| Objective: To construct a rat model of post-infection cough(PIC)with lung heat and yin deficiency syndrome,to study the therapeutic effect of Xiebai Zengye Decoction on cough sensitivity,airway inflammation and airway hyper reactivity of model rats,and to further test its effect on intestinal flora and short-chain fatty acids,and the ERK signal pathway of model rats,in order to preliminarily clarify the mechanism of Xiebai Zengye decoction in treating PIC and provide scientific basis for its clinical application.Methods:(1)Forty juvenile SD rats were randomly divided into normal group,model group,Xiebai Zengye Decoction group and Juhong Tanke Liquid group,with 10 rats in each group.Except for the normal group,rats were exposed to smoke and given lipopolysaccharide nasal drops and capsaicin atomization to prepare PIC juvenile rat model.After successful modeling,rats in each group were given corresponding drug intervention for 7 days.During the modeling stage,the changes of anal temperature,limb paw heart temperature,precordial area temperature,body weight,fecal water content and drinking water volume of rats in the normal and model groups were dynamically observed.After medication,observe the changes of the above indicators in each group;cough sensitivity was measured;the indexes of lung function measured by animal ventilator include TE,PENH,EEP and RT;detection of cell classification count and TNF-α,IL-1β level in BALF by ELISA;to detect the content of serum cAMP and cGMP,and calculate the cAMP/cGMP value.(2)Forty juvenile SD rats were randomly divided into normal group,model group,Xiebai Zengye Decoction group and Montelukast sodium group,with 10 rats in each group.Except for the normal group,rats were exposed to smoke and given lipopolysaccharide nasal drops and capsaicin atomization to prepare PIC juvenile rat model.After successful modeling,rats in each group were given corresponding drug intervention for 7days.Measurement of airway reactivity index in rats;detection of TNF-α,IL-1β level by ELISA;Detection of IL-4,IL-6 and IL-10 protein expression in rat lung tissue by IHC;HE and PAS staining methods were used to observe the histopathological changes of lung and colon in rats.(3)To detect the expression level of p-MEK1/2,p-ERK1/2,p-C-FOS protein in lung and colon tissues of rats by IHC.Detection of ERK and p-ERK protein content in lung and colon tissues of rats.(4)Analysis of community species information in rat feces by 16 S rRNA and targeted metabolomics.Obtain species composition information and inter-group composition differences through OTU cluster taxonomy analysis,inter-sample alpha and beta diversity index analysis and multi-group comparative analysis.Determination of SCFAs in rat feces by gas chromatography.Finally,the correlation between SCFAs and serum inflammatory factors and sample species was analyzed,and to explore the key flora causing the difference.Results:(1)Compared with the normal group,the body mass of rats in the model group decreased,the water content of feces decreased,the amount of drinking water increased,and the temperature of anus,limbs,paw heart and precordial area increased(P<0.05 or P<0.01);the cough latency,TE and EEP of rats were significantly shortened(P<0.05 or P<0.01),and the number of coughs within 5 minutes,RT and PENH were significantly increased(P<0.05 or P<0.01);neutrophil,macrophage,lymphocyte,eosinophil count and IL-1β,TNF-α in BALF were significantly increased(P<0.05 or P<0.01);the level of cAMP and cAMP/c GMP in serum were also increased(P<0.05 or P<0.01).Compared with the model group,the body mass and fecal water content of rats in the Xiebai Zengye Decoction group increased(P<0.05),and the temperature of anus and limbs and paw heart decreased(P<0.05),the amount of drinking water decreased(P<0.05),cough latency,TE and EEP were significantly prolonged(P<0.05),RT and PENH were decreased,and cough times within 5 minutes were reduced(P<0.05);neutrophil,macrophage,lymphocyte,eosinophil count and IL-1β 、 TNF-α in BALF,the level of serum cAMP and cAMP/c GMP were decreased(P<0.05);only the cough latency,TE and EEP were prolonged,the number of coughs within 5 minutes were decreased,and the number of neutrophils,lymphocytes and TNF-α were reduced in Juhong Tanke Liquid group(P<0.05).