Roles For Derlin-1 In The Endoplasmic Reticulum Stress Induced By Cigarette Smoke Extract In AECⅡ

Objective Verify whether inhibition of Derlin-1 expression increased cigarette smoke (CSE) induced RLE-6TN cell apoptosis rate,and its relation with endoplasmic reticulum stress (ERS) mechanism, thus preliminary exploration the role of derlin-1 in cigarette smoke induced RLE-6TN cells in the endoplasmic reticulum stress induced apoptosis..Methods:The rat AECII training;Interference with carrying Derlin-1 sequence of siRNA infect AECII Derlin-1 gene silence;CSE preparation;Treated with 10% CSE AECII 0 h,3 h,6 h,12 h,24 h:flow cytometry instrument testing before intervention apoptosis rate; Before and after the intervention,the expression of P-IRE1, Hrd1, P-JNK and CHOP was measured by Immunofluo rscence technique, Western blot, Realtime PCR technique.Results:1.Flow cytometry technology to detect cell apoptosis rate: RLE-6TN cells in 10% CSE processing Oh,3h,6h,12h,24h after type Ⅱ lung epithelial cells of rats in no-load group of early apoptosis rate is: 1.3%,2.2%,5.6%,8.3%,5.6%;12.2%, Silent group of early apoptosis rate was 15.4%,15.7%,33%,43.3%,48.9%;Light and silence group rate of cell apoptosis with the extension of time is on the rise, cell apoptosis in silent group was obviously higher than that of light group, with significant difference (P< 0.05).2.1mmunofluorescence technique showed that the natural growth of RLE-6TN cells by CSE, Derlin-1 protein expression levels (0.117±0.011) higher than that of control group (0.095±0.005), with significant difference (P<0.05); Derlin-1 silent group of RLE-6TN cells by CSE, Derlin-1 protein expression levels (0.0640±.003) higher than that of control group (0.014±0.001), with significant difference (P< 0.05).Derlin-1 before and after the interference CSE Derlin-1 protein expression level processing group were increased.3.Westen blot results showed that light group of RLE-6TN cells in CSE 0 h,3 h,6h,12h,24h. Derlin-1 express respectively (1.76±0.19, 1.97±0.223.75±0.23,4.85±0.70,1.01±0.11),the highest protein expression in 12h,24h group reduced expression, different time points between two groups of comparison, had statistical significance (P< 0.05).Silent group of Derlin-1 protein expression, respectively (0.18±0.05,0.34±0.08,0.49±0.07,0.66±0.09,0.89±0.12), early role in CSE Derlin-10 h,3 h,6 h expression has no obvious change, there was no statistically significant difference (P>0.05), the late 12 h,24 h group higher expression, with statistical significance (P<0.05);Rt-PCR results show that the Derlin-1 mRNA test results are consistent with the Westen blot.4.Westen blot results showed that light group total IRE1, P-IRE1, Hrdl, total JNK, P-JNK, CHOP protein in CSE 0 h,3 h,6 h,12 h,24 h group of expression (15.08±2,17.42±1.44,15.38±2.02,17.24±1.24,14.18±1.14; 0.95±0.01,1.62±0.02,2.26±0.03,7.13±0.31,9.12±0.20; 0.21±0.01,0.69±0. 08,1.21±0.23,1.70±0.34,2.18±0.40; 0.97±0.07,0.94±0.11,1.11±0.06,1.05 ±0.04,1.12±0.09; 0.29±0.02,0.66±0.02,0.63±0.01,0.60±0.11,0.71±0.15; 1.32±0.40,2.92±0.50,4.53±0.60,10.54±1.04,9.01± 1.4),the total IRE1,total JNK expression of different time points were no obvious difference (P< 0.05), P-IRE1, Hrdl, P-NK, CHOP the CSE prolonged increasing trend, compared at different time points between two groups of time, all have statistical significance, (P<0.05).Silent group total IRE1, P-IRE1, Hrdl, total JNK, P-JNK, CHOP protein expression with respectively CSE role in different time (1.67±0.14,1.62±0.21,1.74±0.45,1.78±0.39,1.69±0.25; 0.33±0.02,0.48±0.02,0.84±0.04,0.36±0.01,0.32±0.03; 0.32±0.01,0.38 ± 0. 03,0.42±0.04,0.48±0.05,0.56±0.07; 0.87±0.24,0.65±0.031,0.85±0.15,0.8 2± 0.033,0.872±0.19; 0.22±0.01;0.46±0.07,0.64±0.08,0.24±0.03,0.28± 0. 04; 0.41±0.06,0.80±0.08,1.70±0.19,1.99±0.21,1.60±0.19), the total IRE1, JNK protein expression compared with control group, no significant changes, statistics between each time point group no significant difference (P> 0.05);After gene silence Derlin-IP-IRE1, P-JNK,CHOP early higher expression, advanced gradually decline, P-IRE1, P-JNK in 3 h,6 h group compared with the other groups between two had statistical significance (P<0.05), while in 0 h,12 h,24 h group there was no statistically significant difference between (P> 0.05).Hrdl in CSE early 0 h,3 h,6 h expression has no obvious change, there was no statistically significant difference (P>0.05), the late 12 h,24 h group higher expression, was statistically significant (P<0.05).Silent group CHOP in 3 h,6 h,12 h time expression between with other groups at each time point between two comparison, had statistical significance (P< 0.05).No obvious difference between 0 h,24 h, (P> 0.05).5.RT-PCR results showed that silent group of Derlin-1 the Hrdl, IRE1, JNK, CHOP mRNA expression changes over time with Westen blot results are basically identical.Conclusion:CSE can induce AECII ERS and further mediated ERAD, cut ERAD Derlin-1 gene silencing effect, causes the type Ⅱ alveolar epithelial cell apoptosis induced by CSE increases, the results of the study suggest Derlin-1 May for smoking RLE-6TN cell endoplasmic reticulum stress caused by apoptosis plays a protective role.