| Objective: to observe the effect of docosahexaenoic acid(DHA)on the apoptosis of A549 and HTB-182 cells,and to explore is its mechanism of endoplasmic reticulum stress.Methods:1.A549 and HTB-182 cells were divided into 6 groups,respectively.Then they were exposed to DHA at 0μM,20μM,40μM,80 μM,160μM and 320μM for 24 h,CCK-8 assay was used for cell viability.and then to calculate the IC50.2.A549 and HTB-182 cells were divided into control group and DHA treatment group,respectively.The 80 μM DHA was added to the model group.CCK-8 assay was used for cell viability at 0h,3h,6h,12 h,24h,48 h.and then to calculate the inhibition rate.3.A549 and HTB-182 cells were divided into control group and DHA treated group,respectively.After 24 h incubation,Then the Annexin V-APC/7-AAD Apoptosis Detection Kit was used for detecting the apoptosis of cells.Western blot was applied to detect the expression of BIP and some endoplasmic reticulum stress-related apoptotic factors(like CHOP,JNK,p-JNK,Caspase-4).Meanwhile,Real-time PCR(RT-PCR)technology was applied to detect the mRNA of BIP and CHOP.Caspase-4 Assay was used for the activity.Results:1.Effects of different concentrations of DHA on cell proliferation: In A549 and HTB-182 cells,Compared with the control group,the OD value of 20μM and 40μM in DHA treated group is not statistically significant(P>0.05);While the OD value of 80μM,160μMand 320μM in DHA treated groups are gradually decreasing trend,and differences between each group are statistical significant(P<0.05).These results showed that DHA inhibited the proliferation of A549 and HTB-182 cells in a concentration dependent manner.The IC50 of DHA treated A549 cells and HTB-182 cells was 121.022μM and 138.417μM,respectively.Thus 80μM was chosen for the next experiment.2.Effects of different treatment time of DHA on Cell proliferation: In A549 and HTB-182 cells,Compared with the control group,the inhibitory rate of 12 h,24h and 48 h in DHA treated groups are gradually decreasing trend(P<0.05).The highest inhibitory rate of DHA on cells occured at 24 h,Thus 24 h was chosen for the next experiment.3.The apoptotic rate in each group: In A549 cells,the results showed that compared with the control group(3.30±0.21)%,the apoptotic rate in DHA group(12.85 ± 0.47)% were significantly increased,with significant difference(P<0.05).In HTB-182 cells,the results showed that compared with the control group(3.55±0.12)%,the apoptotic rate in DHA group(9.80±0.48)% were significantly increased,with significant difference(P<0.05).In control groups,the apoptotic rate in A549 cells and HTB-182 cellswere no significant difference(P>0.05).While in DHA treated groups,the apoptotic rate in A549 cells was higher than that of HTB-182 cells(P<0.05).4.Detection of related factors of endoplasmic reticulum: In A549 cells,the results of Western blot showed proteins of BIP was significantly expressed in the control group(0.36±0.06),and decreased in DHA group(0.13 ± 0.02)(P<0.05).While proteins expression of CHOP,p-JNK and Caspase-4 in DHA treated group(0.53±0.14),(0.72±0.12),(0.61 ± 0.11)were increased compared with control group(0.05±0.03),(0.31±0.06),(0.07±0.02)with statistical significance(P<0.05).Compared with the control group(0.90±0.19),the expression of JNK protein in DHA treated group(0.87 ± 0.12)showed no significant difference(P>0.05).The results of RT-PCR showed mRNA of BIP was significantly expressed in the control group(1.00±0.00),and decreased in DHA group(0.11±0.01)(P<0.05).While mRNA expression of CHOP in DHA treated group(3.06±0.21)was increased compared with control group(1.00±0.00)(P<0.05).In HTB-182 cells,the results of Western blot showed proteins of BIP was significantly expressed in the control group(0.35±0.07),and decreased in DHA group(0.17 ± 0.05)(P<0.05).While proteins expression of CHOP,p-JNK and Caspase-4 in DHA treated group(0.70±0.13),(0.73±0.11),(0.53±0.12)were increased compared with control group(0.14±0.03),(0.10±0.02),(0.10±0.03)with statistical significance(P<0.05).While compared with the control group(0.98 ± 0.18),the expression of JNK protein in DHA treated group(0.97±0.19)showed no significant difference(P>0.05).The results of RT-PCR showed mRNA of BIP was significantly expressed in the control group(1.00±0.00),and decreased in DHA group(0.37±0.02)(P<0.05).While mRNA expression of CHOP in DHA treated group(2.59±0.11)was increased compared with control group(1.00±0.00)(P<0.05).5.Detection of the Caspase-4 activity: In A549 cells,the results showed that DHA treated group(572.25±11.73)higher than that of the control group(35.26±2.63),with statistical significance(P<0.05).In HTB-182 cells,the results showed that DHA treated group(635.70±10.40)higher than that of the control group(35.33±1.31),with statistical significance(P<0.05).Conclusion:DHA can induce A549 and HTB-182 cell apoptosis,and inhibit its proliferation.The mechanism may be down-regulation of BIP,thereby inhibiting the unfolded protein response. |
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