Promotion Effect Of Hydrogen Sulfide On Tolerability Of HepG-2Cells To ADM And The Underlying Mechanisms

【Objective】The hepatocellular carcinoma cell lines HepG-2treating with adriamycin（ADM） was deserved as experimental model （ADM-killing HepG-2model）,and then the role of hydrogen sulfide （H₂S） in tolerance of HepG2to ADMand its mechanism were investigated on the basis of experimental model.【Methods】1. HepG-2cell were cultured in vitro, treated with ADM and NaHS. TheIC50value of ADM was analyzed by CCK8assay. In view of ADM IC50,experiment was divided into four groups, containing control, ADM,ADM+NaHS, NaHS group. HepG-2apoptosis was detected by propidiumiodide （PI） staining and flow cytometry （FCM）. Meanwhile, expression levelof caspase-3protein was determined by ELISA. Western blot was used todetermine expression of multidrug resistance （MDR） protein Bcl-2,P-glycoprotein （P-gp）. To known about whether H₂S promoted tolerance ofHepG-2to ADM.2. HepG-2cell were cultured in vitro, treated with ADM, NaHS,glybenclamide (Gly, inhibitor of K_ATPchannel). The IC50value of ADM wasanalyzed by CCK8assay. HepG-2apoptosis was detected by propidiumiodide （PI） staining and flow cytometry （FCM） in five group, containingcontrol, ADM, ADM+NaHS, ADM+NaHS+Gly, Gly group. Western blot was used to determine expression of Bcl-2and P-glycoprotein （P-gp） in four group,containing control, NaHS, NaHS+Gly, Gly. To known about whether K_ATPchannel mediated the role of H₂S promoting tolerance of HepG-2to ADM.3. According to ADM IC50, experiment was divided into five groups,containing control, ADM, ADM+NaHS, ADM+NaHS+K252a （inhibitor ofBDNF-TrKB pathway）, K252group. Apoptosis and cell viability of the fivegroups were determined by CCK8and FCM, respectively. Western blot wasused to determine expression of Bcl-2, P-glycoprotein （P-gp） and BDNF infour groups, containing control, NaHS, NaHS+K252a, K252a. To known aboutwhether BDNF-TrkB pathway mediated the role of H₂S promoting toleranceof HepG-2to ADM.【Results】1. The effects of H₂S on tolerance of HepG-2to ADM1.1IC50of ADMIC50of ADM in NaHS （100、200、400μmol/L） treatment group were0.59±0.05mg/L,0.78±0.07mg/L,1.42±0.08mg/L, higher than control group（only treatment with ADM,0.32±0.03mg/L）（P<0.01, P<0.001）.1.2ApoptosisApoptosis rate of ADM group （77.9±0.9）%was higher than that of controlgroup （16.9±0.6）%（P<0.001）. Comparing with ADM group （77.9±0.9）%,apoptosis rate of ADM+NaHS group （25.7±2.3）%significantly decreased（P<0.001）. Apoptosis rate of NaHS group was lower than that of controlgroup （16.9±0.6）%（P<0.01）.1.3Caspase-3expressionExpression of caspase-3in ADM group （1.09±0.05ng/ml） was higher thanthat of control group （0.53±0.03ng/ml）（P<0.001）. Comparing with ADMgroup （1.09±0.05ng/ml）, expression of caspase-3in ADM+NaHS group（0.81±0.03ng/ml） significantly decreased （P<0.01）. There was no significantdifference between control group and NaHS group （P＞0.05）. 1.4Bcl-2and P-gp expressionExpression of Bcl-2in NaHS（100、200、400μmol/L）group was higher thanthat of control group （P<0.05, P<0.001）. Expression of P-gp in NaHS（200、400μmol/L）group was higher than that of control group （P<0.01, P<0.001）.2. The mediating role of K_ATPchannel for H₂S promoting tolerance ofHepG-2to ADM2.1IC50of ADMIC50of ADM in NaHS group （0.78±0.07mg/L） was higher than that ofADM group （0.32±0.03mg/L）（P<0.001）. IC50of ADM in NaHS+Gly group（0.58±0.01mg/L） was lower than that of NaHS group （0.78±0.07mg/L）（P<0.05）.2.2ApoptosisApoptosis rate of ADM group （77.9±0.9）%was higher than that of controlgroup （16.9±0.6）%（P<0.001）. Comparing with ADM group （77.9±0.9）%,apoptosis rate of ADM+NaHS group （25.7±2.3）%significantly decreased（P<0.001）. Apoptosis rate of ADM+NaHS+Gly group[（50.5±3.9）%] was higherthan that of ADM+NaHS group （25.7±2.3）%（P<0.01）. There was no significantdifference in apoptosis rate between control group and Gly group（P＞0.05）.2.3Bcl-2and P-gp expressionExpression of Bcl-2and P-gp protein in NaHS group was higher than thatof control group（P<0.05, P<0.01）. Expression of Bcl-2and P-gp protein inNaHS+Gly group was lower than that of NaHS group（P<0.05）. There was nosignificant difference in expression of Bcl-2and P-gp protein between controlgroup and Gly group（P＞0.05）.3. The mediating role of BDNF-TrkB pathway for H₂S promoting toleranceof HepG-2to ADM3.1BDNF expressionExpression of BDNF in NaHS （100,200,400μmol/L） group was higherthan that of control group （P<0.05, P<0.001）.3.2Cell viability Cell viability of ADM group （OD value=0.56±0.02） was lower than thatof control group（1.90±0.03）（P<0.001）. Cell viability of ADM+NaHS group（1.39±0.09） was higher than that of ADM group （0.56±0.02）（P<0.001）. Cellviability of ADM+NaHS+K252a group （0.71±0.01） was lower than that ofADM+NaHS group （P<0.001）. There was no significant difference in cellviability between control group and K252group （P＞0.05）.3.3ApoptosisApoptosis rate of ADM group[（77.9±0.9）%] was higher than that ofcontrol group [（16.9±0.6）%]（P<0.001）. Apoptosis rate of ADM+NaSH group[（25.7±2.3）%] was lower than that of ADM group （77.9±0.9）%（P<0.001）.Apoptosis rate of ADM+NaHS+K252a group [（46.0±5.9）%] was higher thanthat of ADM+NaHS group [（25.7±2.3）%]（P<0.05）. There was no significantdifference in apoptosis rate between K252a group and control group（P＞0.05）.3.4Bcl-2and P-gp expressionExpression of Bcl-2and P-gp protein in NaHS group was higher than thatof control group（P<0.05，P<0.001）. Expression of Bcl-2and P-gp protein inNaHS+K252a group was lower than that of NaHS group（P<0.05，P<0.01）.There was no significant difference in expression of Bcl-2and P-gp proteinbetween K252group and control group（P＞0.05）.【Conclusion】1. H₂S promoted the tolerance of HepG-2cell to ADM.2. K_ATPchannel and BDNF-TrkB pathway probably mediated the action ofH₂S promoting tolerance of HepG-2cell to ADM.3. The action of promoting tolerance of HepG-2cell to ADM was associatedwith K_ATPchannel and BDNF-TrkB pathway.