Caspase-3 And Caspase-9 Expressions In The Eosinophils Of Asthmatic Rats Model And Effect Of Glucocorticoid

PrefaceEosinophil(EOS) infiltration in the airway is the characteristic of asthma. Recently, it has been found a failure in EOS apoptosis contributed to the prolonged EOS living, which was related to asthma inflammation, but the exact mechanism of apoptosis is not clear. Caspases are those of the most gene related to apoptosis. They can induce cell apoptosis by changing cell cycles and activing anti - oxide mechanism. They have two varieties : one is regulation caspase, such as caspase-2 ,8,9, 10; another is effect caspase,such as caspase-3,6,7 and so on. Caspase-3 is required for the activation of caspases-2, -6 and -9 and also participates in a feedback amplification loop. Glucocorticoids are potent anti -inflammatory drugs in the asthma therapy, but the mechanism is still unknown .In our study, we remade asthma rat model and treated it using dexametha-sone(DEX).Then the expressions of apoptosis gene caspase-3 and caspase-9 in EOS of BALF were measured to study the effect of caspase-3 and caspase-9 in EOS apoptosis and the role of GCs in changing caspase-3 and caspase-9 expression.MaterialWistar rats; Jsc - Ok ultrasound nebulizer; Universal 32R zentrifugen; Ovalbumin; Dexamethasone; Rabbit anti -caspase-3; Rabbit anti -caspase-9; Percoll.MethodsThirty - six rats were divided into control,asthma and DEX groups. The animals were weighted about 200g at the time of testing. The animals of asthma/ DEX group were immunized by intraperitoneal(i. p. ) injection of 10% OVA on the first day of study, and challenged with an aerosol of 1% OVA on day 15 after the initial sensitization. The challenge was repeated 3 times. The animals of control group received mock sensitization with i. p. injection of sterile normal salt solution (NS) and were challenged with an aerosol of NS. After challenge, the animals of DEX group received i. p. injection of 0.5mg/kg DEX every day. The injection was repeated 5 times.The animals were anesthetived and bronchoaveolar lavage ( BAL) of left lungs (5 ml 6) were performed. And the right - upper lobes were fixed with 4% paraformaldehyde via a trachealcannula and immersion in the fixative for a minimum peroid of 1 week. The lung tissues were embedded in paraffin and cut in 5 jxm sections. The right - lower lobes were cut and stored - 70X1. 3ml BALF of each animals was counted for total cell numbers and different type of cells. The rest BALF was extracted EOS. The sections were stained with hematoxvlin and eosin ( HE). Western blotting was performed to examine caspase-3 and caspase-9 in EOS and lungs.Statistical analysis: SPSS was used to deal with all the data. Data were expressed as mean (SE) values, Analyses of variance (ANOVA) was performed , using the LSD test to adjust for multiple comparisons, A value of P <0. 05 was considered to indicate a significant difference.ResultsThe HE stained section of asthma group saw bronchial extraction and infiltration of inflammatory cells,which was mainly EOS.The results of BALF:The total number of cell and EOS% in asthma group (47.67 ±2.50) xl07/L,EOS%(16.25 ±0.69)%were higher than those ofthe control group(30 ±1.71) x 107/L, ,EOS% (6.69/ ±0. 37)% (P <0.01) ; and the two data of DEX group(33. 33 ± 1. 67) x 107/L, EOS% ( 12. 74 ± 0.97) % were lower than those of asthma group( P <0.01) .Caspase-3 in EOS of BALF:IDV in asthma group( 14186.67 ± 1661.6) was significantly low compared with that of control group (22026. 67 ±225. 57) and DEX group(21093.33 ± 337.21) ( P < 0.05 ). caspase-3 in lung: IDV in asthma group(9048. 00 ± 124. 00) decreased compared with that of control group (14152.00 ±613.81) and DEX group( 12064.00 ±335.01) (P <0.05).Caspase-9 in EOS of BALF:IDV in asthma group(6066.67 ± 202.07 ) was relatively low compared with that of control group(7408. 33 ±564.29)and DEX group(7000.00 ±303.11) (P <0.05). caspase-9 in lung: IDV in asthma group (6762.00 ±530. 02) was low compared with that of control group (8722. 00 ± 449.09)andDEXgroup(9359.00±306.00)(P<0.05).DiscussionAsthma is a chronic disorder of airway and eosinophils is considered to be a major contributor to the chronic inflammation . It has been showed recently that there is a defect in eosinophils apoptosis in asthma. Eosinophils play a major role in the onset and maintenance of bronchial inflammation and tissue injury in asthma. like other leukocytes, eosinophils present in excessive numbers in inflamed tissues are removed by apoptosis. This phenomenon, also called' programmed cell death' , allows elimination of dangerous or redundant cells, thereby ensuring maintenance of tissue homeostasis. It has been suggested that a defect in eosinophil apoptosis would participate in the development and persistence of allergic airways inflammation in asthma. Eosinophil apoptosis are closely regulated by various stimuli, including glucocorticoids. These stimuli have been shown to alter the expression and function of different molecules involved in the cascade of events characterising the apoptotic process, particularly caspase family. Caspases are key molecules in the control of apoptosis, but relatively little is known about their contribution to eosinophil apoptosis.Our study showed that EOS of BALF expressed there is a decrease incaspase-3 and caspase-9. The low expression of caspases played an important role in reducing apoptosis of inflammatory cell. Glucocorticoid has been proven to be one of the most efifective agents in asthma. We found the total number of cells and percent of EOS in BALF of DEX group dropped significantly, it showed glucocorticoid functioned partly by decreasing the number of EOS. The expression of caspase-3 in EOS increased and expression of caspase-9 increased also after using DEX, which indicated that glucocorticoid had a key role in regulating EOS apoptosis.Our study suggested airway inflammation in asthma was correlated with cell apoptosis. Glucocorticoid reduced the airway inflammation by regulating caspase-3 and caspase-9, which might play the important part in resisting asthma airway inflammation.Conclusion1. There was increased EOS in the airway of asthma.2. Glucocorticoid could suppress infiltration of EOS in airway of asthma. This suppression is related to EOS apoptosis induced by glucocorticoid, which might be the explanation of the mechanism of glucocorticoid treatment.3. The expression of caspase-3 in EOS and lung tissue of asthma is low, which suggested caspase-3 curtailed EOS apoptosis in asthma airway, participating in regulating asthma airway inflammation.4. The expression of caspase-9 in EOS of asthma is low also, which indicated caspase-9 induced EOS apoptosis.