| Objective:To investigate whether nicotine(NIC)aggravates podocyte injury induced by high glucose through apoptosis and endoplasmic reticulum stress(ER stress).Methods:After the cells were resuscitated,they were incubated in RPMI-1640medium containing 10%fetal bovine serum,100 IU/ml penicillin,100 UG/ml streptomycin and 4 ng/ml mouse interferon-γ in a 5%CO2 incubator at 33℃.When the cell density in the medium reached about 80%,the cells were digested with 0.25%trypsin and passaged as needed.After passage,10%fetal bovine serum,100iu/ml penicillin and 100ug/ml streptomycin RPMI-1640medium were replaced.The cells were cultured in 37℃ 5%CO2 incubator for 2 weeks to make them mature.The medium was changed every 2 days or so.The cells were divided into five groups:normal control group(NC group):containing 5.6 mmol/L D-glucose;mannitol group(Mn group):containing 5.6 mmol/L D-glucose+19.4 mmol/L D-Mannitol;high glucose group(Hg group):containing 30 mmol/L D-glucose;nicotine group(NIC group):containing 5.6 mmol/L D-glucose+0.8 mmol/L nicotine;high glucose+nicotine group(NIC+Hg group):containing 30 mmol/L D-glucose D-glucose+0.8 mmol/L nicotine.The treatment time of each group was 24 hours.Cell counting Kit(CCK-8)was used to detect the cell activity.The production of reactive oxygen species(ROS)was detected by DCFH-DA.Apoptosis was detected with FITC annexin V apoptosis detection kit(BD Biosciences,New York,USA)according to the manufacturer’s instructions.The expressions of chop,BiP,Bcl-2 and Bax were detected by Western blot.Results:1.Compared with NC group,the cell viability of Hg group was lower;NIC group was lower;Hg + NIC group was lower.2.Compared with NC group,the expression of ROS increased in Hg group,NIC group and NIC+Hg group.3.Compared with NC group,apoptosis increased in Hg group;apoptosis increased in NIC group;apoptosis increased in NIC+Hg group.4.Compared with NC group,the expressions of chop,BiP,Bcl-2,Bax and other proteins in Hg group and NIC group were increased;the expressions of chop,BiP,Bcl-2,Bax and other proteins in Hg+NIC group were further increased(all P<0.05).Conclusion:Nicotine aggravates the podocyte injury induced by high glucose,which may be related to the synergistic induction of endoplasmic reticulum stress and apoptosis by nicotine and high glucose. |
