Roles For Hrd1in The Endoplasmic Reticulum Stress Induced By Cigarette Smoke Extract In AECⅡ

[Objective] To observe the apoptosis rate of primary cultured rats aleolar type IIepithelial cells (AECII) exposed to cigarette smoke extract (CSE) at different time. Toevaluate the expression change of Hrd1，endoplasmic reticulum stress (ERS) relatedfactors and ERS induced apoptosis(ERSIA) related factors of AECII. To observe theimpact of Hrd1gene intervention to the expression levels of factors above in AECIIexposed to CSE. To explore the effect of hrd1in the pathogenesis of COPDconcerning lung structure cell apoptosis.[Methods] Primary and subcultured of AECII; Preparation of CSE;Silence orupregulate Hrd1gene by infect AECII via lentivirus carried interference or encordingsequence.At different times of rat AECII exposed to10%CSE,apoptosis rate weredetected before and after infection by hoechst；Hrd1, PERK, P-PERK, eIF2α, P-eIF2α,IRE1, P-IRE1, ATF6, caspase12, JNK, P-JNK and CHOP expression were assayed atdifferent times of AECII exposed to10%CSE before and after cell infection. ThemRNA expression was measured by Realtime-PCR and protein expression levelsassayed by Western blot technique inspection.[Results]：1. There is no obvious morphologic and physiologic difference between theoriginal generation and the third generation of AECII.2. Hoechst staining shows: apoptosis rate of AECII exposed to10%CSE rising.The apoptosis rate of infected cell increased significantly compared with wild type atevery different time point. The apoptosis rate of infected cells was lower than wildtype cells at corresponding time group.3.Westen blot results showed that the total PERK,eIF2α and IRE1proteinexpression had no statistically significant differences between the control group and the CSE groups(P>0.5). P-PERK expression beginning to rise from3h group, the12hgroup is peak, then gradually reduced but still higher than the control group, P-eIF2α,P-IRE1and ATF6expression keep increasing with the extension of time exposed toCSE. After successfully knocked down hrd1gene,P-PERK protein expression with alittle increase at3h group then decreased to the bitter end, P-eIF2α and ATF6αprotein expression was a consistent decline With prolonged CSE stimulation, P-IRE1expression gradually began to rise from3h group, the12h group of peak,24h,48hexpress decreased.Under the condition of Hrd1gene upregulated, P-PERK proteinexpression with the role the extension of time was a consistent decline; P-eIF2gradually began to rise from3h group, the12h group of peak,, then according to theextension of CSE effect time significantly reduced; P-IRE1expression increasing astime goes by; ATF6express gradually began to reduce from3h group,6h group isminimum,12h group start increasing and hold on in24h and48h groups.4. The total JNK protein expression is similar to PERK while P-JNK and CHOPto P-IRE1,caspase12to P-PERK. After successfully knocked down hrd1gene, CHOPprotein expression from3h group beginning to rise, the12h is highest,with role afterthe extension of time of CSE gradually declined but expression always higher thanthose in the control group. Under the condition of Hrd1gene upregulated, CHOPprotein expression continue to rise by CSE effect.[Conclusion]：1. As supportted with appropriate in vitro culture conditions, the3rd generationAEC Ⅱ is capable to maintain relatively stable diferentiation phenotype, and couldbe used in vitro experiments.2. CSE could induce AECII suffered ERS and apoptosis, PEAK/IRE1/ATF6three transmembrane signal pathways are engaging in the CSE induced ERS,Caspase12/JNK/CHOP three signaling pathways are involved in the AECIIapoptosis induced CSE mediated.3. Hrd1plays an important role in protecting AECII from apoptosis inducedby CSE.