| ObjectiveIn this study, we investigated the mechanism underlying the attenuation of cigarette smoke (CS)-induced respiratory inflammation by icariin, astragaloside Ⅳ baicalin and catapol in cigarette smoke-induced inflammatory models in vivo and in vitro.MethodsIn vivo, mice were exposed to smoke of15cigarettes for1h/day,6days/week for3months and dosed with icariin (25,50and100mg/kg) or astragaloside Ⅳ (10,20and40mg/kg) or baicalin (25,50and100mg/kg) or catapol (5,10and20mg/kg) or dexamethasone (1mg/kg). Basal lung function was measured in unrestrained mice using a whole-body plethysmography (WBP) system. Lungs were stained with hematoxylin and eosin (H&E) before histopathological assessment of the bronchus and the parenchyma, including inflammation score, the mean linear intercepts (MLI) and destructive index (DI), being examined. Interleukin (IL)-8, tumor necrosis factor-α (TNF-α), and matrix metalloproteinase (MMP-9) levels in both BALF and serum were measured using ELISA kits. The protein expression of HDAC2and GR was investigated using both immunohistochemistry (IHC) stain and Western blot. The protein expression of phosphor-HDAC2, P65, phosphor-P65and IκB-α were investigated using Western blot. HDAC2activity in the mice lung tissue was measured using an HDAC2activity assay kit.In vitro, A549cells were incubated with icariin (1,10and50μM) or astragaloside Ⅳ (1,10and50μM) or baicalin (1,10and50μM) or catapol (1,10and50μM) or dexamethasone (10-12,10-10,10-8and10-6M) followed by treatments with cigarette smoke extract (CSE,2.5and5%), or TNF-α (10ng/ml). The influences of CSE, icariin, astragaloside Ⅳ, baicalin, or catapol on cell proliferation were measured using CCK-8kit. The levels of Interleukin (IL)-6, IL-8and TNF-α in A549cells culture supernatant were measured using ELISA kits. The protein expression of HDAC2, GR and P65were measured using Western blot. HDAC2activity in the mice lung tissue was measured using an HDAC2activity assay kit. The expression of HDAC2mRNA and GR mRNA was measured using real-time PCR.Results1. CS-exposed mice demonstrated marked and consistent decreases in peak inspiratory flow (PEF), peak expiratory flow (PIF) and minute ventilation (MV) as compared to controls (P<0.05). Inflammatory cells were significantly (P<0.05) evident in the airway and the lung parenchyma of CS-exposed mice and CS-exposed mice demonstrated marked increases in MLI and DI as compared to controls. Significant (P<0.05) increases of TNF-α, IL-8and MMP-9levels in both BALF and serum, NF-κB activation, including P65phosphorylation and decreased expression of IκB-α, in lung tissue were observed following treatment with3months of CS-exposure compared with controls. Furthermore, CS-exposure impared HDAC2activity and protein expression, decreased GR protein expression in the lung. Moreover, in vitro CSE increased the levels of IL-6、IL-8and TNF-α, induced HDAC2degradation, decreased protein and gene expression of GR and activated NF-κB cell signaling pathway.2. As observed in vivo, icariin showed significantly (P<0.05) growth on the breathing parameters, including PIF PEF and MV, compared with CS-exposed mice. Icariin significantly decreased the inflammatory cells, MLI and DI of lung in response to CS. Levels of TNF-α, IL-8and MMP-9in both BALF and serum were significantly decreased after treatment with icariin. Furthermore, icariin restored CS-impared HDAC2activity, HDAC2protein expression and GR expression, suppressed HDAC2phosphorylation and inhibited NF-κB activation in CS-exposed mice.3. As observed in vivo, astragaloside Ⅳ significantly decreased MLI and DI, inhibited inflammatory cells in lung tissue in response to CS. MMP-9in both BALF and serum and TNF-α in BALF were significantly decreased after treatment with astragaloside Ⅳ. Furthermore, astragaloside IV inhibited NF-κB activation by suppressing P65phosphorylation in CS-exposed mice.4. As observed in vivo, baicalin showed significantly (P<0.05) growth on the breathing parameters, including PIF PEF and MV, compared with CS-exposed mice. Baicalin significantly decreased the inflammatory cells, MLI and DI of lung in response to CS. Levels of TNF-α, IL-8and MMP-9in both BALF and serum were significantly decreased after treatment with baicalin. Furthermore, baicalin restored CS-impared HDAC2activity and protein expression, suppressed HDAC2phosphorylation and inhibited NF-κB activation in CS-exposed mice.5. As observed in vivo, catapol significantly decreased the inflammatory cells, MLI and DI of lung in response to CS. Levels of TNF-α, IL-8and MMP-9in both BALF and serum were