Effect Of Theophyline On Glucocorticoid Resistance Of Mononuclear Cells Exposed To Tobacco Smoke Exposure And Mechanism Research

PART IEffects of Theophylline on IL-8release of Human mononuclearcells exposed to tobacco smoke exposureOBJECTIVE：To observe whether tobacco smoke exposure can cause humanmononuclear cells glucocorticoid resistance or not; whether theophylline canreduce smoke exposure human mononuclear cells exposed to tobacco smokeexposure glucocorticoid resistance or not.METHODS: U937cells were the research objects. Oxidative stress cellmodel of tobacco smoke exposure was made by cigarette smoke extract (CSE).The cells were grouped theophylline+CSE groups, CSE groups and controlgroups. To observe effect of Interleukin-8（IL-8）of each group induced sevendifferent concentrations of dexamethasone and Tumor necrosis factor-α （TNF-α）（10ng/L）.IL-8Levels in supernatant were measured by Enzyme-linkedimmunosorbent assay (ELISA).IL-8inhibition rate and dexamethasone halfinhibitory concentration (IC50-Dex) were calculated by Microsoft Excel2003.RESULTS：IL-8inhibition rates and IC50-Dex:The IL-8inhibition rates oftheophylline+CSE groups were lower than the control groups, but higher thanthat of CSE groups. The IC50-Dex of theophylline+CSE groups were higherthan the control groups but lower than that of the CSE groups (P<0.05)..Tobacco smoke exposure can increase the production of IL-8.Theophylline candecrease the production of IL-8significantly.CONCLUSION:1. Tobacco smoke exposure decreased the glucocorticoidsensitivity of Human mononuclear cells, resulting in glucocorticoid resistance.2.Theophylline can reduce the glucocorticoid resistance of Human mononuclearcells under tobacco smoke exposure, increase glucocorticoid anti-inflammatoryeffects. PART IIEffect of theophyline on HDAC2expression and PI3K-δ/AKT pathway ofHuman mononuclear cells exposed to tobacco smoke exposureOBJECTIVE：To explore whether theophylline can through inhibit PI3K-δ/AKT pathway that was activated by tobacco smoke exposure to increase theexpression of HDAC2resulting in reverse the glucocorticoid resistance ofhuman mononuclear cells under tobacco smoke exposure or not.METHODS: U937cells were the research objects. Oxidative stress cellmodel of tobacco smoke exposure was made by CSE and HDAC2–siRNA wastransfectioned into the cells to interference HDAC2expression. The cells weregrouped (1) theophylline+CSE groups, CSE groups and control groups.(2)HDAC2–siRNA groups, HDAC2-siRNA+theophylline groups, emptyplasmid missense oligonucleotide (scrambled oligonucleotide, SC) groups andcontrol groups.(3) theophylline+CSE groups, IC87114+CSE groups, CSEgroups and control groups.To observe effect of IL-8of (2)、（3） group induced seven differentconcentrations of TNF-α（10ng/L）.IL-8Levels in supernatant were measured by ELISA.IL-8inhibition rateand IC50–Dex were calculated by Microsoft Excel2003.The gene expression levels of histone deacetylase2(HDAC2) were analyzedby real-time fluorescence quantitative polymerase chain reaction (RT-PCR). The protein expression levels of Phosphoinositide-3-kinase (PI3K)、Phosphorylase-Protein Kinase B （P-Akt）、Protein Kinase B （AKT） andHDAC2were analyzed by western blotting.The protein expression levels of PI3K were analyzed byimmunofluorescence. The protein expression levels of P-Akt、 AKT wereanalyzed by Immunohistochemistry.RESULTS：1.IL-8inhibition rates and IC50-Dex:(1)The IL-8inhibitionrates of HDAC2-siRNA+theophylline group were lower than SC groups andcontrol groups but higher than that of HDAC2-siRNA, The IC50-Dex ofHDAC2-siRNA+theophylline group were higher than SC groups and controlgroups but lower than that of HDAC2-siRNA groups (P <0.05).(2) The IL-8inhibition rates of IC87114+CSE groups were lower than the control groups, buthigher than that of CSE groups. The IC50-Dex of IC87114+CSE groups werehigher than the control groups but lower than that of the CSE groups (P<0.05).Compared with IC87114+CSE group and theophylline+CSE group, thedifference of IL-8inhibition rate and IC50Dex had no statistical significance（P>0.05）. Knockdown HDAC2can increase the IL-8production of cells; buttheophylline or selective PI3K-delta inhibitors IC87114can decrease theproduction of IL-8significantly.2. HDAC2mRNA expression:(1) The HDAC2mRNA expression levels oftheophylline+CSE groups were higher than the CSE groups but lower than that of the control groups (P <0.05).(2) The HDAC2mRNA expression levels ofHDAC2-siRNA+theophylline group were higher than HDAC2-siRNA groupsbut lower than that of SC groups and control groups (P <0.05). Tobacco smokeexposure can decrease the gene expression of HDAC2; Knockdown HDAC2also can decrease the gene expression of HDAC2; Theophylline or IC87114cansignificantly increase the gene expression of HDAC2.3. PI3K protein, P-Akt protein and Akt protein and HDAC2proteinexpression levels:(1) The protein expression levels of PI3K had no statisticallysignificant differences in each group（P>0.05）.(2) The protein expression levelsof AKT had no statistically significant differences in each group（P>0.05）.(3)The P-AKT protein expression levels of theophylline+CSE groups were lowerthan the CSE groups but higher than that of the control groups (P <0.05).(4)The P-AKT protein expression levels of IC87114+CSE groups were lower thanthe CSE groups but higher than that of the control groups (P <0.05).Comparison of IC87114+CSE groups and theophylline+CSE groups, theprotein expression levels of AKT had no statistically significant differences.（P>0.05）.(5) The HDAC2protein expression levels of theophylline+CSEgroups were higher than the CSE groups but lower than that of the controlgroups (P <0.05).(6) The HDAC2protein expression levels of HDAC2-siRNA+theophylline group were higher than HDAC2-siRNA groups but lower thanthat of SC groups and control groups (P <0.05).(7) The HDAC2protein expression levels of IC87114+CSE groups were higher than the CSE groupsbut lower than that of the control groups (P <0.05). Comparison of IC87114+CSE groups and theophylline+CSE groups, the protein expression levels ofHDAC2had no statistically significant differences（P>0.05）. Tobacco smokeexposure can increase the protein expression of P-AKT, theophylline orIC87114can significantly decrease P-AKT protein expression levels. Tobaccosmoke exposure and knockdown HDAC2can decrease the protein expression ofHDAC2; Theophylline or IC87114can significantly increase the proteinexpression of HDAC2.CONCLUSION: Tobacco smoke exposure can activate the PI3K/AKTpathway to reduce the expression of HDAC2resulting in glucocorticoidresistance; theophylline and selective PI3K-delta inhibitors IC87114can throughinhibit PI3K/AKT pathway that was activated by tobacco smoke exposure toincrease the expression of HDAC2resulting in reverse the glucocorticoidresistance of human mononuclear cells under tobacco smoke exposure.