Resveratrol reduces the apoptosis induced by cigarette smoke extract by upregulating MFN2

Abstract

Purpose: The aim of this study was to investigate the effect of resveratrol (RSV) on cigarette smoke extract (CSE)-induced cell apoptosis and mitochondrial dysfunction in vitro, as well as changes in the MFN2 expression level.

Methods: Cultured human bronchial epithelial (HBE) cells were initially treated with CSE to induce apoptosis, followed by incubation either with or without RSV. Numerous techniques were used to evaluate the outcomes of the present study, including a cell counting kit-8 assay, real-time quantitative polymerase chain reaction (real-time qPCR), western blotting, JC-1 fluorescence, Hoechst 33342 staining, Annexin V-PI flow cytometry apoptosis analyses, and siRNA technology.

Results: A 24 h incubation in 3.5% CSE induced apoptosis in HBE cells, and pretreatment of HBE cells with RSV (20 μM) significantly suppressed the CSE-induced apoptosis, prevented the CSE-induced decrease in MFN2 levels, suppressed BAX translocation to the mitochondria, and prevented mitochondrial membrane potential loss and cytochrome C release. However, following the transfection of MFN2 siRNA, the anti-apoptotic effects of RSV were significantly attenuated.

Conclusion: The results of the present study demonstrated that RSV may protect bronchial epithelial cells from CS-induced apoptosis in vitro by preventing mitochondrial dysfunction, and MFN2 may be associated with the anti-apoptotic functions of RSV in HBE cells.

Fig 1. Effects of CSE on cell viability and apoptosis in HBE cells.

(A) HBE cells undergoing 0% and 3.5% CSE exposure for 24 h showed typical characteristics of apoptosis, as detected by Hoechst 33342 staining. Apoptotic cells showed strong fluorescent signals (bright turquoise color) outlining the chromatin-condensed nuclei, whereas normal cells appeared weakly stained. (B) CCK-8 assay showed the influence of different concentrations of CSE at different times on HBE cell viability, and the 24 h of treatment with 3.5% CSE was the optimal choice. h, hours.

Fig 2. Effect of different concentrations of RSV on MFN2 protein expression in HBE cells and on the MFN2 mRNA levels in each group and the examination of apoptosis by Hoechst33342 staining fluorescence imaging as well as cell viability by CCK-8 assay.

(A) With the increase of the RSV concentration, the expression of MFN2 increased, except for the 40 μM RSV group,there were no statistically significant differences in other RSV groups when compared with the control group (p>0.05). (B) The MFN2 mRNA level in the 5 μM RSV+CSE and 20 μM RSV+CSE groups was increased compared with that in the CSE group, and the 20 μM RSV+CSE group had a higher expression level than the 5 μM RSV+CSE group (p<0.05). However, the MFN2 mRNA level was not significantly affected by 1 μM RSV+CSE compared with that in the CSE group. (C) a; Control group; b: RSV group; c: CSE group; d: RSV+CSE group. Hoechst staining of HBE cells. HBE cells pretreated with RSV exhibited fewer apoptotic cells (d) than that of the non-RSV-treated group (c), following a 24 h exposure to 3.5% CSE. Apoptotic cells are strongly stained bright turquoise. (D) CCK-8 assay. The RSV+CSE group has a higher cell viability than CSE group. *: p<0.05, #: p>0.05.

Fig 3. The protective effects of RSV against CSE-induced apoptosis and the activity of MFN2 and its preventative effects of the release of cytochrome C from mitochondria to cytosol and BAX translocation.

(A) BAX began to translocate to mitochondria in the CSE group, cytochrome C in the cytoplasm began to increase in the CSE group compared with the normal group (p<0.05). However, RSV pretreatment prevented the release of cytochrome C from the mitochondria to cytosol and prevented the translocation of the BAX from the cytosol to mitochondria. (B) MFN2 levels were decreased in the CSE group compared with those in the control group (p<0.05). However, RSV pretreatment prevented the decrease of MFN2 expression (p<0.05). (C) Mitochondrial membrane potential (ΔΨm) analysis by JC-1 fluorescence. In the CSE group, ΔΨm levels were lower than those in the control groups (p<0.05). However, ΔΨm was significantly increased in the RSV+CSE group compared with the CSE group (p<0.05).

Fig 4. MFN2 siRNA attenuates the protective effects of RSV against the apoptosis induced by CSE in HBE cells.

(A) The transfection efficiency of HBE cells was detected by fluorescence microscopy, which showed that more than 90% of cells were labeled (red fluorescence). (B-C) Real-time qPCR and western blot analysis were used to test three MFN2 RNA interference target sequences for gene silencing efficiency. The results show that the first silence sequence (si-h-mfn2_001: CGGCAAGACCGACTGAAAT) exhibited the highest efficiency, and the optimal intervention time was 72 h. Therefore, the first RNA interference target sequence was selected for the following experiments. (D) CSE-induced apoptosis was markedly attenuated by RSV pretreatment (RNAi + RSV + CSE); however, this protective effect was abolished by MFN2 gene knockdown (MFN2 siRNAi+ RSV + CSE). (E) Western blot analysis showed apoptosis marker molecules in all of the groups. BAX began to translocate to mitochondria in the CSE group, and cytochrome C in the cytoplasm began to increase in the CSE group compared with that in the MFN2 siRNA group (p<0.05). However, the effects of RSV to prevent the release of the cytochrome C from the mitochondria to the cytosol and prevent the translocation of the BAX from the cytosol to the mitochondria were decreased. There were no differences between the MFN2 siRNA+ CSE group and the MFN2 siRNA+ RSV+CSE group (p>0.05). (F) Western blot analysis showed that the RSV pretreatment did not increase the MFN2 levels in the MFN2 siRNA groups compared with the negative control siRNA groups. *: p<0.05, **: p<0.01, ***; p<0.001, #: p>0.05.

Fig 5. The protective effects of RSV were markedly reduced following the siRNA-mediated knockdown of MFN2 compared with those in the negative RNAi groups, as shown in the MFN2 siRNA+RSV+CSE group and the RNAi+RSV+CSE group.

(A-B) As for the MFN2 siRNA groups, an increased number of apoptotic cells was detected, together with a much higher apoptotic rate than the control RNAi groups (B), which was consistent with the changes in the mitochondrial membrane potential (ΔΨm) analyzed by JC-1 fluorescence (A). Group 9 had a lower red/green rate than group 8. *:p<0.05, **:p<0.01, #:p>0.05.