| Objective: Heart is the first organ which formed during the process ofembryonic development, and it can not regenerate as a highly specializedorgan. The scar repair is the main replacement for the damage of heart, whichseriously impacts on myocardial contractile function and is also thepathological basis of irreversible heart failure. In2011, Porrello et al removed15%of the ventricular muscle of1-day-old mice by surgical operation, while21days later the heart of mice completely regenerated and functionednormally without any scar formation. This study proved that the myocardial ofmammals could regenerate. In2013, Senyo et al demonstrated thatmammalian pre-existing myocardial cells are the main source of maintainingmyocardial cell daily to turnover and regenerative after injury.Nkx2-5and its downstream gene Tbx5are the transcription factors forthe early development of heart, which are the genetic markers for cardiacprogentior cells and closely related with function. The levels of Nkx2-5andTbx5expression are decreased with the development of myocardial. The studyfound that the regeneration of myocardial injury is related with the increasingexpressions of Nkx2-5and Tbx5. So the increasing expressions of Nkx2-5andTbx5are the important factors to participate in the cardiac regeneration.High glucose and hypoxia are common clinical factors for myocardialdamage, leading to cardiac hypertrophy and myocardial fibrosis, finallycausing the irreversible damage of heart function. It is the relationshipbetween high glucose or hypoxia and the injury that has been clearlyunderstood, but there is no clear understanding of the relationship betweenhigh glucose or hypoxia and myocardial regeneration. It is still unclearwhether high glucose and hypoxia can stimulate upregulation of the earlytranscription factor Nkx2-5and Tbx5in myocardial cells and whether it is involved in myocardial cell regeneration.Both high glucose and hypoxia can stimulate the activity of induciblenitric oxide synthase (iNOS) activity, while iNOS could catalyzes substrates toproduce a large amount of nitric oxide (NO). As all known, the rapid reactionbetween excessive NO and superoxide anion can generate peroxynitrite(ONOO-) that is one of the most active oxidative substances, while this is alsothe most important causes of oxidized stress damage in vivo. However, thechanges of iNOS expression in myocardial cells exposed to high glucose andhypoxia in different time of are still unclear. It has not been reported whetherthe level of ONOO-can change expression of Nkx2-5and Tbx5.Tongxinluo, a compounded Chinese medicinal prescription whichapproved by SFDA, is widely used for the treatment of heart disease.Theinfluence of different concentrations of Tongxinluo on the expression of iNOS,Nkx2-5, Tbx5of cardiomyocytes which stimulated by high glucose andhypoxia is not clear before.Our study aimed to investigate whether high glucose and hypoxia canmake the gene expression of Tbx5, Nkx2-5of primary myocardial cellsincreased; whether the level of ONOO-is the important mechanism for theinfluence of Nkx2-5,Tbx5expression;the intervention of Tongxinluo indifferent concentrations.In this experiment, neonatal rats which were born within three days wereused. Take their hearts out to make myocardial cells primary cultured. Themethod for identifying cardiomyocytes is immunofluorescence techniques.Respectively detected the gene expression of iNOS, Nkx2-5, Tbx5in highglucose or hypoxia at different times; then detected gene expression of iNOS,Nkx2-5Tbx5after using different concentrations of Tongxinluo under thestimulation of high glucose or hypoxia; detect gene expression of Nkx2-5,Tbx5under the application of SIN-1and FeTTPs.Methods:1The culture of primary myocardial cells Primary cardiomyocytes cells isolated and cultured: Take out7to8SDrats which born within three days, disinfected with75%alcohol, open thechest by ophthalmic scissors in the second rib sternal angle, squeeze the