(2)Compared with the model group,the airway resistance of rats was significantly reduced(P<0.05),serum TNF-α、IL-8 level were decreased(P<0.05 or P<0.01),the expression of IL-4 and IL-6 protein in lung tissue decreased,while the expression of IL-10 protein increased(P<0.05)in Xiebai Zengye Decoction group.Compared with the model group,inflammatory cell infiltration in and around the bronchus decreased or even disappeared,goblet cell proliferation in the airway mucosa significantly reduced,and lung tissue inflammation score decreased(P<0.05 or P<0.01),the percentage of airway goblet cells decreased(P<0.05)in Xiebai Zengye Decoction group and Montelukast sodium group.Compared with the model group,the destruction of colonic mucosa tissue was reduced,the crypt structure was restored,the infiltration of inflammatory cells was reduced,and the number of colonic goblet cells was increased in Xiebai Zengye Decoction group(P<0.05),no significant changes were found in Montelukast sodium group.(3)Xiebai Zengye Decoction can down-regulate the expression of ERK signal pathway related proteins in lung and colon tissues.Compared with the model group,the expression of p-MEK1/2,p-ERK1/2 and p-C-FOS protein in rat lung tissue were decreased in Xiebai Zengye Decoction group and Montelukast sodium group(P<0.05 or P<0.01);the expression of p-ERK1/2 and p-C-FOS protein in colon tissue of Xiebai Zengye decoction group decreased(P<0.05 or P<0.01),but the change of p-MEK1/2 was not obvious;no significant changes were found in Montelukast sodium group.Compared with the model group,the relative expression of p-ERK protein and the expression of ERK m RNA in the lung and colon tissues of rats in the Xiebai Zengye decoction group and in the lung tissues of rats in the montelukast sodium group were significantly reduced(P<0.05 or P<0.01);the protein change in colon tissue of Montelukast sodium group was not obvious.(4)Compared with the normal group,the content of acetic acid,propionic acid,butyric acid and total SCFAs in model rats decreased significantly(P<0.05 or P<0.01);the structure of intestinal flora changed significantly,and the sobs index,chao index and shannon index decreased(P<0.05 or P<0.01),simpson index increased significantly(P<0.01);the relative abundance of Lactobacilli and Allobaculum decreased,while the relative abundance of Romboutsia,Turicibacter and unclassified＿f＿Oscillospiraceae increased in the model group decreased.Xiebai Zengye Decoction can up-regulate the content of SCFAs(P<0.01);improve the diversity of Alpha and Beta flora;increase the beneficial flora represented by Turicibacter,recall the declining Lactobacilli and Allobaculum,and correct the imbalance of flora.The correlation analysis showed that there was a positive correlation between the acetic acid and Allobaculum(P<0.05),and there was a negative correlation between acetic acid and the unclassified＿f＿Oscillospiraceae(P<0.05);propanoic acid was positively correlated with Lactobacilli et al(P<0.01 or P<0.05),and negatively correlated with Rombousia et al(P<0.05);butanoic acid is positively correlated with Akkermansia,Lactobacilli,etc.(P<0.01 or P<0.05),and negatively correlated with unclassified＿f＿Oscillospiraceae,Eubacterium＿xylanophilum＿group(P<0.01 or P<0.05);TNF-α was negatively correlated with Allobaculum(P<0.05);there was a significant positive correlation between IL-8 and Rombousia,Turiciactor(P<0.01 or P<0.05).Conclusions:(1)The post-infectious cough juvenile rat model prepared by combination of smoke exposure,lipopolysaccharide nasal drops and capsaicin atomization has the characteristics of lung heat and yin deficiency syndrome.(2)Xiebai Zengye Decoction can not only effectively improve the airway inflammation and airway hyper reactivity of model rats,but also improve the pathological changes of colon tissue.(3)Xiebai Zengye Decoction can synchronously down-regulate the expression of MEK1/2,ERK1/2,C-FOS protein in ERK signal pathway of lung and colon tissues of model rats,thus playing the role of lung and intestine combined therapy.(4)Xiebai Zengye Decoction can regulate the abundance and diversity of intestinal flora in model rats,increase the beneficial flora represented by Zurich bacilli,callback the decreased lactobacillus and heterobacillus to correct the imbalance of flora,increase the content of SCFAs,and play the role of combined treatment of lung and intestine. |
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