intranasal prevention of respiratory infectious diseases,with various methods,high public acceptance and relative safety.Xiangzhu Fanggan formula by nasal administration(hereinafter referred to as "Xiangzhu Fanggan formula")is a Chinese medicine compound formula(National Invention Patent CN202110132855.X)developed and optimized for intranasal use after systematically combing ancient and modern external anti-infection prescriptions and establishing a database of influenza prevention and treatment prescriptions(2019SR1114775).Clinical pre-test(registration number:ChiCTR1800014674)confirmed that the drug has the effect of preventing and relieving influenza-like symptoms through intranasal sniffing.In order to further clarify the intranasal prophylactic and immunomodulatory effects of Xiangjiao Anti-Influenza Formula,this study was conducted to select Influenza A virus A/PR/8/3 strain(H1N1)and use two intranasal administration methods,namely non-direct contact inhalation by aromatic bursa and direct contact inhalation by nasal drip,and set up two positive methods,namely zanamivir,which is mainly antiviral,and yupingfeng,which is mainly immunomodulatory.Two positive control groups,zanamivir,which is the main antiviral agent,and yupingfeng,which is the main immune regulator,were administered to normal mice before influenza infection and model mice after virus infection.The preventive effects and immune mechanisms of the nasal anti-infection formula were investigated by using HE,Western Blot(WB),Polymerase Chain Reaction(PCR)and Enzyme Linked Immunosorbent Assay(ELISA),in order to provide reference for influenza prevention and related research.ObjectiveTo observe the preventive effects and related immunological mechanisms of the intranasal administration of Xiangzhu Fanggan formula in normal mice before infection with influenza A/PR/8/3 strain and in mice after infection with influenza A/PR/8/3 strain,and to clarify the effects and related immunological mechanisms of different intranasal administration methods.Methods1.Effect of Xiangzhu Fanggan formula on normal miceBALB/c mice were randomly divided into seven groups:normal group,zanamivir group,yupingfeng group,aromatic formula high and low dose group,nasal drip high and low concentration group,8 mice in each group,half of each sex.All groups,except the normal group,were given prophylactically for 5 consecutive days and were dissected and taken on the morning of the 6th day.During the prophylactic administration,the general conditions of mice such as mental,hair,movement and body mass were observed by using the general symptom scale.The lung tissues were stained with HE to observe the pathological changes;the expression level of immunoglobulins A(IgA)in lung tissues was measured by Western Bolt method;the upper respiratory lavage fluid was measured by Enzyme Linked Immunosorbent Assay(ELISA).The expression and secretion of interleukin-6(IL-6),interleukin-2(IL-2),and interleukin-6(IL-6).2.Effects of Xiangzhu Fanggan formula on influenza A miceBALB/c mice were randomly divided into eight groups:normal group,model group,zanamivir group,yupingfeng group,aromatic anti-infection formula high and low dose group,nasal drip high and low concentration group,8 mice in each group,half of each sex.All groups except the normal and model groups were administered prophylactically for 5 consecutive days;on the morning of day 6,mice in each group except the normal group were infected with 15LD50 influenza virus strain APR8 viral solution in nasal drops at 35μl/each.The general conditions of mice such as mental,hair,movement and body mass were observed using the general symptom scale for 5 consecutive days after infection.The mice were dissected and sampled in the morning of the 10th day,and the lung tissues were observed by HE staining to do pathological changes;the viral load in lung tissues was detected by PCR method;the IgA expression level in lung tissues was detected by WB method;the SIgA in upper respiratory lavage fluid,and the expression and secretion of TNF-α,IFN-λ,IL-2 and L-6 in serum were detected by ELISA method.Results1.Effects of Xiangzhu Fanggan formula on the body mass and general symptoms of normal and influenza A model miceEffects on normal mice:(1)Body mass:compared with the normal group,there was no significant change in body mass in the high and low dose groups of Xiangjiao,and the difference was not statistically significant;the body mass in the zanamivir,yupingfeng and nasal drip high and low concentration groups was significantly reduced,and the difference was statistically significant(P<0.05);compared with the zanamivir group,there was no significant change in body mass in the high and low dose groups of Xiangjiao and nasal drip high and low concentration groups,and the difference was not statistically The difference was not statistically significant.