| Objective:The aims of this study were to test if Querelated protection against Dox-induced cardiotoxicity involved（1）upregulation of 14-3-3c;（2）suppression of oxidativestress;and（3）improvement of mitochondrial function.Methods:1.The primary neonatal rat cardiomyocytes culture.All experimental protocols were carried out following the U.S.National Institutes of Health Guide for the Care and Use of Laboratory Animals（NIH Publication No.85-23,revised 1996）,and approved by the Ethics Committee of Nanchang University（No.2015-0112）.Cardiomyocytes from 2-day-old Sprague Dawley rats were prepared as previously described.Briefly,the ventricles were digested with pancreatin（1 mg/ml）in the D-Hanks balanced salt solution.After harvested,resuspended The non-adherent cardiomyocytes were plated on the gelatin-coated 60-mm Primaria culture dishes at 1×106 cells per dish.18hours,the cardiomyocytes were washed and cultured in the serum-free maintenance mediumfor the duration of the experiments.2.Experimental grouping.The experimental groups were designed as follows:（1）Control group:the cardiomyocyte were incubated in normal conditions;（2）Dox group:the cardiomyocyte were exposured to 1μM doxorubicin for 24 hours;（3）Que+Dox group:the cardiomyocytethat were treated with Que（10,20,40,80μM）for22 hours prior to Dox exposure;（4）Que+pAD/14-3-3γ-shRNA+Dox group:the cardiomyocytethat were additionally treated with pAD/14-3-3γ-shRNA for 2hours before Que treatment,and exposure Dox after 22 hours’co-incubation;（5）Que+pAD/scrRNAi+Dox group:the cardiomyocytethat were additionally treated with pAD/scrRNAi for 2hours before Que treatment,and exposure Dox after 22hours’co-incubation.3.Adenovirus transfection.Constructs of pAD/14-3-3γ-shRNA or pAD/scrRNAi were transfected into the cardiomyocyte that were cultured in fresh DMEM medium,Transfected cells were incubated before Que treatment,and start exposure Doxexperiment after 22 hours’co-incubation.4.MTS assay.A colorimetric MTS is a pale yellow substrate that produces a dark blue formazan product,which directly dissociate in medium when incubated with living cells.the absorbance of each well was measured at 490nm by a micro plate reader,and the amount of absorbance,which is directly proportional to the number of living cells in culture.5.Measurements of the release of Lactate dehydrogenase（LDH）.Thus after experiment treatment,we collected supernatant and detected the activity of LDH by a microplate reader（Bio-rad 680,USA）according to the specifications of LDH assay kit（Jiancheng,Nanjing,China）.6.Western blot assay.Harvested cells were lysed with Radio Immunoprecipitation and incubated at 4°C for 2 hours.the membranes were saturated with an enhanced chemiluminescence mixture for 1 min and viewed by autography using preflashed X-ray film（Fujifilm,Tokyo,Japan）for 300 s.The bands were analyzed with densitometric scanning using Quantity One software.7.Measurement of intracellular ROS.DCFH-DA was converted by intracellular esterases into DCFH.Which was then oxidized by ROS into highly fluorescent DCF.The assay was performed according to the protocol provided by the manufacturer.the fluorescence intensity of each group was determined using a flow cytometer（Becton Dickinson,USA）at excitation and emission wavelengths of 485 and 528 nm,respectively.8.Assessment of the activities of antioxidant enzymes,the levels of non-enzymatic antioxidants,and lipid peroxidation.Catalase,SOD,and GP-x are important anti-oxidant enzymes in the body that provide a first line of defense against oxidative damage.Supernatant of cellular lysis were collected after treatment and measurement according to the manufacturers’instructions.At last,all collections were tested by a microplate reader（Bio-rad 680,USA）Reduced glutathione is the most abundant intracellular thiol,and the intracellular redox state,as reflected by levels of oxidized（GSSG）and reduced（GSH）glutathione as well as the GSH/GSSG ratio,is considered to be an important indication of cellular health.Supernatant of cellular lysis were collected after treatment and measurement according to the manufacturers’instructions.9.Assessment of mitochondrial membrane potential（MMP）.The fluorescence was then measured by a Flow Cytometer（Becton-Dickinson,USA）with first at excitation and emission wavelengths（ex/em）of 530 and 580 nm（red）,and then at ex/em of485/530 nm（green）respectively.The ratio of red to green fluorescence intensity of cells reflects the level of MMP.10.Opening of Mitochondrial Permeability Transition Pores（mPTP）.mPTP opening plays a major role of cell apoptosis.Ca2+induced mitochondria swelling assay is used to determine mPTP opening.The absorbance at 520nm was measuredevery minute until stable values were observed.The changes in absorbance were used to measure the extent of mPTP opening.11.Activity of caspase-3.the activity of caspase-3 could be analyzed by each experimental group absorbance.The assay was performed in accordance with the protocol provided by