Regulatory Mechanism Of Sequential Therapy Of TLR4 / NF-κB Signaling Pathway On Inflammatory Response In "AECOPD-Dangerous Window" Rats

ObjectiveTo observe the dynamic inflammatory characters and define the duration of sequential acute exacerbation (AE) and risk-window (RW) phases in an acute exacerbation of chronic obstructive pulmonary disease (AECOPD) rat model, and provide basis for sequential treatments. To explore the effects and remote impacts in the secondary AE of sequential treatments of Tongsai granules (TSG), clearing heat and dissipating phlegm, and Bufei Yishen granules (BYG), reinforcing the deficiency in vital qi, in AE and RW, respectively, in AECOPD rats, and disscuss the mechanism involving regulating the Toll-like receptor (TLR)-4/nuclear factor (NF)-κB signaling.MethodsExperiment 1:Forty rats were randomized into Control, COPD, AE1 and AE2 groups. COPD model induced by cigarette-smoke and bacterial exposures was intratracheally challenged with Klebsiella pneumonia on the 6th day (Day 1) of week 9 after a 5-day heat exposure; the second challenges were performed in AE1 and AE2 groups on Day 15 and 23, respectively. Rats were sacrificed on Day 44. Pulmanory function tests were performed weekly and 24 h post the AEs challenge. Body temperature, cytological analysis, serum amyloid A (SAA) and C-reactive protein (CRP) were determined each 2 days after challenged. Forced vital capacity (FVC), forced expiratory volume 0.3s (FEV0.3) and FEV0.3/FVC were tested before sacrificed and pulmonary histomorphological changes were determined after sacrificed. Mean linear intercept (MLI) and mean alveolar numbers (MAN) were calculated.Experiment 2:A hundred and twelve rats were randomized into Control, COPD, AECOPD, TSG/normal saline (TSG/NS), moxifloxacin+salbutamol/NS (MXF+STL/NS), TSG/BYG, MXF+STL/STL and TSG+MXF+STL/BYG+STL groups. AECOPD rat model was established as above mentioned in "Experiment 1". Rats were treated with sequential and non-sequential treatments with appropriate medicine(s) 2 days before the first challenge and ended on Day 15. The second challenge was performed on Day 23. Rats were sacrificed on Day 15 and 24, respectively. Body temperature, cytological analysis, SAA and CRP in peripheral blood were determined each 2 days while peak expiratory flow (PEF) were measured weekly after challenge. FVC, FEV0.3, FEV0.3/FVC were determined before sacrifice. Myeloperoxidase (MPO), polymorphonuclear (PMN) elastase, interleukin (IL)-1β, IL-6, IL-10, tumor necrosis factor (TNF)-a in BALF, serum and lung tissue were tested. Pulmonary histomorphological changes were analyzed after sacrificed. The mRNA and protein expressions of TLR4, NF-κB, the inhibitor of NF-κB (IκB), p-NF-KB and p-IκB were determined after sacrificed.ResultExperiment 1:Body weight gain, PEF, FVC, FEV0.3, FEV0.3/FVC, MAN decreased in COPD rats, while the population of white blood cell (WBC), neutrophils, monocyte, SAA, CRP and LMI raised; marked inflammatory cell infiltration, bronchial wall thickness and fusion of pulmonary alveoli was also observed, when compared to Control group. Compared to COPD rats, body temperature, WBC, neutrophils, SAA and CRP increased in AECOPD rats 24 hours after challenge, and declined sharply in the following 4～6 days, and then smoothly in 10-12 days; PEF declined in 24 hours after challenged, and then recovered in 3 weeks; the changing range of PEF, WBC, neutrophils, SAA and CRP in AE1 group on Day 16 was greater than the other exacerbations, and the recovery time was 2 days longer, while the indicators in AE2 group on Day 23 were fimaliar to Day 2. When sacrificed, FVC, FEV0.3 and FEV0.3/FVC MAN decreased, especially in the AE1 group, while LMI increased; more marked airway inflammation, remodeling and emphysema were observed in AECOPD group, especially in AE1 group.Experiment 2:Body weight gain, PEF, FVC, FEV0.3, FEV0.3/FVC, MAN decreased in COPD rats, while the population of WBC, neutrophils, monocytes, SAA, CRP, IL-6, IL-10, IL-1β, TNF-a, MPO, IL-10, LMI, TLR4, NF-κB, IκB mRNA expressions and p-NF-κB、 p-IκB protein expression elevated. Compared to COPD rats, body temperature, the population of WBC, neutrophils, monocytes, SAA and CRP increased in AECOPD rats while PEF declined on Day 2, and recovered to the level of COPD group on Day 16 and 22. Compared to AECOPD group, all biomarkers improved in administrated groups on Day 2, and sequential groups (TSG/BYG, MXF+STL/STL and TSG+MXF+STL/BYG+STL group) returned to baseline of COPD earlier on Day 6～8 than non-sequential groups on Day 10-12, especially in the TSG+MXF+STL/BYG+STL group. FVC, FEV0.3, FEV0.3/FVC and MAN in AECOPD were lower than COPD group on Day 15, while serum MPO, BALF PMN elastase, IL-6, IL-10, lung tissue IL-1β, TNF-a, MPO, IL-10 and MLI were higher than COPD group, and more marked airway inflammation, remodeling and emphysema were observed in AECOPD group. Compared to AECOPD group, above-mentioned indicators reduced in administrated groups at different degree, especially in TSG+MXF+STL/BYG+STL group. Twenty-four hours after the secondary AE, the population of WBC and neutrophils, SAA, CRP,MPO、PMN elastase, IL-6 and IL-10 were observed higher in AECOPD rats than COPD rats, while monocyte, IL-1β, TNF-a in serum, BALF and lung tissue, and the mRNA and protein expression of TLR4, NF-κB, IkB, p-NF-KB and p-IκB decreased.The impaiement of AECOPD group was more severe than COPD group. Compared to AECOPD group, WBC, neutrophils, SAA, CRP, MPO、PMN elastase, IL-6 and IL-10 reduced in administrated groups at different degree, while IL-1β, TNF-a in serum, BALF and lung tissue, the relative expression of TLR4, NF-κB, IκB raised. Airway inflammation, remodeling and emphysema were improved in administrated groups, while sequential groups were better than non-sequntial groups, especially in the TSG+MXF+STL/BYG+STL group;Conclusion1. AECOPD rat model was established and RW, a period about 10 ～ 12 days after AE phase (approximately 5 ～ 7 days), was observed successfully.2. RW was characterized by incompletely recovered lung function and inflammatory responses, and more severe inflammation reaction and longer recovery time of the acute exacerbation happened in the RW.3. Sequential treatment of Tongsai granule and Bufei Yishen granule during AECOPD-RW period can improve pulmonary function and alleviate inflammation in AECOPD rats, shorten recovery course, and decrease the inflammatory response in the secondary exacerbation, with significantly long-term effects, especially the integrated sequential treatments (Tongsai+ moxifloxacin+salbutamol/salbutamol+Bufei Yishen granule). The curative effects of Tongsai/Bufei Yishen granule and moxifloxacin+salbutamol/salbutamol sequential treatment were second to the integrated sequential treatments.4. The mechanisms of the sequential Chinese medicine treatments might involve down-regulating TLR4/NF-κB signaling, reducing imflammtory responses, and improving monocytes-macrophage impairments.