红芪多糖通过Keap1-Nrf2通路抑制脂肪变性肝细胞铁死亡的研究OA

Objective To explore the mechanism by which hedysarum polybotrys polysaccharide(HPS)improves ferroptosis of steatotic hepatocytes through the kelch-like ECH-associated protein 1-Nuclear factor-erythroid 2-related factor 2(Keap1-Nrf2)signaling pathway.Methods Lentivirus transfection interfered with the expression of Nrf2 in LO-2 cells.The cells were cultured with 0.6 mmol·L-1 fatty acids for 12 hours to replicate the steatotic cell model.The cells were divided into group A(normal cells),group B(transfected with empty lentivirus),group C(normal cells+0.6 mmol·L-1 fatty acids for modeling),group D(transfected with empty lentivirus+0.6 mmol·L-1 fatty acids for modeling),group E(transfected with Nrf2 silencing lentivirus+0.6 mmol·L-1 fatty acids for modeling)and group F(transfected with Nrf2 silencing lentivirus+0.6 mmol·L-1 fatty acids for modeling+50 mg·L-1 HPS).All groups of cells were cultured for 24 hours.Oil red 0 staining was used to analyze the positive area of cellular lipid deposition(％Area);enzyme-linked immunosorbent assay(ELISA)was used to measure the contents of cellular lipid peroxides(LPO),4-hydroxynonenal(4-HNE),glutathione(GSH)and superoxide dismutase(SOD);fluorescent probes were used to detect cellular Fe2+and reactive oxygen species(ROS);real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR),Western blot and immunofluorescence were used to detect the expression levels of Nrf2,Keap1,glutathione peroxidase 4(GPX4)and solute carrier family 7 member 11(SLC7A11)messenger RNA(mRNA)and protein.Results The％Area of cell lipid deposition in hepatocytes of group A,group B,group C,group D,group E and group F were(10.97±1.57)％,(11.80±2.76)％,(30.59±1.72)％,(28.00±1.71)％,(42.04±2.98)％and(24.46±2.16)％,the contents of LPO in these six groups were(0.22±0),(0.23±0.02),(0.32±0.04),(0.34±0.04),(0.42±0.02)and(0.34±0.05)nmol·mgprot-1,the contents of SOD in these six groups were(101.26±0.12),(95.31±5.97),(61.08±8.55),(57.81±4.07),(20.03±3.82)and(44.26±8.90)U·mgprot-1,the contents of GSH in these six groups were(3.01±0.05),(3.27±0.10),(2.34±0.24),(2.25±0.13),(1.85±0.21)and(2.06±0.11)mgGSH·gprot-1,the contents of 4-HNE in these six groups were(15.95±0.63),(15.94±0.50),(23.12±2.40),(23.64±1.05),(26.35±0.69)and(24.18±1.20)ng·L-1,the contents of MDA in these six groups were(1.39±0.11),(1.50±0.01),(2.07±0.26),(2.15±0.04),(2.75±0.05)and(2.52±0.22)nmol·mgprot-1,respectively;Fe2+relative fluorescence intensity in these six groups were 9.65±2.82,11.66±2.53,32.11±1.60,33.32±1.27,34.91±1.26 and 20.00±2.35,the levels of ROS in these six groups were 22.73±1.17,23.90±0.26,31.67±0.95,30.40±0.53,46.87±0.98 and 37.80±0.78,respectively;the levels of Nrf2 mRNA in these six groups were 1.00±0.06,0.97±0.07,0.55±0.09,0.64±0.08,0.37±0.05 and 0.77±0.08,the levels of Keap1 mRNA in these six groups were 1.00±0.10,0.98±0.05,1.61±0.04,1.57±0.04,2.07±0.06 and 1.41±0.09;the levels of GPX4 mRNA in these six groups were 1.00±0.11,1.08±0.10,0.50±0.09,0.48±0.06,0.23±0.03 and 0.69±0.13;the levels of SLC7A11 mRNA in these six groups were 1.00±0.07、1.07±0.00、0.66±0.09、0.67±0.04、0.38±0.13 and 0.84±0.10,respectively.Compared with the group A and B,there were statistically significant differences in the above indexes of group C and D(all P＜0.05);compared with the C,D group,there were statistically significant differences in the above indexes of group E(all P＜0.05);compared with the group E,there were statistically significant differences in the above indexes of group F(all P＜0.05).Conclusion HPS promotes Nrf2 expression through the Keap1-Nrf2 pathway,enhances cellular antioxidant capacity,reduces lipid peroxidation in hepatocytes,and thereby inhibits ferroptosis,protecting hepatocytes.