| Objective Chronic obstructive pulmonary disease(COPD)is a complex disease which affects the whole body.As it progresses,the acute exacerbation may occur frequently,leading to rising hospitalization rate and morbidity,rapid decline in lung function,deteriorating quality of life and causes economic burden.In recent years,it has been found that a weakened alveolar macrophage(AM)phagocytosis is an important pathologic factor for acute exacerbation of COPD.Such phagocytic dysfunction is regulated by Toll-like Receptors(TLR)/ Nuclear factor ?-light chain-enhancer of activated B cells(NF-?B)signal transduction pathway.However,trials are scarse which focus on the effect of Chinese medicinal compound on the pathway above.Therefore,our experimental study attempts to study how Feikang Formula(FKF)intervenes phagocytosis of pathogenic organisms by lung AM and the receptor regulation associated with such process.Moreover,clinical trials were carried out to observe the Traditional Chinese Medicine pattern,pulmonary function,numbers of exacerbation episodes,quality of life,interleukin-1?(IL-1?),tumor necrosis factor ?(TNF-?),IL-6?IL-17 concentrations in induced sputum.Methods 1.Clinical research protocol: Patients who were eligible for the trial were randomly divided into the experimental group and control group.The experimental group was treated with FKF granule combined with inhalational tiotropium bromide,while the control group patients were given only tiotropium bromide inhalation.After a 3-month-treatment,we recorded theindice mentioned above along with simplified St.George Respiratory Questionnaire(SGRQ),Six Minutes Walking Distance(6MWD).2.Animal experimental protocol:60 Sprague-Dawley(SD)male rats were randomly divided into 6 groups,including blank group(no intervention was given),COPD model group(no medicine was given),positive control groupand FKF groups(high-,medium-and low-dosage groups).Rats were induced by a 4-week cigarette smoke inhalation and Lipopolysaccharides(LPS)tracheal instillation.Then the rats of the latter 4 groups were treated with different dosage of FKF granule and DXM for 16 weeks respectively.The general status of rats and lung function were obseverd.The therapeutic effect of FKF granule on COPD rats was determined by the levels of IL-6/TNF-?/IL-1?/IL-17 in AM and bronchoal veolar lavage fluid(BALF),using enzyme-linked immunosorbent assay(ELISA).The m RNA levels of TLR2/TLR4/pNF-?B/ NF-?B/p65 in AM and the m RNA expressions of TLR4/TLR2/pIkappa B/Ikappa B and p65 in lung tissue were measured by Quantitative real time polymerase chain reaction(Q-PCR).The levels of TLR2/TLR4/pIkappa B/Ikappa B/ p65 proteins in AM and lung tissue were detected by Western blot.The expressions of TNF-?/IL-6/p65 were assayed by immunohistochemical staining.Results 1.Clinical Observation(1)There were no statistical differences,obtained from SPSS 19.0 analysis,between the 2 groups in aspects of age,gender,BMI,smoking habit,time-span of disease,severity,pulmonary function,which indicated an acceptable comparability.(2)Changes in TCM syndrome scaling: all symptoms of FKF group were relieved after administration of the TCM granule(P < 0.05),whereas statistical improvement in symptom scores of control group was only reported in coughing,sputum quantity and wheezing.All symptom scores showed statistical difference between two groups of patients apart from coughing and wheezing.(3)Numbers of exacerbation episodes and their severity were similar between the two groups(P>0.05)(4)Statistical difference could be seen in pre-BDT FEV1,FVC,IC(P<0.05)between the two groups after a 3-month treatment.While post-BDT indexes were similar of the two were similar(P>0.05).(5)Improvement in simplified SGRQ scores,symptom scores,activity scores,disease affecting scores in both groups were all statistically decreased after the 3-month treatment,with the improvement more significant in the group given FKF granule(P<0.05).(6)6MWD scores increased in both groups after the 3-month medication(P<0.05),with the increase more evident in FKF group compared with control group(P<0.05).(7)IL-1??TNF-a?IL-6?IL-17 concentration in sputum of the patients in both groups decreased significantly(P<0.05).Additionally,TNF-? and IL-17 sputum concentration reported a more evident downturn in FKF group.2 Animal Experiment:(1)General status: blank group of rats had white smooth fur,good activity and appetite,and smooth breaths.By contrast,the fur of those of COPD model group turned dull and sallow,with deteriorated activity and appetite and more rapid breaths but generally unchanged body weight.Sneezing could be seen in some of the smoking rats.(2)Pulmonary function: PIF,PEF,EF50 decreased significantly in the smoking COPD model group(P<0.01).FKF granule-fed rats reported improved PIF,PEF,EF50 compared with those of the model group,with the improvement positively correlated with the dosage.However,DXM group scored similarly with the model group after medication.(3)Pathological changes: no inflammatory exudate,abnormal enlargement was microscopically observed in the alveoli of the blank group of rats,while the alveoli could be seen enlarged,fused,and the alveolar walls thinner in the lung slices of COPD model rats.The pathologic changes above were seen generally recovered in the lung tissue of DXM group,whereas such recovery was less evident in those of the FKF group.