| Objective1.The purpose of this study was to evaluate the efficacy of JPYFⅡ in the treatment of COPD at early stage.2.The aim of this study was to investigate the regulation of immune cell of bronchoalveolar lavage fluid(BALF)in IAV infection in CS mice.3.In order to clarify the effect of JPYFⅡ that can reduce influenza virus-induced airway inflammation in CS-exposed mice may have clinical implications for the treatment of AECOPD associated with IAV infection.Methods1.A randomized,double-blind,placebo-controlled clinical trial:COPD stable stageⅠ-Ⅱ patients were randomly divided into the treatment group and the control group,the treatment group was given JPYFⅡ granules;control group was given JPYFⅡ placebo granules.The screening period was 2 weeks,treatment period was 12 weeks,and follow-up period was 12 weeks.Assessment team between the frequency of exacerbations and acute exacerbation rate,and pulmonary function,SGRQ,CAT,mMRC score,BODE index of the two groups.2.IAV-regulated immune cell of BALF in CS exposed mice.BALB/c mice were subjected to CS for 4 days and mice were infected with IAV on day5.The group were randomly divided into four groups:sham+MEM+saline,CS+MEM+saline,sham+IAV+saline and CS+IAV+saline.The following parameters were evaluated 5 post infection:cell count,superoxide production in BALF,and mRNA expression of cytokines,chemokines,interferons and Influenza matrix in mouse lungs.3.Effects of JPYFⅡ prescription on airway immune cells induced by CS and IAV.Mice were treated with JPYFⅡ formulation(24 g/kg)or saline via oral gavage 1h before CS exposure or IAV infection daily until they were culled at day 5 post infection.The group were randomly divided into eight groups:sham+MEM+saline,sham+MEM+JPYFⅡ,CS+MEM+saline,CS+MEM+JPYFⅡ,sham+IAV+saline,sham+IAV+JPYFⅡ and CS+IAV+saline,CS+IAV+JPYFⅡ.The following parameters were evaluated 5 post infection:cell count,superoxide production in BALF,and mRNA expression of cytokines,chemokines,interferons,NP and Influenza matrix in lungs,protein expression of gp91,HO-1 and lung function,viral titer of mice.4.Pre-infection treatment of JPYFⅡ formulation on airway immune cells induced by CS and IAV.BALB/c mice were subjected to CS for 4 days and mice were infected with IAV PR8 on day5.Mice were treated with JPYFⅡ formulation(24 g/kg)or saline until they were culled at day 3 and 5 post infection.The group were randomly divided into six groups:sham+MEM+saline,CS+IAV(3d)+saline,CS+IAV(3d)+JPYFⅡ;sham+MEM+saline,CS+IAV(5d)+saline,CS+IAV(5d)+JPYFⅡ.The following parameters were evaluated cell count,superoxide production in BALF and mRNA expression of cytokines,chemokines,and Influenza matrix.Results1.Clinical research:A total of 62 patients were enrolled in this study.There was no significant difference between the treatment group and control group at baseline(P>0.05).Compared with control group,the treatment group could effectively reduce CAT,SGRQ and MMRC score,reduce the frequency of AECOPD.However,the improvement of BODE and SpO2 was no better than that of placebo.Compared with placebo,JPYFⅡ had a certain degree of improvement in post FVC and post FEV1/FVC,but the difference between groups was not significant.There was no significant difference between JPYFⅡand placebo group in adverse reactions.2.Experimental research(1)IAV-regulated immune cell of BALF in CS exposed mice.①The number of inflammatory cells in BALF in mice exposed to CS and infected with IAV.The number of total cells,macrophages were significantly increased in BALF of CS+MEM+saline,CS+IAV+saline compared to sham+MEM+saline(P<0.05);the number of total cells,neutrophils were increased in CS+IAV+saline compared to sham+MEM+saline and CS+MEM+saline(P<0.05);the number of total cells,macrophages were increased in CS+IAV+saline compared to sham+MEM+saline and sham+IAV+saline(P<0.05).