Protection Effects And Mechanisms Of Rhizoma Aneamarrhenae-Phellodendron Herb Pair And Effective Component Mangiferin Against H₂O₂-induced Oxidative Damage In INS-1 Cells

ObjectiveTo establish H2O2-induced oxidative model in INS-1 cells,and to study cell viability,oxidative damage maker,apoptosis,mitochondria protection and insulin related gene after administration of Rhizoma Aneamarrhenae-Phellodendron herb pair and mangi ferin,to explore the possible protection mechanism and provide clinical base.Methods1.ORAC method was used to evaluate oxyradical scavenging capacity of different concentrations of Rhizoma Aneamarrhenae-Phellodendron(YD:1.56,3.13,6.25,12.5,25,50,100,200,400,800,1600,3200?g/mL).2.Different concentrations of H2O2(25,50,100,200 ?M)were used to establish INS-1 oxidation model by MTT method.3.Cell viabilities after administration of different concentrations of Rhizoma Aneamarrhenae-Phellodendron(YD:1.56,3.13,6.25,12.5,25,50,100,200,400,800,1600,3200 ?g/mL)for 24 h were evaluated using MTT method.4.Cell viabilities after administration of dif ferent concentrations of Rhizoma Aneamarrhenae-Phellodendron(YD:1.56,3.13,6.25,12.5,25,50,100,200,400,800,1600,3200 ?g/mL)and mangiferin(MGN:50,100,500 ?M)for 24 h were evaluated using MTT method.5.INS-1 cells were cultured and divided into control,model,positive drug group and administration groups.Cells were incubated with DCFH-DA probe to detect ROS generation after administration of YD 50,100,200 ?g/mL or MGN50,100,500 ?M for 24 h.Lipid peroxidation MDA assay kit was used to detect MDA concentration after administration of YD or mangiferin.6.Changes of mitochondrial membrane potential were detected by Flow Cytomery and UCP2 mRNA expression was detected by RT-qPCR.7.Apoptosis assay were measured by Flow Cytomery with Annexin V-FITC double staining after administration of YD or mangiferin.And each group was stained with Hoechst33342 to observe cellular morphology.8.The activation of Nrf2-Keapl-ARE after administration of YD was measured by RT-qPCR.SOD2 mRNA after administration of mangiferin was detected.9.The expression of PDX-1 mRNA after administration of YD was measured by RT-qPCR.Results1.The results of ORAC showed that,YD displayed oxyradical scavenging capacity from 12.5 u g/mL,and the value increased in a dose dependent.2.Cell viabilities were 1.01 ±0.14,0.97±0.03,0.64±0.01 and 0.10±0.01 respectively after 25,50,100,200 ?M H2O2 stimulation for 1 h.Among which 100 and 200 u M group had statistical significance compared with control group(P<0.001).3.When the concentration of YD ranged from 1.56?g/mL to 200 ?g/mL,the herb pair had no cytotoxicity after administration for 24 h.Compared with MOD group,YD50,100,200 ?g/mL showed significant protection effect(P<0.05).50,100,500 uM MGN had no protection effect.4.The results of ROS showed that,ROS in MOD group significantly increased compared with CON group(P<0.001).YD50,100 and 200 ?g/mL could significantly decreased ROS production compared with MOD group,the positive cell rates were 9.00±2.05%,6.93±3.03%and 7.27±0.87%(P<0.01,P<0.001,P<0.001).MGN 50,100 and 500 ?M groups could significantly decreased ROS production compared MOD group(P<0.001,P<0.05,P<0.001),positive cell rates were 23.10±3.46%,26.80±2.72%and 18.57±2.44%,respectively.The results of MDA showed that,MDA concentration in MOD group significantly increased after H2O2 induction compared with CON group(P<0.01).Low,medium and high dose of YD presented downward trend,among which,YD50 and YD200 showed statistical significance compared with MOD group(P<0.01,P<0.05).MDA decreasing degree of MGN 50,100 and 500 ?M were similar.The values were 1.24±0.22,1.15±0.13,1.24±0.20,respectively and they all showed statistical significance compared with MOD group(P<0.01).5.Mitochondrial protection experiment:(1)mitochondrial membrane potential experiment showed that mitochondrial membrane potential of MOD group significantly decreased compared with CON group(P<0.001),YD100 and YD200 could significantly increase mitochondrial membrane potential compared with MOD group(P<0.001).YD50 had no positive influence on mitochondrial membrane potential.MGN50 and MGN100 could significantly increase mitochondrial membrane potential compared with MOD group(P<0.001),while MGN500 had no effect.