(2)General symptom scores:compared with the normal group,the general symptom scores of zanamivir group and yupingfeng group,the high and low dose groups of the scented capsule and the high and low concentration groups of the nasal drip did not change significantly,and the difference was not statistically significant;compared with the zanamivir group,the high and low dose groups of the scented capsule and the high and low concentration groups of the nasal drip did not change significantly,and the difference was not statistically significant.Effects on influenza A model mice:(1)Body mass:compared with the normal group,the body mass of the model group was significantly reduced,and the difference was statistically significant(P<0.001);compared with the model group,the body mass of the zanamivir group and the nasal drip high concentration group was significantly increased,and the difference was statistically significant(P<0.05).Compared with the zanamivir group,the body mass in the zanamivir low-dose group and the nasal drip low-concentration group was significantly reduced,and the difference was statistically significant(P<0.05).(2)General symptom scores:compared with the normal group,the general symptom scores in the model group were significantly higher,and the difference was statistically significant(P<0.001);compared with the model group,the general symptom scores in the zanamivir group,the scented capsule high and low dose group and the nasal drip low concentration group were significantly lower,and the difference was statistically significant(P<0.05);compared with the zanamivir group,the scented capsule high and low dose group and the nasal drip high and low concentration group had significantly higher general The difference was statistically significant(P<0.0001).2.Effects of Xiangzhu Fanggan formula on infection-related indexes of normal and influenza A model miceEffect on normal mice:(1)Pulmonary index:compared with the normal group,the lung index of the nasal drip high and low concentration group was significantly higher,and the difference was statistically significant(P<0.01);compared with the zanamivir group,there was no significant change in the lung index of the incense bursa high and low dose group and the nasal drip high and low concentration group,and the difference was not statistically significant.(2)Physiological structure of lung tissues:compared with the normal group,there was no significant change in the physiological structure of lung tissues in all groups;compared with the zanamivir group,there was no significant change in the physiological structure of lung tissues in the high and low dose groups of the balm and the high and low concentration groups of the nasal drip.Effects on influenza A mice:(1)Pulmonary index:compared with the normal group,the lung index of the model group was significantly higher,and the difference was statistically significant(P<0.001);compared with the model group,the lung index of mice in the high-dose group of the balm capsule was significantly lower,and the difference was statistically significant(P<0.001);compared with the zanamivir group,the lung index of mice in the high-dose group of the balm capsule was significantly higher,and the difference was statistically significant(P<0.001).(2)Pulmonary viral load:compared with the normal group,the viral load of lung tissue in the model group was significantly higher,and the difference was statistically significant(P<0.01);compared with the model group,the pulmonary viral load in the low-dose group of the balm capsule was significantly lower,and the difference was statistically significant(P<0.05);compared with the zanamivir group,the pulmonary viral load in the high and low-dose groups of the balm capsule and the high and low concentration groups of the nasal drip did not change significantly,and the difference was not statistically significant.The difference was not statistically significant.(3)Lung histopathological changes:compared with the normal group,the model group produced severe lung inflammation and caused pathological damage;compared with the model group,lung histopathological changes were significantly reduced in the fragrance capsule low-dose group and the nasal drip high-low concentration group;compared with the zanamivir group,there were no significant changes in lung histopathology in the fragrance capsule high-low dose group and the nasal drip high-low concentration group.Effect on lung index before and after influenza A infection:compared with the pre-infection groups,the lung index of the model group,the yupingfeng group,the fragrant capsule high and low dose group and the nasal drip high and low concentration group were significantly higher after virus infection,and the difference was statistically significant(P<0.01).Compared with the difference in lung index before and after infection in the model group,the difference in lung index in the zanamivir group,the yupingfeng group,the shamrock high and low dose group and the nasal drip high concentration group was significantly lower,and the difference was statistically significant(P<0.05);compared with the difference in lung index before and after infection in the zanamivir group,the difference in lung index in the shamrock high and low dose group and the nasal drip low concentration group was significantly higher,and the difference was statistically significant(P<0.01).3.Effects of Xiangzhu Fanggan formula on immune-related indexes of normal and influenza A model miceEffects on normal mice:(1)SIgA of upper respiratory lavage fluid:compared with normal group,SIgA of upper respiratory lavage fluid in zanamvir group,yupingfeng group,incense bursa high dose group and nasal drip high and low concentration group was significantly reduced,and the difference was statistically significant(P<0.05);compared with yupingfeng group,SIgA of upper respiratory lavage fluid in incense bursa high and low dose group and nasal drip high and low concentration group was not significantly changed,and the difference was not statistically significant.The difference was not statistically significant.