②mRNA expression of cytokines,chemokines,and ROS in lungs.The Real-time PCR results showed that the expression of IL-6 in CS+MEM+saline was significantly higher than the sham+MEM+saline(P<0.01).The expression of IL-6 and MMP12 were increased in sham+IAV+saline mice compared to CS+MEM+saline(P<0.05).The expression of MMP12 was increased in CS+IAV+saline compared to CS+MEM+saline and sham+IAV+saline(P<0.01).The expression of ROS was increased in sham+MEM+saline and CS+IAV+saline compared to CS+MEM+saline and sham+MEM+saline(P<0.05);the expression of ROS was increased in CS+IAV+saline compared to sham+MEM+saline(P<0.05).The Real-time PCR results showed that the expression of CXCL1,CXCL5,CCL2 in CS+IAV+saline were significantly higher than the sham+MEM+saline and CS+MEM+saline(P<0.05);the expression of CXCL5 in CS+IAV+saline was significantly higher than the sham+IAV+saline(P><0.01).③ mRNA expression of Influenza matrix and interferons in mouse lungs.The Real-time PCR results showed that the expression of IFNα,IFNβ in sham+MEM+saline,sham+IAV+saline and CS+IAV+saline were significantly higher than the CS+MEM+saline(P<0.05);the expression of Influenza matrix and NP were increased in CS+IAV+saline compared to sham+MEM+saline and CS+MEM+saline(P<0.05).(2)Effects of JPYFⅡ prescription on airway immune cells induced by CS and IAV.①JPYFⅡ formulation treatment reduces the number of inflammatory cells in BALF in mice exposed to CS and infected with IAV.Compared with the CS+IAV+saline,the number of total cells,neutrophils in JPYFⅡwere significantly lower than the CS+IAV+saline(P<0.01).②JPYFⅡ formulation inhibits mRNA expression of cytokines,chemokines in lungs.The Real-time PCR results showed that compared with the CS+IAV+saline,the expression of IL-6 in JPYFⅡ was significantly lower than the CS+IAV+saline(P<0.05).The Real-time PCR results showed compared with the CS+IAV+saline,the expression of CXCL1,CXCL2 in JPYFⅡ were significantly lower than the CS+IAV+saline(P<0.05).③JPYFⅡ formulation alleviates superoxide production of BALF cells.The expression of ROS was significantly increased in lung of sham+MEM+saline,sham+IAV+saline and CS+IAV+saline compared to CS+MEM+saline(P<0.05);compared with the CS+IAV+saline,the expression of ROS in JPYFⅡ was significantly lower than the CS+IAV+saline(P<0.05).The Western Blot results showed that compared with the CS+IAV+saline,the expression of HO-1 and gp91 in JPYFⅡ were significantly lower than the CS+IAV+saline(P<0.05).④JPYFⅡ formulation suppresses replication of IAV.JPYFⅡ formulation treatment significantly reduced viral titer and NP expression at the mRNA level in CS+IAV+saline(P<0.05).⑤JPYFⅡ increased IC and VC in mice.FRC was significantly increased in CS+IAV+saline compared with sham+MEM+saline(P<0.01),JPYFⅡ significantly reduced IC and VC expression in CS+IAV+saline(P<0.05).(3)Pre-infection treatment of JPYFⅡ formulation on airway immune cells induced by CS and IAV.①Pre-infection treatment of JPYFⅡ formulation does not alleviate the number of inflammatory cells in lungPre-infection treatment of JPYFⅡ formulation didn’t reduce the number of BALF cells both at Day 3 and Day 5 after IAV infection compared to CS+IAV+saline.②Pre-infection treatment of JPYFⅡ formulation does not alleviate inflammation and Influenza matrix in lungPre-infection treatment of JPYFⅡ formulation didn’t reduce the expression of IL-6,TNF-α,IL-1β and Influenza matrix both at Day 3 and Day 5 after IAV infection compared to CS+IAV+saline.Conclusion1.This study manifested that JPYFⅡ can reduce the frequency and incidence of acute exacerbations,improve the quality of life and clinical symptoms of COPD,with better efficacy and good safety than placebo.Based on this finding,we will further explore the potential mechanism of JPYFⅡ in reducing the occurrence of AECOPD.2.Taken these into together,this research manifested that CS and IAV infection are pathogenic factors in COPD;it also shows the immune changes of IAV infection in the process of AECOPD.3.The present study showed that JPYFⅡ formulation can reduce airway inflammation induced by IAV infection in CS-exposed mice.4.Pre-infection treatment of JPYFⅡ formulation does not alleviate inflammation in lung. |
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