Mechanisms Of Tiao-bu Fei-shen Therapies And Its Long-term Effect In COPD Rats Through Regulation Of JAK/STAT Signaling

ObjectiveTo evaluate the theapeutic effect and its long-term effect of the Bu-Fei Jian-Pi granule,Bu-Fei Yi-Shen granule and Yi-Qi Zi-Shen granule in treating stable chronic obstructivepulmonary disease (COPD), and to investigate its function characteristics and mechanisms.MethodsRats were randomly divided into six groups: Control group, Model group, Bu-FeiJian-Pi group, Bu-Fei Yi-Shen group, Yi-Qi Zi-Shen group and Aminophyline group. Thestable COPD rat model was duplicated by methods of combination of repeated smokeinhalations and bacterial infections. From the9thto20thweek, the rats with COPD wereadministrated with normal saline (in control and model group), Bu-Fei Jian-Pi granule(Bu-Fei Jian-Pi group), Bu-Fei Yi-Shen granule (Bu-Fei Yi-Shen group), Yi-Qi Zi-Shengranule (Yi-Qi Zi-Shen group) and aminophyline (aminophyline group). At the20thweek,half of the animals were sacrificed, the survival were breed under normal conditions up to32weeks, and sacrificed. The lung function (TV, PEF, EF50) were measured. Lungpathology and ultrastructure were detected. The levels of interleukin (IL)-1β, IL-6, IL-8,IL-10, tumor necrosis factor (TNF)-α, soluble tumor necrosis factor receptor (sTNFR),expressions of JAK2, STAT1, STAT3, STAT5were detected by immunohistochemistrymethods. Expressions of SOCS3mRNA and JAK2mRNA were detected by fluorescencequantitative PCR.Results1General statusModel group had a emaciation, a poor spirit, reduced activities, decreased food andwater intake, loose stool. The three TCM therapy groups (Bu-Fei Jian-Pi granule, Bu-FeiYi-Shen granule, Yi-Qi Zi-Shen granule) and Aminophylline granule can all alleviate thesymptoms above, especially Bu-Fei Jian-Pi granule and Bu-Fei Yi-Shen granule. Bodyweight of the three TCM therapy groups and Aminophylline increased quickly from the 12thweek, but among the groups was no significant statistical difference (P>0.05).2Lung functionThe TV, PEF, EF50level of Model group was significantly lower than Control groupfrom the4thweek (P<0.05, P<0.01). Compared with Model group, PEF and EF50ofBu-Fei Jian-Pi group, Bu-Fei Yi-Shen group and Yi-Qi Zi-Shen group was clearly higherfrom the16thweek (P<0.05, P<0.01). TV increased significantly from the20thweeks(P<0.05, P<0.01). PEF of Aminophylline group was significantly higher only at the20thweek and the28thweek (P<0.05). In the32weeks observation period, the level of TV, PEF,EF50of Bu-Fei Jian-Pi group, Bu-Fei Yi-Shen group, Yi-Qi Zi-Shen group andAminophylline group was no significant difference(P>0.05). PEF of three TCM therapygroups was higher at the32ndweek compared with the20thweek (P<0.05)，PEF ofAminophylline group was generally increasing (P>0.05).3Lung pathologyAt the end of the20thweek, lung histological impaired obviously in Model group. Thethree TCM therapy groups and Aminophylline group alleviated the pathological damage indifferent degree, especially in that treated with the three TCM therapy groups. At the end ofthe32ndweek, the three TCM therapy groups can improve the pathological state better thanAminophylline group.At the end of the20thand32ndweek, inflammatory cells of alveolar interstitial ofmodel group were significantly higher than the control group (P<0.01). The inflammatorycells of Bu-Fei Jian-Pi group, Bu-Fei Yi-Shen granule group, Yi-Qi Zi-Shen granule andAminophylline group were significantly lower compared with the model group (P<0.01).At the end of the20thweek, inflammatory cells of Bu-Fei Yi-Shen granule group werelower than Aminophylline group（P<0.05）. The inflammatory cells of Bu-Fei Yi-Shengranule group and Yi-Qi Zi-Shen granule was lower than the Aminophylline group, butthere were no significant differences (P>0.05). At the end of the32ndweek, inflammatorycells of Bu-Fei Jian-Pi group, Bu-Fei Yi-Shen granule group, Yi-Qi Zi-Shen granule weresignificantly lower than Aminophylline group (P<0.01). Compared with the20thweek, theinflammatory cells in each group were no significant differences at the32ndweek (P>0.05).4Lung ultrastructureAt the20thweek, lung ultrastructure in Model groups changed significantly, and Bu-FeiJian-Pi granule, Bu-Fei Yi-Shen granule, and Yi-Qi Zi-Shen granule could repair thedamages better than aminophylline. At the32ndweek, Bu-Fei Jian-Pi granule, Bu-FeiYi-Shen granule, Yi-Qi Zi-Shen granule alleviated the pathological damages, especially the Bu-Fei Jian-Pi granule.5Expressions of cytokines of lungAt the20thand32ndweek, the expressions of IL-1β, IL-6, IL-8, IL-10, TNF-α, sTNFRin Model group were significantly higher compared with Control group (P<0.01). Thelevels were lower in the three TCM treated groups