Antioxidant Activity Of Total Flavonoids From Callicarpa Macrophylla And Its Mechanisms Of Protective Effect On Rotenone-induced Apoptosis In Human Neuroblastomas SH-SY5Y Cells

Objectives: To determin the antioxidant activity of Total Flavonoids fromCallicarpa macrophylla(TFCM) in vitro and evaluate the possible mechanisms onrotenone-induced apoptosis in human neuroblastomas SH-SY5Y cells。Methods:1．Determination of TFCM and antioxidant activity evaluation:(A) Content ofTFCM were determined by NaNO2––Al(NO3)3–NaOH method.(2) Antioxidantactivity in vitro was evaluated by DPPH·、O2-、·OH radical and H2O2scavengingactivity and Fe3+reducing power and vitamin C and BHT are used as controls.2. The effect of TFCM against SH-SY5Y cells damage induced by rotenone:SH-SY5Y cells were divided into control group，rotenone-treated group，TFCM low、middle and high dose-groups. The TFCM low、middle and high dose-groups weretreated with different concentrations of TFCM (30mg/L、60mg/L、90mg/L)respectively for24h,and then treated with20μM Rotenone for24h.The cellviability was assessed by MTT reduction assay and the apoptotic rate of cells was wasevaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)assay.3. The effects of TFCM on the expressions of genes related to the damage ofSH-SY5Y cells and the activity of SOD、GSH-Px and the content of MDA:SH-SY5Y cells were divided into control group，rotenone-treated group, TFCM low、middle and high dose-groups. The TFCM-treated group were treated with differentconcentrations of (30mg/L、60mg/L、90mg/L)respectively for24h,and then treatedwith20μM Rotenone for24h together with rotenone-treated group.. SOD activity wasdetermined with xanthine oxidase method and MDA content was determined withthiobarbituric acid method, GSH-Px were assayed by quantifying the rate of oxidationof the reduced glutathione to the oxidized glutathione.Mcl-1and Cleaved caspase-3were detected by immunofluorescence and Western Blot assay.Results:1.(1) Content of TFCM determined by NaNO2––Al(NO3)3–NaOH method.was81.19%with Vitamin as control.(2)In the experients of antioxidant activity invitro,in the Fe3+reducing power experiment,reducing power activity wasTFCM<BHT≤Vitamin C.In the DPPH scavenging experiment,the scavenging activity was TFCM≤BHT<Vitamin C.In the O2-free radicalscavengingexperiment,the scavenging activity was TFCM<VitaminC. In the H2O2scavengingexperiment, the scavenging activity was TFCM<VitaminC,but they nearly has thesame scavenging activity.2. The effect of TFCM against SH-SY5Y cells damage induced byrotenone:SH-SY5Y cells were treated with different concentrations of rotenone for24h. MTT reduction assay was used to assessed the cell viability.It showed thatcell viability of1μM、5μM、10μM、15μM、20μM、25μM groups are respectively（75.20±4.29）%、(67.20±8.43)%、(63.40±8.15)%、(58.30±5.94)%、(50.50±5.85)%、(39.00±2.69)%. Compared to control group, cell viability wassignificantly reduced, differences are statistically significant (P<0.01),So we chosethe terminal concentration of rotenone20μM in the following experiments.SH-SY5Y cells were cultured with TFCM (30mg/L、60mg/L、90mg/L) for24h，and then exposed in rotenone(20μM) for24h. MTT reduction assay was used toassessed the cell viability and TUNEL assay was used to detected the apoptotic rate.MTT reduction assay showed that compared to the model group,the cell viability ofgroups which was significantly increased in a dose-dependent manner（P<0.05）.TUNEL assay showed that the number of TUNEL-positive cells in model group ishigh, and the apoptotic rate were（61.8±6.0）%. Apoptotic rate were（28.2±8.0）%、（16.7±6.0）%respectively in groups(60mg/L、90mg/L) respectively;Comparedto model group,apoptotic rate was significantly （P<0.01） reduced in adose-dependent manner.3. SOD、GSH-Px activity and MDA content measurement showed that Rotdecreased the levels of SOD and GSH-Px and increased the concentration of MDAcompared to control group and differences are statisticallysignificant （P<0.01）.SOD and GSH-Px activities were significantly increased andthe MDA content was significantly reduced in TFCM groups(60mg/L、90mg/L) in adose-dependent manner compared to the model group (P<0.01or P<0.05). WesternBlot assay releaved that Mcl-1expression was obviously decreased by Rottreatment，while Cleaved caspase-3expression was increased（P<0.01）, differencesare statistically significant. Pretreatment with TFCM (60、90mg/L) down-regulatedthe expression of Cleaved caspase-3（P<0.05or P<0.01），but increased Mcl-1expression（P<0.05or P<0.01）compared to Rot treatment group in a dose-dependentmanner. Immunofluorescence assay showed that Mcl-1was located on nucleus membrane and the expression is high, but the CC3expression is low. Cell nucleuswere unnormol in Rot-treatment group, Mcl-1expression was significantlyreduced,CC3expression was significantly high in nucleus. Compared to modelgroup,TFCM groups（60、90mg/L）gradually up-regulated the expression of Mcl-1and down-regulated the expression of CC3in a dose-dependent manner.Conclusions:1. TFCM has antioxidant activity in vitro to a certain extent.2. In the Rot treatment models, TFCM can increase the cell viability andreduce the cell apoptotic rate, indicating that TFCM can suppress SH-SY5Y celldamage induced by Rot.3. The mechanisms of TFCM suppressing SH-SY5Y cell damage inducedby Rot may be related to oxidative stress and the up regulation of Mcl-1and downregulation of Cleaved caspase-3which are related to apoptosis.