caspase-3 activity assay kit（BestBio,China）.12.Assessment of Apoptosis.Assessment of apoptosis was performed by Annexin V-EGFP/PI Apoptosis Detection Kit（KeyGENBioTECH,Nanjing,China）.The stained cells were analyzed using flow cytometry analysis（Becton-Dickinson,USA）to test DCF fluorescence,which is an index of cellular damage.Results1.Que pretreatment protects the cardiomyocyteagainstDox-induced toxicity.The viability of Que-pretreated groups in a dose-dependent manner was higher than that of Dox group（P<0.01,）.And the optimal concentration of Que was 20μM（P<0.01）,the cell viability of the group pretreated with 20μMQue+pAD/14-3-3γ-shRNAwas decreased（P<0.01,）.The LDH activities of Que-pretreated groups in a dose-dependent manner were lower relativeto that observed in Dox group（P<0.01,）.LDH viability of the cells pretreated with 20μMQue+pAD/14-3-γ-shRNAwas much higher than that of Que-treated cells.It is suggested that the addition of pAD/14-3-3γ-shRNAattenuates the cardioprotection of Que against Dox-induced injury（P<0.01,）.2.Que pretreatment upregulates the expression of 14-3-3γin the cardiomyocyte against Dox-induced toxicity.The expression of 14-3-3γin cardiomyocyte upregulated in Que+Doxgroup and Que+pAD/scrRNAi+Dox group than in Dox group.However,the effects were inhibited by the addition of pAD/14-3-3γ-shRNA.It was indicated that Quecould upregulate the 14-3-3γexpression.3.Que pretreatment reduces ROS generation in the cardiomyocyte against Dox-induced toxicity.The peak of ROS levels was moved to the right markedly,which indicated an obvious increase of ROS generation in Dox group compared with control group（P<0.01）.Que pretreatment caused a significant shift of the peak of ROS levels to the left,which indicated a significant decrease in ROS generation compared with Dox group（P<0.01）.The peak of ROS in the cardiomyocyte induced by Dox in the group pretreated with Que and pAD/14-3-3γ-shRNA was shifted to the right compared with Que+Dox group,which indicated a significantly more ROS generation than Que+Dox group（P<0.01）.4.Que pretreatment preserves the activities of antioxidant enzymes,the levels of non-enzymatic antioxidants,and reduces lipid peroxidation in the cardiomyocyte against Dox-induced toxicity.Compared to those in control group,the activities of SOD,GP-x,and catalase were lower and MDA level was higher Dox group（P<0.01）.Que pretreatment,however,exhibited significant increasing in SOD,GP-x,and catalaseactivities,anddecreasingMDAlevel（P<0.01）.But pAD/14-3-3γ-shRNAcould cancel the effects of Que pretreatment（P<0.01）.Dox led to significant reductions in the GSH content,and GSH/GSSG ratiocompared with those in control group（P<0.01）.Surprisingly,Que pretreatment exhibited significant increasing in these parameters（P<0.01）,suggesting that Que can reverse oxidative stress due to Dox-induced toxicityby increasing levels of non-enzymatic antioxidants.5.Que pretreatment maintains MMP in the cardiomyocyte against Dox-induced toxicity.The peak of MMP levels was moved to the left markedly,which indicated an obvious loss of MMP in Dox group compared with control group（P<0.01）.Que pretreatment caused a significant shift of the peak of MMP to the right.The peak of MMP in the cardiomyocyte induced by Dox in the group pretreated with Que+pAD/14-3-3γ-shRNA was shifted to the left compared withQue+Dox group,which indicated a significantly loss of MMP than Que+Dox group（P<0.01）.6.Que pretreatment inhibits the opening of mPTP in the cardiomyocyte againstDox-induced toxicity.Dox group showed the steepest downward trend,followed by Que+pAD/14-3-3γ-shRNA group（P<0.01）.Quepretreatment reduced the decline in absorbance（P<0.01）.These results show that Que reduced the openness of mPTP;no reduction in absorbance was observed after co-administration of pAD/14-3-3γ-shRNA（P<0.01）.7.Que pretreatment restrains the activity of caspase-3 in the cardiomyocyte against Dox-induced toxicity.The activity of caspase-3 in Dox group was significantly increased compared with control group（P<0.01）,while Que pretreated group had a significant inhibiting the activity of caspase-3 compared with Doxgroup（P<0.01）.The activity of caspase-3 increased again in Que+pAD/14-3-3γ-shRNA group（P<0.01）.8.Que protects the cardiomyocyte against Dox-induced apoptosis.The number of apoptotic cells was notably higher in Doxgroup than in control group（P<0.01）.Pretreatment with Que lowered the percentage of apoptotic cells（P<0.01）.However,the addition of pAD/14-3-3γ-shRNA rendered the difference in apoptosis insignificant（P<0.01）.Conclusion:From fruits,vegetables,leaves,and grains a flavonoid,Que not merely possess itself antioxidant property,but also upregulate 14-3-3γexpression of the cardiomyocyteagainst Dox-induced toxicity.Thus,there is an urgent need for available options in the prevention and treatments of associated cardiotoxicity to improve efficacy of cancer chemotherapy,survival and quality of life. |
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