compared to Model group (P<0.01). Atthe20thweek, the expressions of IL-1β, IL-6, IL-10, TNF-α, sTNFR in Aminophyllinegroup were lower compared to Model group (P<0.05,P<0.01), while the levels IL-1β、IL-8、IL-10was significantly higher than Bu-Fei Jian-Pi group(P<0.05,P<0.01), sTNFRwas significantly higher than Bu-Fei Yi-Shen granule group, Yi-Qi Zi-Shen granule. TheIL-10levels of Bu-Fei Jian-Pi group was significantly lower than Bu-Fei Yi-Shen granulegroup and Yi-Qi Zi-Shen granule (P<0.05). At the32ndweek, the expressions of IL-1β,IL-6, IL-10of Aminophylline group declined notably compared to Model group (P<0.05,P<0.01).The levels of IL-1β, IL-6, TNF-α in the three TCM treated groups weresignificantly lower than Aminophylline group (P<0.01), the levels of IL-8, IL-10, TNF-α ofBu-Fei Jian-Pi group and Bu-Fei Yi-Shen group were lower compared with Aminophyllinegroup (P<0.05, P<0.01); the level of IL-8of Bu-Fei Jian-Pi group was significantly lowerthan Yi-Qi Zi-Shen granule and Bu-Fei Yi-Shen granule group (P<0.01); IL-10of Bu-FeiJian-Pi group and Bu-Fei Yi-Shen group were significantly lower than Yi-Qi Zi-Shengroup (P<0.05, P<0.01). There was no significant statistical difference in three TCMtreated groups at the level of IL-1β, IL-6, TNF-α, sTNFR (P>0.05).Compared with the20thweek, at the32ndweek, the levels of IL-6, IL-8of Bu-FeiJian-Pi group was significantly lower(P<0.05,P<0.01), the levels of IL-6, IL-8, TNF-α ofBu-Fei Yi-Shen group was significantly lower(P<0.05,P<0.01), the levels of IL-6of Yi-QiZi-Shen group was significantly lower(P<0.05).6Expressions of JAK2, STAT1, STAT3, STAT5, SOCS3mRNA and JAK2mRNAAt the20thand32ndweek, the expressions of p-JAK2, JAK2mRNA, p-STAT1,p-STAT3, p-STAT5in Model group increased significantly compared with Control group(P<0.01). Compared with Model group, the expressions of p-JAK2, JAK2mRNA,p-STAT1, p-STAT3, p-STAT5in the three TCM treated groups and Aminophylline groupwere significantly lower (P<0.01). At the20thweek, the expressions of p-JAK2, JAK2mRNA in the three TCM treated groups were lower than Aminophylline group (P<0.05,P<0.01). The expressions of p-STAT1, p-STAT3of Bu-Fei Jian-Pi group were lower thanAminophylline group (P<0.05, P<0.01), the level p-STAT5were lower than Yi-Qi Zi-Shengranule (P<0.05). At the32ndweek, the expressions of p-JAK2, JAK2mRNA, p-STAT1, p-STAT3, p-STAT5in the three TCM treated groups were significantly lower thanAminophylline group (P<0.01). The level of p-STAT5of Bu-Fei Jian-Pi group wassignificantly lower than Yi-Qi Zi-Shen group and Bu-Fei Yi-Shen group (P<0.01).At the20thand32ndweek, the expression of SOCS3mRNA in Model group washigher than Control group (P<0.01), the level was higher in the three TCM treated groupsand Aminophylline group compared to Model group (P<0.01). SOCS3mRNA in the threeTCM treated groups is higher than Aminophylline group (P<0.05, P<0.01).Compared with the20thweek, at the32ndweek, the levels of p-JAK2, JAK2mRNA,p-STAT3, p-STAT5of Bu-Fei Jian-Pi group were significantly lower(P<0.05, P<0.01), thelevels of p-STAT3of Bu-Fei Yi-Shen group were significantly lower(P<0.01), the levels ofp-STAT3, p-STAT5of Yi-Qi Zi-Shen group were significantly lower (P<0.05, P<0.01).There were no significant statistical difference in Aminophylline group (P>0.05).7Correlation analysisThere is significant negative correlation between degree of TV and level of lung IL-1β,IL-6, IL-8, IL-10, TNF-α, sTNFR, p-JAK2, p-STAT1, p-STAT3, p-STAT5(r=-0.580,-0.611,-0.601,-0.670,-0.507,-0.521,-0.660,-0.598,-0.728,-0.738, P<0.01）).There is negativecorrelation between PEF and level of lung IL-6, IL-8, IL-10, TNF-α, p-JAK2, p-STAT1,p-STAT3, p-STAT5(r=-0.488,-0.442,-0.524,-0.404,-0.526,-0.499,-0.572,-0.527,P<0.05,P<0.01). There is significant negative correlation between EF50and level of lungIL-1β, IL-6, IL-8, IL-10, TNF-α, sTNFR, p-JAK2, p-STAT1, p-STAT3, p-STAT5(r=-0.531,-0.686,-0.526,-0.489,-0.573,-0.540,-0.686,-0.502,-0.717,-0.723, P<0.01).Conclusion1Bu-Fei Jian-Pi granules, Bu-Fei Yi-Shen granules and Yi-Qi Zi-Shen granules canobviously improve the whole condition, pulmonary function and pathological changes inRats of stable COPD, as well the long-term effect.2There are good effect and long-term effect of Bu-Fei Jian-Pi granules, Bu-Fei Yi-Shengranules and Yi-Qi Zi-Shen granules in reducing the expression of JAK/STAT signalingpathways related factors and inflammatory factor significantly, the mechanism may beinvolved in regulating JAK/STAT pathways and the level of inflammatory cytokines.3The effect and long-term effect of Tiao-Bu Fei-Shen Therapies are far better thanAminophylline in regulating JAK/STAT pathways and the level of inflammatory cytokines,especially Bu-Fei Jian-Pi granules, Bu-Fei Yi-Shen granules.