The Protective Role Of Sulforaphane And N-Acetyl-L-Cysteine On RLE-6TN Cell Against Injury Caused By Cigarette Smoke Extract

Background:Cigarette smoke is a main risk factor of COPD in the world. It leads to damage of lung epithelial cells and there involves variety of mechanisms. Oxidative stress is one of the mechanisms. Sulforaphane can protect cells from injury by activating the Nrf2-ARE pathway. N-Acetyl-L-Cysteine has been known for decades as its antioxidant ability and free radical cleaning ability. We used both SFN and NAC in our experiment.Objective:We observed the cytoprotective effect of SFN and NAC, and explored the relationship between Nrf2and them.Method:We used RLE-6TN cells and divided them into4groups, a control group without any treatment, a5%CSE group with merely CSE treatment, a SFN+5%CSE group with SFN pre-treatment for12h, and a NAC+5%CSE group with N-Acetyl-L-Cysteine and CSE co-treatment. MTT analysis was used to determine the cell viability and choose the fit concentration to treat cells. RT-PCR and western blot analysis were used to quantify the mRNA expression and protein level of Nrf2. Flow cytometry was used to detect cell cycle and cell apoptosis. ROS level was detected by using DCFH-DA. Cell nuclei were stained with Hoechst33258. Besides,1GFBP-2,1GFBP-3, IGFBP-5and P53, P21mRNA were detected and quantified by RT-PCR analysis.Result:According to the MTT assay, we choosed5%CSE and0.5μM SFN to treat or pre-treat RLE-6TN cells. The RT-PCR assay showed an obvious promotion of Nrf2(about8fold) by protection of sulforaphane and the Western Blot assay showed that sulforaphane and N-Acetyl-L-Cysteine can both promote Nrf2protein level. Flow cytometry assay indicated that CSE stimulation can cause the block of G1phase of cell cycle, while the situation was reduced from(%)68.89to50.47and56.09by pre-treatment of sulforaphane and adding N-Acetyl-L-Cysteine. After pre-treatment of sulforaphane and adding N-Acetyl-L-Cysteine, cell apoptosis caused by CSE was reduced from(%)78.08±0.97to6.12±0.33and12.35±1.16. After pre-treatment of sulforaphane and adding N-Acetyl-L-Cysteine, ROS level caused by CSE was reduced from(%)86.63±1.21to28.03±0.26and58.27±2.77. Quantification of IGFBP-2, IGFBP-3, IGFBP-5and P53, P21mRNA showed that IGFBP-3、1GFBP-5and P53mRNA promoted after stimulation of CSE. After pre-treatment of sulforaphane only IGFBP-5mRNA reduced significantly, while after adding N-Acetyl-L-Cysteine, IGFBP-3, IGFBP-5and P53mRNA reduced significantly.Conclusions:ln conclusion, we observed CSE can cause lung epithelial cells injury and apoptosis,also nuclei fragment and oxidative stress. Sulforaphane and N-Acetyl-L-Cysteine can reduce the injury and alleviate the cell oxidant burden. Cell cycle regulation may be linked with Nrf2expression. We also observed that sulforaphane and N-Acetyl-L-Cysteine can reduce IGFBP-3, IGFBP-5and P53mRNA level which may lead to influence of cell cycle and apoptosis.