(2)IgA in lung tissue:compared with the normal group,the expression of IgA in lung tissue of mice in the Yu Ping Feng group,the high and low dose groups of Xiang Cai and the low dose group of nasal drip was significantly higher,and the difference was statistically significant(P<0.001);compared with the Yu Ping Feng group,the expression of IgA in lung tissue of the high dose group of Xiang Cai was significantly higher,and the difference was statistically significant(P<0.001).(3)Serum IFN-λ:compared with the normal group,serum IFN-λ was significantly elevated in the zanamivir group,the yupingfeng group,the shamrock high and low dose group and the nasal drip high and low concentration group,and the difference was statistically significant(P<0.001);compared with the yupingfeng group,serum IFN-λ secretion was significantly elevated in the shamrock high and low dose group and the nasal drip high and low concentration group,and the difference was statistically significant(P<0.001).Effects on influenza A mice:(1)SIgA of upper respiratory lavage fluid:compared with the normal group,SIgA of upper respiratory lavage fluid was significantly lower in the model group,and the difference was statistically significant(P<0.001);compared with the model group,SIgA secretion of upper respiratory lavage fluid was significantly higher in the zanamivir group,yupingfeng group,scented capsule low-dose group and nasal drip high-low concentration group,and the difference was statistically significant(P<0.01);compared with the yupingfeng group,there was no significant change in SIgA in the upper respiratory tract lavage fluid of the fragrant bursa high and low dose group and the nasal drip high and low concentration group,and the difference was not statistically significant.(2)Lung tissue IgA:compared with the normal group,lung tissue IgA was significantly lower in the model group,and the difference was statistically significant(P<0.001);compared with the model group,lung tissue IgA expression was significantly higher in the zanamivir group,the yupingfeng group,the fragrant bursa high and low dose group and the nasal drip high and low concentration group,and the difference was statistically significant(P<0.001);compared with the yupingfeng group,the fragrant bursa high and low dose group and the The difference was statistically significant(P<0.01).(3)Serum IFN-λ:compared with the normal group,serum IFN-λ was significantly increased in the model group,and the difference was statistically significant(P<0.001);compared with the model group,serum IFN-λ secretion was significantly decreased in the zanamivir,yupingfeng,shamrock high and low dose and nasal drip high and low concentration groups,and the difference was statistically significant(P<0.01);compared with the yupingfeng group,shamrock high and low dose and nasal drip high and low concentration groups,and the difference was statistically significant(P<0.01).The difference was statistically significant(P<0.001).Effects on immune-related indexes of mice before and after influenza A infection:(1)SIgA of upper respiratory lavage fluid:compared with each group before infection,SIgA of upper respiratory lavage fluid was significantly lower in the model group after virus infection,and the difference was statistically significant(P<0.001),and SIgA of upper respiratory lavage fluid was significantly higher in the nasal drip high concentration group,and the difference was statistically significant(P<0.001);compared with The difference in SIgA of upper respiratory lavage fluid before and after infection in the model group was significantly higher in the zanamivir group,the yupingfeng group,the shamrock high-dose group and the nasal drip high and low concentration group in terms of lung index,and the difference was statistically significant(P<0.05).(2)Lung tissue IgA:compared with the pre-infection groups,after viral infection,the lung tissue IgA levels in the model group,the yupingfeng group and the shamrock low-dose group were significantly lower,and the difference was statistically significant(P<0.05),while the lung tissue IgA levels in the zanamivir group and the nasal drip high concentration group were significantly higher,and the difference was statistically significant(P<0.01);compared with the difference in lung tissue IgA levels in the model group before and after infection Compared with the difference of lung tissue IgA levels before and after infection in the model group,the difference of lung tissue IgA levels in the zanamivir group,the balm low dose group and the nasal drip high and low concentration groups were significantly higher,and the difference was statistically significant(P<0.01).