Effects Of Bushen Chinese Herbal Component Icariin On Eosinophils Apoptosis In Bronchial Asthmatic Mice

ObjectivesTo study the effects of bushen Chinese herbal component icariin on eosinophils apoptosis in asthmatic mice. And Further effects of icariin on apoptosis relative factors:IL-1, IL-5, GM-CSF, Bcl-2, Bax and NF-KB p65. From the way of eosinophils apoptosis, we discuss the effective mechanisms of the treatment of asthma by Bushen Chinese herbal component icariin.Methods(1)Animals:48 female Balb/c mice,3-6 months old, illumination time was fixed, water and food were taken unconventionally. (2)Drug preparation:Icariin was provided by Shanghai Ronghe Pharmaceutical Technology Co.,Ltd. Batch No.: 091209.(3)48 Balb/c mice were randomly divided into 6 groups. Group A was normal control group. Group B was allergic asthma medel group, which was sensitized by ovalbumin(OVA) at the 1st and the 8th day and began to challenged by OVA at the 15th day once a day for 6 weeks. Group C, D, E, F were sensitized and challenged by OVA as same as Group B. (4)The group D, E, F were intragastric administrated with respective dose of icariin(25mg/kg,50mg/kg, 100kg/kg) at fifteenth day once a day and for 6 weeks. Group C was introperitioneal injected with Dexamethasone 0.5ml/kg. Group A and B were introperitioneal injected with physiological saline. All the mice were anesthetized 6-12 hours later after the last challenge by OVA, then samples were taken. (5) Lung histological slices, bronchoalveolar lavage fluid (BALF) eosinophils counting. Using ELISA th test the level of serum OVA-IgE in mice. Eosinophils apoptosis were detected by the TUNEL method, the Bcl-2 and Bax protein expression were examined by the immunohistochemistry method. The expression of NF-KB p65 protein was tested by Western blotting and the serum cytokine level was measured by lumenex 200. Then we use SPSS 17.0 to carry on data statistical analysis processing to the data.Results (1).observation of asthmatic clinical symptoms Compared to the normal group, the mice of asthma model presented asthmatic clinical symptoms, such as itching, sneeze, mouth breathing, abdominal muscle contraction, incontinence, some symptoms like withered hair and emaciation had also performed.(2).Airway hyperresponsiveness(Penh%) The mice in normal control group showed polypnea, mouth breathing,opisthotonos, sialorrhea and cyanosis at the dose of Mch 25-50mg/ml.however, compared to the normal control group, the mice in asthma model group performed these symptoms early, most of the mice showed the symptoms at the dose of Mch6.25-12.5mg/ml,sepecially at the dose 12.5mg/ml. Airway hyperresponsiveness in Dexamethasone group and icariin groups were significantly decreased.(3).The observation of lung histopathology. Compared to the control group, the inflammation of main airway of asthma model group had been changed obviously, and the infiltration of inflammatory cells especially eosinophils markedly increasd.(4).The serum OVA-IgE level The serum level of OVA-IgE of mice in asthma groups had been increased (P<0.05). The OVA-IgE level of Dexamethason group, icariin low-dose group, icariin middle-dose group and icariin high-dose group are significantly lower than asthma model group (P<0.05). Among the groups icariin middle-dose group is the lowest (P<0.05).(5).The counts of eosinophils in BALF in each group mice The counts of eosinophils in BALF in mice treated with Bushen Chinese herbal components were decreased than those in asthmatic model group (P<0.05). Among the groups, the counts of eosinophils of BALF in icariin middle-dose group mice decrease the most (P<0.05).(6).The serum level of apoptosis-related cytokines The serum level of GM-CSF:The serum level of GM-CSF of normal control group is higher than other five groups (P<0.05). Compared with the serum level of GM-CSF of asthmatic model group, the serum levels of GM-CSF of thr four treatment groups are significantly lowered.The serum level of IL-5:Compared with normal control group, the serum level of I1-5 in asthmatic model group is higher. The serum levels of IL-5 of treatment groups (Dexamethasone group, icariin low-dose group, icariin middle-dose group and icariin high-dose group) are lower than asthmatic model group.(7).The eosinophils apoptosis index As compared with the model group, The apoptosis ratio of eosinophils were higher in the four treatment groups (P<0.05); compared with dexamethasone group, icariin middle and high dose group showed no significant difference (P>0.05).(8). Bcl-2, Bax protein expression in lung of each group mice Expression of Bcl-2 protein:normal control group in mice lung tissue and airway wall area of low Bcl-2 positive, the model group lung expression of Bcl-2 positive area was significantly higher than normal control group(P<0.05); with the model group, the lung tissue of treated mice and Bcl-2 positive airway area was significantly lower (P<0.05); the expression of Bcl-2 protein in icariin low dose group in mice lung was higher than those in the dexamethasone group (P<0.05); dexamethasone group Bcl-2 positive area was lower than icariin low-dose and high-dose group, but the difference was not statistically significant (P>0.05).Expression of Bax protein:normal control group mice lung expression of Bax protein was high, compared with the normal group, model group mice lung Bax-positive area was significantly lower (P<0.05); the lung tissue of treated mice and Bax positive area was significantly higher than model group (P<0.05); dexamethasone group lung expression of Bax protein were larger than those of icariin low-dose group (P<0.05); there were no significant difference between icariin middle and high dose group and dexamethasone group(P>0.05).Bcl-2, Bax expression ratio of each group mice:compared with normal control group, the expression of Bcl-2 protein in asthmatic group mice were high, expression of Bax protein were low, Bcl-2, Bax ratios were high (P<0.05); dexamethasone group and icariin low, medium and high dose group were lower than the ratio of Bcl-2/Bax lung asthma model group (P<0.05); Epimedium glycosides low, medium and high dose groups dose Bcl-2/Bax ratio was lower than in the dexamethasone group, icariin low dose group was significantly lower than dexamethasone group(P<0.05). (9). NF-κB p65 protein expression in the lung of each group miceCompared with the control group, asthmatic mice lung tissue NF-κB p65 protein expression increased (P<0.05); dexamethasone in lung tissue expression of NF-κB p65 protein reduction; with model group, low icariin, middle and high dose group in asthmatic mice lung tissue NF-κB p65 protein expression was decreased(P<0.05); which doses of icariin in lung tissue of mice NF-κB p65 protein was the lowest(P<0.05).ConclusionsFrom our study we do observe the Chinese herbal component icariin could significantly promote the eosinophils apoptosis in the lungs of asthmatic mice. It may reduce eosinophil apoptosis inhibitory cytokine IL-5 and GM-CSF levels. It may also reduce anti-poptotic protein Bcl-2 expression and increase apoptosis gene Bax gene protein expression and transcription factor NF-κB p65 protein expression. We observe that the medium dose groups’ therapeutic effect is better than the low and high groups.It indicate that icariin’ promoting eosinophils apoptosis effect may acts on a dose dependent manner.however it need a further study in vitro to prove it.