(4)Cell types and counts in BALF: WBC count increased significantly in the BALF of COPD model rats compared with that of blank ones(P<0.01).In terms of cell types,neutrophils and lymphocytes increased more significantly,with the number of monocytes unchanged.In the BALF samples of COPD model group,neutrophil count rose but monocyte count decreased,while lymphocyte numbers were statistically similar.These inflammatorycells decreased in numbers in the BALF of FKF and DXM rats(P<0.05),and constituted,however,a similar proportion compared with those of COPD model rats.This decrease was most evident in DXM group,but not as evident in FKF groups.Such decrease positively correlated with FKF granule dosage.(5)Compared with those of the blank group,phagocytosis of microorganism and mean fluorescent intensity(MFI)decreased drastically(P<0.01).By comparison,the positive rate of AM FIC-E.coli and MFI were improved in the lungs of rats of FKF and DXM groups(P<0.01).Such effectiveness of DXM was similar to high-dosage FKF in tested rats.(6)Concentration of inflammatory mediators in BALF and AM: BALF IL-1?,TNF-?,IL-6,IL-17 concentration of COPD model rats increased significantly compared with that of blank group.By comparison,the BALF concentration of these mediators decreased in the lungs of rats after a 4-month administration of FKF granule and DXM(P<0.01).What was more,high dosage of FKF granule induced a similar decrease in BALF IL-1?,TNF-?,IL-6 concentration compared with DXM(P=0.1231;P=0.299;P=0.210).The AM of COPD model rats emitted much more IL-1?,TNF-?,IL-6,IL-17 than those of blank rats did(P<0.001).FKF granule and DXM proved to be capable of down-regulating such emission.The higher dosage of FKF granule given,the stronger the down-regulation was.Additionally high-dosage FKF had a similar down-regulating power compared with DXM(P>0.05).(7)Immunohistochemical results: TNF-?,IL-6 and protein P65 expression rose in lung tissue of COPD model rats compared to those of blank group.FKF granule and DXM feeding both led to decrease in the above expressions.In specific,such inhibitory effect in FKF groups positively correlated with its dosage.(8)Western blot results: a)AM cyto-plasmatic TLR4,TLR2,I?B quantity decreased in the lungs of COPD model rats compared with blank group,while increase was detected in p-I?B and nuclear P65,the levels of which dropped and TLR4,TLR2,I?B level rose in lung AM of DXM-fed rats.FKF granule proved effective in inhibiting p-I?B and nuclear P65 expression and boost TLR,TLR2,I?B expression.Such effect positively correlated with dosage.b)Lung cells cyto-plasmatic TLR4,TLR2,I?B quantity decreased in the lungs of COPD model rats compared with blank group,while increase was detected in p-I?B and nuclear P65,the levels of which dropped and TLR4,TLR2,I?B level rose in lung cells of DXM-fed rats.FKF granule proved effective in inhibiting p-I?B and nuclear P65 expression and boost TLR,TLR2,I?B expression.Such effect positively correlated with dosage.(9)q PCR results: TNF-? and IL-6 m RNA expression was boosted while m RNA of TLR4 and TLR2 was inhibited in lung AM of COPD model rats compared with blank rats.Fed with FKF granule,TNF-? and IL-6 m RNA expression could be ingibited while m RNA of TLR4 and TLR2 expression was increased in lung AM.Such change in expression positively correlated with dosage of the tested TCM granule.Similar effect on TNF-?,IL-6,TLR4 and TLR2 m RNA expression was detected in DXM-fed rats.Additionally,TNF-?,IL-6 and TLR2 reported no statistical difference from high-dosage FKF rats(P=0.502,P=0.396,P=0.091).TLR4 m RNA expression was statistically more active in DXM-fed rats compared to high-dosage FKF granule-fed rats(P<0.001).Similarly,TNF-? and IL-6 m RNA expression was boosted while m RNA of TLR4 and TLR2 was inhibited in lung tissue of COPD model rats compared with blank rats.Fed with FKF granule,TNF-? and IL-6 m RNA expression could be ingibited while m RNA of TLR4 and TLR2 expression was increased in lung AM.Such change in expression positively correlated with dosage of the tested TCM granule.Similar effect on TNF-?,IL-6,TLR4 and TLR2 m RNA expression was detected in DXM-fed rats.Additionally,TNF-?,IL-6 and TLR2 reported no statistical difference from high-dosage FKF rats(P=0.502,P=0.396,P=0.091).TLR4 m RNA expression was statistically more active in DXM-fed rats compared to highdosage FKF granule-fed rats(P<0.001).Conclusions 1.Clinical Observation(1)FKF proved effective in relieving lung and spleen qi deficiencyrelated clinical manifestations in non-exacerbating COPD patients,especially in the following symptoms: sputum emission,susceptibility to catching cold,spontaneous sweating,fatique,poor appetite,abdominal distension,fecal abnormalities.(2)FKF proved effective in improving pulmonary function and preventing acute exacerbation in COPD patients.(3)FKF improved quality of life in COPD patients,especially in relieving wheezing.(4)FKF decreased significantly IL-1??TNF-a?IL-6?IL-17 concentrationin sputum of the patients.Considering its clinical efficacy,it might berelated to controlling the airway inflammation by reducing the level ofairway inflammatory factors.2.Animal Experiments(1)20-week smoking plus intra-tracheal LPS injection could induce COPDrelated pathological changes in lung tissue and AM phagocytic dysfunction.(2)TLR/NF-?B transduction pathway affected AM phagocytosis,which,plus inflammation-related genes expression,could be better activated byproperly regulating the pathway above,(3)FKF proved to be effective in enhancing lung AM phagocytosis possiblyby regulating TLR/NF-?B transduction pathway and inhibiting inflammationrelated genes expression.3.FKF,which was invented by following the rule of “reinforcing earthto generate metal”,proved effective in enhancing lung AM phagocytosis,therefore improving COPD related conditions. |
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