(3)Serum IFN-λ:compared with the pre-infection groups,the serum IFN-λ,level was significantly higher in the model group after viral infection,and the difference was statistically significant(P<0.001);compared with the difference of serum IFN-λ level before and after infection in the model group,the difference of serum IFN-λ level was significantly lower in the zanamivir group,yupingfeng group,shamrock high and low dose group and nasal drip high and low concentration group,and the difference was statistically significant(P<0.001).4.Effects of Xiangzhu Fanggan formula on inflammatory cytokines in normal and influenza A model miceEffects on normal mice:Serum TNF-α,IL-2,IL-6:compared with the normal group,serum TNF-α,IL-2,IL-6 secretion was significantly higher in the zanamivir group,the yupingfeng group,the incense bursa high and low dose group and the nasal drip high and low concentration group,and the difference was statistically significant(P<0.001);compared with the yupingfeng group,serum TNF-α,IL-2,IL-6 secretion was significantly higher in the incense bursa high and low dose group and the nasal drip high and low concentration group TNF-α and IL-2 secretion were significantly increased in the high and low dose groups of the incense bursa and the low concentration group of the nasal drip,and the difference was statistically significant(P<0.05).Effects on influenza A model mice:Serum TNF-α,IL-2,IL-6:compared with the normal group,serum TNF-α,IL-2,IL-6 were significantly increased in mice of the model group,and the difference was statistically significant(P<0.001);compared with the model group,serum TNF-α,IL-2,IL-6 secretion was significantly decreased in the zanamivir group,yupingfeng group,shamrock high and low dose group and nasal drip high and low concentration group,and the difference was statistically significant(P<0.001).6 secretion was significantly reduced,and the difference was statistically significant(P<0.01).Compared with the Yu Ping Feng group,the secretion of serum TNF-α increased significantly in the nasal drip low concentration group,and the difference was statistically significant(P<0.01),and the secretion of serum IL-2 and IL-6 increased significantly in the high and low dose groups of the Xiang Bao and nasal drip high and low concentration groups,and the difference was statistically significant(P<0.001).Effects on inflammatory cytokines in mice before and after influenza A infection:(1)Serum TNF-α:compared with the groups before infection,the serum TNF-α levels in the model group and zanamivir group were significantly increased after virus infection,and the difference was statistically significant(P<0.05);compared with the difference in serum TNF-α levels in the model group before and after infection,the serum TNF-α levels in the zanamivir group,yupingfeng group,shamrock high and low dose group and nasal drip The difference of serum TNF-αlevels in the high and low concentration groups was significantly lower,and the difference was statistically significant(P<0.001).(2)Serum IL-2:compared with the pre-infection groups,the serum IL-2 levels in the model group,zanamivir group and nasal drip high concentration group increased significantly after the infection,and the difference was statistically significant(P<0.05);compared with the difference in serum IL-2 levels in the model group before and after the infection,the difference in serum IL-2 levels in the zanamivir group,yupingfeng group,shamrock high and low dose group and nasal drip high and low concentration group was significantly lower,and the difference was statistically significant(P<0.001).(3)Serum IL-6:compared with the pre-infection groups,the serum IL-6 levels in the model group,zanamivir group and nasal drip high concentration group increased significantly after the infection,and the difference was statistically significant(P<0.05);compared with the difference of serum IL-6 levels in the model group before and after the infection,the difference of serum IL-6 levels in the zanamivir group,yupingfeng group,shamrock high and low dose group and nasal drip high and low concentration group significantly decreased,and the difference was statistically significant(P<0.001).Conclusion1.Xiangzhu Fanggan formula has certain preventive effects on influenza,mainly for inhibiting influenza virus replication and alleviating lung lesions.2.The possible mechanism of action of Xiangzhu Fanggan formula in preventing influenza:regulating the process of related adaptive immunity and inflammatory response by upregulating IgA,SIgA and IFN-λ,and regulating pro-inflammatory and chemotactic cytokines such as TNF-α,IL-2 and IL-6.3.Compared with direct contact nasal drip inhalation,non-direct contact balloon inhalation and sniffing has less effect on the general condition and body mass of normal mice,and has better effect on inhibiting influenza virus replication,suggesting that non-contact transnasal inhalation method has better preventive effect and safety,and can be further developed. |
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