The Study Of Icariin Attenuating Inflammation Of Rat Chronic Obstructive Pulmonary Disease

Objective:Chronic obstructive pulmonary disease (COPD) is a common, recurrent attacking and progressive aggravating disease. The anti-inflammatory effects of icariin on COPD models in vitro and in vivo were evaluated from aspects of lung function, pathology of lung tissue, inflammatory cell, pro-inflammatory cytokines and transcription factor. The mechanism of icariin attenuating the Inflammation of COPD Models was futher explained.Materials and Methods:1. In vivo, COPD rat model was established by cigarette smoke (CS) exposure. Thirty-six Sprague-Dawley (SD) rats were randomly divided into six groups: Normal group, Model group, DXM group, Low-dosage-Icariin group, Middle-dosage-Icariin group, High-dosage-Icariin group. HE staining pathological section of lung tissue was made to observe the pathological changes and average alveolar area under the optical microscope, and pulmonary function in these groups was mesured by small animal spirometer. Total and differential leukocytes counting in BALF were determined under the optical microscope. The production of cytokines in serum and bronchoalveolar lavage fluid (BALF), CRH and CORT concentration in serum were measured by ELISA. The expression of NF-κB p65 protein in lung tissue was detected by immunohistochemistry. Real time PCR was used to test NF-κB p65 mRNA and GR mRNA levels in lung, CRH mRNA levels in hypothalamus, CORT mRNA levels in adrenal glands.2. In vitro, COPD model was established by using cigarette smoke extract (CSE) to stimulate SD rat typeⅡpneumocytes (ATⅡ). Cells were also divided into six groups:Control group, CSE group, PDTC group,40μg/ml Icariin group,80μg/ml Icariin group, and 160μg/ml Icariin group. The typeⅡpenumonocytes were identified by improved pap staining and electron microscope methods. The influence of Icariin to the cell proliferation was detected with CCK-8 kit. Cytokines levels in cell culture supernatant were measured by ELISA. Expression of IκB-αand p65 phosphorylation was detected by western blot. NF-κB DNA binding activity was detected by EMSA.Results:1. In vivo.Respiratory function in model group decreased significantly than normal group (P<0.05). However, after intervention of icariin, the respiratory function of rats recovered gradually, and there was no significant difference between normal group and high-dosage-icariin group (P>0.05). HE staining pathological section of lung tissue in model group showed alveolar septa destruction, enlargement of alveoli, infiltration of alveolar macrophages and neutrophils, and increasing of goblet cell. However, the pathological changes lessened gradually after intervention of icariin, and the pathological change of high-dosage-baicalin group was the closest to the normal group. Under the optical microscope, average alveolar area of model group or DXM group was obviously larger than that of normal group (P<0.01). Middle or high-dosage-icariin group has not statistically significant change compared with normal group (P>0.05), but it has statistical significance compared with model group (P<0.05). Compared with normal group, total leukocyte and neutrophils percentage of model group increased obviously (P<0.05), the proinflammatory cytokines (IL-8, IL-6 and TNF-α) levels in serum and BALF also increased significantly (P<0.05), but anti-inflammatory cytokines (IL-10) decreased (P<0.05) and the expression of NF-κB p65 protein or mRNA in lung tissue decreased significantly (P<0.05). The results of DXM group were similar to model group (P>0.05). However, in three icariin groups, the proinflammatory cytokines (IL-8, IL-6 and TNF-α) levels in serum and BALF decreased gradually (P<0.05), as well the expression of NF-κB p65 protein or mRNA in lung tissue. IL-10 levels in serum and BALF of icariin groups increased gradually compare to model group (P<0.05). CRH and CORT levels in the serum of icariin groups increased gradually, as well GR mRNA level in lung and CRH mRNA in Hypothalamus, and CORT mRNA in adrenal gland. The high-dosage-icariin group was close to nomal group, and there was no statistical significance (P>0.05).2. In vitro.Identified by staining and electron microscope, the primary cultured cells were the Type II pneumocytes (ATⅡ). Result of CCK-8 exhibited that icariin at the doses of 40μg/ml,80μg/ml and 160μg/ml had no cytotoxicity to the ATⅡ, while CSE inhibited ATⅡProliferation in a concentration-and time-dependent manner. ELISA results showed that proinflammatory cytokines (IL-8, IL-6 and TNF-α) expression in CSE group increased significantly compared with control group(P<0.05), while their expression in PDTC group was lower, similar to control group(P>0.05). IL-10 levels in CSE group decreased significantly compared to control group (P<0.05). However, IL-8, IL-6 and TNF-a in Icarrin groups decreased, IL-10 increased, and it shows dose-effect relationship. There was low P-p65 level and high quantity of IκB-αin control group examined by western blotting. CSE stimulating, the P-p65 increased, and IκB-αdecreased markly. After icariin intervention, P-p65 decreased, and IκB-αincreased gradually with the icarrin dosage rising. NF-κB DNA binding activity in CSE group increased markly compared with control group, whereas icarrin attenuated it.Conclusions:①Icariin has protective effect on SD rats'lung function against CS exposure induced COPD.②In vivo, Icariin can inhibit the infiltration of inflammatory cells, expression of proinflammatory cytokines and NF-Kb, but promote anti-inflammatory cytokines expression in CS exposure induced rat COPD model.③In vitro, Icariin can inhibit expression of proinflammatory cytokines and activation of NF-κB (phosphorylation of p65, degradation of IκB-αand NF-κB DNA binding activity), but promote anti-inflammatory cytokines expression in CSE stimulated ATⅡ.④Icariin has inhibitory action on CS exposure-induced inflammation of rat COPD and inflammation of CSE stimulated rat ATⅡ, it potentially achieves through inhibiting NF-κB signaling Pathways. The effectiveness increases with the icarrin dosage rising.⑤The potential mechanism of Icariin inhibition on rat COPD model is Icariin can activate HPA axis to increase endogenous Hormone secretion, increase GR expression in lung, inhibit NF-κB expression and its inflammation, thus attenuate rat COPD inflammation.