significantly decreased after treatment with catapol. Furthermore, catapol restored CS-impared GR expression and inhibited NF-κB activation by increased expression of IκB-α in CS-exposed mice.6. As observed in vivo, Compound group showed significantly (P<0.05) growth on the breathing parameters, including PIF PEF and MV, compared with CS-exposed mice. Compound group significantly inhibited the inflammatory cells of lung, suppressed levels of TNF-α, IL-8and MMP-9in both BALF and serum compared to CS-exposed mice.7. As observed in vivo, Dexamethasone failed to protect CS-reduced pulmonary function and suppress inflammatory cells in lungs compared to CS-exposed mice. Dexamethasone significantly inhibited levels of TNF-α and IL-8, but failed to decrease MMP9, in both BALF and serum. Dexamethasone inhibited NF-κB P65phosphorylation, but failed to up-regulate IκB-α expression. Dexamethasone restored CS-reduced HDAC2activity, but the effects of up-regulating the expression on HDAC2or GR are uncertain.8. As observed in vitro, icariin significantly decreased levels of TNF-α and IL-8in A549supernatant. Furthermore, icariin significantly restored CS-impared HDAC2activity and protein expression, up-regulated GR expression and inhibited P65protein expression.9. As observed in vitro, astragaloside Ⅳ significantly decreased levels of TNF-α and IL-8in A549supernatant and inhibited P65protein expression. Furthermore, astragaloside Ⅳ significantly restored CS-impared HDAC2activity but the effect on HDAC2expression was not clear.10. As observed in vitro, baicalin significantly decreased levels of TNF-α and IL-8in A549supernatant. Baicalin significantly restored CS-impared HDAC2activity and protein expression, and inhibited P65protein expression.11. As observed in vitro, catapol significantly decreased levels of TNF-α IL-6and IL-8in A549supernatant. Catapol significantly restored CS-impared GR gene and protein expression. Furthermore, catapol significantly restored CS-impared HDAC2activity but the effect on HDAC2expression was not clear. 12. As observed in vitro, effects of dexamethasone in CSE-induced inflammatory response is weak than that in TNF-α-induced inflammatory response. Furthermore, dexamethasone up-regulated HDAC2protein expression in CSE-exposed A549cells, however this was not observed in vivo.Conclusion1. Cigarette smoke (CS) could induce COPD inflammatory responses, impaired pulmonary function of mice, increased inflammatory cells and developed emphysema in lung tissue of mice. CS significantly increased TNF-α, IL-8and MMP-9levels in both BALF and serum, impared HDAC2function, decreased GR protein expression in the lung and activated NF-κB cell signaling pathway. A549exposed to CSE induced similar inflammatory responses including increased TNF-α, IL-8and IL-6levels, impared HDAC2function, decreased GR expression and activated NF-κB cell signaling pathway.2. Icariin has anti-inflammatory effects in cigarette smoke induced inflammatory models in mice and A549cells. The mechanism of its anti-inflammatory effects might be through inhibiting NF-κB activation, restoring HDAC2effect and up-regulating GR expression.3. Astragaloside Ⅳ has weak anti-inflammatory effects in cigarette smoke induced inflammatory models in mice and A549cells. The mechanism of its anti-inflammatory effects might be through inhibiting NF-κB activation.4. Baicalin has anti-inflammatory effects in cigarette smoke induced inflammatory models in mice and A549cells. The mechanism of its anti-inflammatory effects might be through inhibiting NF-κB activation and restoring HDAC2effect.5. Catapol has anti-inflammatory effects in cigarette smoke induced inflammatory models in mice and A549cells. The mechanism of its anti-inflammatory effects might be through inhibiting NF-κB activation and up-regulating GR expression.6. Compound of icariin, astragaloside Ⅳ and catapol has anti-inflammatory effects in cigarette smoke induced inflammatory responses by significantly inhibiting the inflammatory cells of lung, suppressing levels of TNF-α, IL-8and MMP-9in both BALF and serum compared to CS-exposed mice.7. Icariin, astragaloside Ⅳ, baicalin, catapol and compound of icariin, astragaloside IV and catapol can inhibit the inflammatory responses in cigarette smoke induced inflammatory models in mice and A549cells in different degrees. It seemed that the anti-inflammatory effect of astragaloside Ⅳ is weaker than icariin, baicalin and catapol.8. The effects of dexamethasone to attenuate CS-induced inflammatory response are not completely. |
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