(2)After H2O2 stimulation,UCP2 mRNA of MOD group significantly decreased compared with CON group(P0.01).After administration of Rhizoma Aneamarrhenae-Phellodendron,the three doses could increase expression to some degree,and only YD50 had statistical significance compared with MOD group(P<0.01).6.Apoptosis study showed that,apoptosis rate of MOD group significantly decreased compared with CON group(P<0.001).YD50,YD100 and YD200 could significantly decreased apoptosis rate compared with MOD group(P<0.001).The values were 12.67±1.00%?11.10±0.87%and 13.80±1.42%,respectively.MGN 50,MGN100 and MGN500 significantly decreased apoptosis rate compared with MOD group(P<0.001,P<0.05,P<0.001).The values were 11.47±0.81%?13.70±0.14%?10.40±0.36%,respectively.Hoechst33342 resul ts showed that INS-1 cell nucleus was densely stained in MOD group,and cells in MGN or YD group almost all returned to normal shape.7.RT-qPCR results of Nrf2-Keapl-ARE:Nrf2 mRNA expression:Compared with CON group,relative Nrf2 mRNA expression decreased to 0.76±0.07(P<0.05)in MOD group.YD could increase Nrf2 mRNA in different degree.The expression in low,medium and high dose were 1.11±0.17,1.07±0.07 and 1.03±0.14,respectively.And all showed statistical significance compared with MOD group(P<0.01).Keapl mRNA expression:Compared with CON group,relative Keapl mRNA expression decreased to 0.77±0.06(P<0.01).YD could increase Keapl mRNA in different degree.The expression in low,medium and high dose were 1.14±0.11,1.20±0.13 and 1.36±0.14,respectively.And all showed statistical significance compared with MOD group(P<0.001).SOD2 mRNA expression:Compared with CON group,relative SOD2 mRNA expression decreased to 0.83±0.15(P<0.05).YD could increase SOD2 mRNA in different degree.The expression in low,medium and high dose were 1.05±0.18,1.15±0.25 and 1.33±0.25,respectively.And all showed statistical significance compared with MOD group(P<0.05,P<0.05,P<0.001).50,100 and 500?M mangiferin could all increase SOD2 mRNA expression and showed statistical significance compared with MOD group(P<0.05?P<0.05?P<0.01).The expression was 1.09±0.06,1.10±0.17 and 1.12±0.11.YD showed no influence on NQ01 mRNA.8.RT-qPCR results of insulin related gene PDX-1:Compared with CON group,relative PDX-1 mRNA expression decreased to 0.50±0.03(P<0.01).YD could increase PDX-1 mRNA in dose dependent.The expression in low,medium and high dose were 0.76±0.14,0.94±0.27 and 1.42±0.50,respectively.Among three doses,YD100 and YD200 showed statistical significance compared with MOD group(P<0.05,P<0.001).ConclusionIn summary,Rhizoma Aneamarrhenae-Phellodendron herb pair showed oxyradical scavenging capacity to some degree in vitro.It could improve oxidative damage induced by H2O2 in rat INS-1 cell,probably by improving cell viability,decreasing ROS and MDA production,improving mitochondrial membrane potential and regulating UCP2 mRNA expression,improving apoptosis,activating Nrf2-Keapl-SOD2 pathway and increasing PDX-1 mRNA expression.Mangiferin had no effect on cell viability,but it could protect oxidative INS-1 probably by decreasing ROS and MDA,regulating SOD2 mRNA and apoptosis and improving mitochondrial membrane potential.