Protective Effects Of Magnesium Isoglycyrrhizinate Against Simulated Ischemia-reperfusion Injury In Hepatocytes And The Related Mechanisms

Objective:This study is aimed to investigate the protective effects of Magnesiumisoglycyrrhizinate(MgIG) preconditioning against simulated hepatocytes ischemia-reperfusion injury and research the possible mechanisms,provide experimentalfoundation and theoretical basis for the application of MgIG against hepatic ischemia-reperfusion injury in the future.Methods:The human liver cell line HL-7702were cultured in vitro until they were inlogarithmic growth phase:1.The cells were inoculated into96-well plates,and wererandomly divided into ischemia-reperfusion group(IR group) and preconditioninggroups of MgIG， accordingly preconditioned with different concentration(0,10-2,10-1,1,10,100mg/ml) of MgIG for24h after the cells adhered,and then after6h ofsimulated ischemia and4h of simulated reperfusion conditions,the absorbance(A)were measured by MTT test;2.The cells were inoculated into culture plates、costars oron slides,and then were randomly divided into IR group,low,medium and highconcentration group,correspondingly preconditioned with different concentration(0,0.1,1,10mg/ml) of MgIG for24h after the cells adhered, and then after6h ofsimulated ischemia and4h of simulated reperfusion conditions,the cells andcellculture supernatant fluid were collected and used for the following tests：①Observe the apoptosis of HL-7702cells by making AO/EB staining;②Detected theALT,SOD, MDA,IL-1β,TNF-α levels of every group by the corresponding kits;③Detected the expression level of bcl-2、Bax、NF-ΚB mRNA in every group by Realtime quantitative fluorescence PCR test.Results:1.MTT assay indicated that MgIG could improve the absorbance(0.285±0.017、0.320±0.008、0.341±0.019) of HL-7702cells which suffered from ischemia-reperfusion injury in the corresponding concentrations(0.1、1、10mg/mL) groups,compared with the IR group(0.227±0.013),the difference were statistically significant (P<0.01),When the concentration of MgIG increased to100mg/ml,the HL-7702cellswere observed death,When the concentration of MgIG decreased to0.01mg/ml,although the absorbance(0.235±0.014) increased compared with the IR group(0.227±0.013),but there was no statistical significance(P>0.05);2.The results of AO/EB staining showed that the apoptosis rate(0.5763±0.0405)of low-concentration experimental group was lower than the IR group (0.7124±0.0581),The difference was statistically significant(P<0.05),the apoptosis rate of themiddle-concentration experimental group and the high-concentration experimentalgroup(0.2866±0.0619、0.1261±0.0439) were both lower than the IR group(0.7124±0.0581),and the difference were statistically significant(P<0.01);3.In the low,middle and high concentration groups,ALT(21.27±1.58、17.03±0.87、14.79±1.54u/L) were lower than the IR group(24.65±1.22u/L),the differencewere statistically significant(P<0.01);4.In the low,middle and high concentration groups,SOD(16.99±1.25、20.22±1.35、23.76±0.99u/mL) were higher than the IR group(12.52±1.80u/mL),thedifference were statistically significant(P<0.01);5.In the low,middle and high concentration groups,MDA(9.84±1.14、5.64±0.87、3.93±0.59nmol/mL) were lower than the IR group(11.89±0.82nmol/mL),thedifference were statistically significant(P<0.01);6.In the low,middle and high concentration groups,IL-1β(33.34±1.30、30.51±0.76、28.35±1.24pg/ml) and TNF-α(58.75±4.05、40.04±4.74、29.22±2.31pg/ml) wereboth lower than the IR group(37.07±1.18pg/ml)、(66.43±3.28pg/ml),the differencewere statistically significant(P<0.01);7.The results of Real time quantitative fluorescence PCR showed that theexpression of bcl-2mRNA(3.350±0.151、9.279±0.350、15.290±0.756) were higher inthe low,middle and high concentration groups than that in the IR group(0.928±0.068),the difference were statistically significant(P<0.01),the expression ofbax mRNA(0.743±0.035、0.265±0.065、0.119±0.019) and NF-ΚB mRNA (0.597±0.062、0.248±0.067、0.141±0.029) were both lower in the low,middle and highconcentration groups than that in the IR group(0.945±0.063)、(0.962±0.034),and thedifference were statistically significant(P<0.01). Conclusion:MgIG could alleviate hepatocytes ischemia-reperfusion injury,the mechanismsmay be related to that MgIG could improve hepatocytes’ vitality,inhibit lipidperoxidation,restrain inflammation reaction, reduce the cells apoptosis.