chestgently until saw the heart fully exposed then take the heart off by tweezersinto Petri dishes and cut it into pieces, digested the myocardial cells tissue bytrypsin a small number of times, adopt physical and chemical methods toremove fibroblasts in order to purify cardiomyocytes. We recoveredmyocardial cells with DMEM (high glucose) containing15%fetal bovineserum, and then inoculated in six-well plates. We cultured the cells inincubator with37°C and5%CO2for7days for the experiment.2GroupsExperiment is divided into high glucose group and hypoxia group. Thecells were exposed to25mM high glucose DMEM in the high glucose group,while they were stimulated with100uM CoCl2in the hypoxia group.(1) Detect the transcription level of iNOS, Nkx2-5, Tbx5in myocardialcells at different times under the stimulation of high glucose. The cells weredivided into normal group, high glucose1h,2h,3h,4h,5h group.(2) Detect the transcription level of Nkx2-5, Tbx5under decreasing orincreasing the level of ONOO-, the cells were divided into normal group, highglucose group, high glucose+FeTTPs group,SIN-1group;(3) Detect the transcription level of iNOS, Nkx2-5, Tbx5in myocardialcells with different concentrations of Tongxinluo under high glucosestimulated, the cells were divided into groups of high glucose, Tongxinluo360μg/ml group, Tongxinluo540μg/ml group, Tongxinluo720μg/ml group;(4) Detect transcription level of iNOS, Nkx2-5, Tbx5in myocardial cellsby hypoxic stimulus at different times, the cells were divided into normalgroup (ie,0h group), hypoxia1h,2h,3h,4h,5h group.(5) Detect the transcription level of Nkx2-5, Tbx5under decreasing orincreasing the level of ONOO-, the cells were divided into normal group,hypoxia group, hypoxia+FeTTPs group, SIN-1group. (6) Detect the transcription level of iNOS, Nkx2-5, Tbx5with differentconcentrations of Tongxinluo under stimulation of hypoxic. The cells weredivided into groups of hypoxia, Tongxinluo360μg/ml group, Tongxinluo540μg/ml group, Tongxinluo720μg/ml group.3The observation of morphologicalThe myocardial cells which extracted justly is spherical,12hours later,the cells become adherent to the bottom of culture bottle and gradually extendpseudopodia, the cells began to establish connection with each other; after48hours, the cells gradually appear beating and the beat become into pieces inconsistent frequency.4Detect the expression of cTNT which extracted from myocardial cells byimmunofluorescence for the identification of myocardial cells.5Under different stimulation of the myocardial cells then cultured them,detect gene transcription level of iNOS, Nkx2-5, Tbx5.Total RNA was isolated using a one-step Trizol reagent incardiomyocytes; Real-time PCR analysis for the expression of mRNA foriNOS, Nkx2-5and Tbx5genes β-actin as a control reference were carried out.Results:1The purification and culture of primary myocardial cellsWith the collagenase digestion solution, at first, we cut the heart tissueinto the tissue blocks; next, we successfully isolated the tissue blocks into asingle cardiomyocytes following digested by collagenase. In order to removefibroblasts, we used selective plating technique repeatedly to purify thecardiomyocytes. Cardiomyocytes cultured with high glucose DMEM.Cardiomyocytes were sphericity, bright and three-dimensional under the phasecontrast microscope. In addition, they have high survival rate and longsurvival time, after48hours, the cells gradually appear beating and the beatbecome into pieces in consistent frequency, cTnT was expressed in themyocardial cells with immunofluorescence assay, indicating that the primarycardiomyocytes cultured successfully.2.1The changes of the transcription levels of iNOS, Nkx2-5, Tbx5in cardiomyocytes cultured with the stimulation of high glucose by0h,1h,2h,3h,4h and5h.2.1.1The relative expression level of mRNA for iNOS was significantlyincreased in the1h group (0.1511±0.0078, P<0.01),2h group (0.2193±0.0300,P <0.01),3h group (0.1370±0.0079, P <0.01),4h group (0.0792±0.0106, P<0.05), and5h group (0.1076±0.0102, P <0.01) than that in0h group (0.0198±0.0023) in cardiomyocytes with HG.2.1.2The relative expression level of mRNA for Nkx2-5was no overtsignificance in the1h group (0.0899±0.3012),2h group (5.0721±0.3238, P<0.01),3h group (3.9528±0.4833, P <0.01),4h group (3.6183±0.4822, P<0.01), and5h group (3.3206±0.3590, P <0.01) than that in0h group incardiomyocytes with HG.2.1.3The relative expression level of mRNA for Tbx5was significantlyincreased in the1h group (2.4470±0.2681, P<0.01),2h group (6.8240±0.3612,P <0.01),3h group (3.3203±0.3412, P <0.01),4h group (3.0649±0.3478, P<0.01), and5h group (2.4626±0.3227, P <0.01) than that in0h group incardiomyocytes with HG.2.2Decreasing or increasing the level of ONOO-, the changes of thetranscription levels of Nkx2-5, Tbx5in normal group, high glucose group,high glucose+FeTTPs group, SIN-1group.2.2.1The relative expression level of mRNA for Nkx2-5was significantlyincreased in the high glucose group (7.8980±1.3253, P<0.01), highglucose+FeTTPs group(3.2280±0.8611) was no overt significance than that innormal group,SIN-1group(6.8342±0.3039， P<0.01) was significantlyincreased.2.2.2The relative expression level of mRNA for Tbx5was significantlyincreased in the high glucose group (5.6467±0.3538, P<0.01), highglucose+FeTTPs group(1.4675±0.2194) was no overt significance than that innormal group,SIN-1group(5.7920±0.1311， P<0.01) was significantlyincreased.2.3The changes of the transcription levels of iNOS, Nkx2-5, Tbx5in cardiomyocytes in high glucose stimulated with different concentrations ofTongxinluo treated (360μg/ml,540μg/ml and720μg/ml)2.3.1The relative expression level of mRNA for iNOS was no overtsignificance in the360μg/ml group (0.1666±0.0318),540μg/ml group(0.0908±0.0090, P<0.01),720μg/ml group (0.1182±0.0140, P<0.05) wassignificantly lower than that in control group (0.1697±0.0128) incardiomyocytes with TXL.2.3.2The relative expression level of mRNA for Nkx2-5was no overtsignificance in the360μg/ml group (0.0452±0.0031),540μg/ml group(0.0255±0.0013, P<0.01),720μg/ml group (0.0313±0.0025, P<0.05) wassignificantly lower than that in control group (0.0436±0.0038) incardiomyocytes with TXL.2.3.3The relative expression level of mRNA for Tbx5was no overtsignificance in the360μg/ml group (0.1087±0.0197),540μg/ml group(0.0342±0.0040, P<0.01),720μg/ml group (0.0481±0.0059, P<0.05) wassignificantly lower than that in control group (0.1287±0.0253) incardiomyocytes with TXL.3.1The changes of the transcription levels of iNOS, Nkx2-5, Tbx5incardiomyocytes with the stimulation of hypoxia by0h,1h,2h,3h,4hand5h.3.1.1The relative expression level of mRNA for iNOS was significantlyincreased in the1h group (0.3834±0.0468, P<0.01),2h group (0.0282±0.0216,P<0.01),3h group (0.1534±0.0139, P<0.05),4h group (0.0745±0.0154), and5h group (0.0679±0.0145) was no overt significance than that in0h group(0.0541±0.0071) in cardiomyocytes with hypoxia.3.1.2The relative expression level of mRNA for Nkx2-5was significantlyincreased in the1h group (0.1929±0.0186, P<0.01),2h group (0.1191±0.0142,P<0.01),3h group (0.0888±0.0124, P<0.05),4h group (0.0396±0.0063), and5h group (0.0328±0.0021) was no overt significance than that in0h group(0.0356±0.0050) in cardiomyocytes with hypoxia.3.1.3The relative expression level of mRNA for Tbx5was significantlyincreased in the1h group (0.3750±0.0173, P<0.01),2h group (0.2353±0.0135, P<0.01),3h group (0.1413±0.0134),4h group (0.1556±0.0055), and5h group(0.1767±0.0249) was no overt significance than that in0h group(0.1339±0.0059) in cardiomyocytes with hypoxia.3.2Decreasing or increasing the level of ONOO-, the changes of thetranscription levels of Nkx2-5, Tbx5in normal group, hypoxia group,hypoxia+FeTTPs group, SIN-1group.3.2.1The relative expression level of mRNA for Nkx2-5was significantlyincreased in the hypoxia group (5.4382±0.4141, P<0.01), hypoxia+FeTTPsgroup (1.7654±0.4388) was no overt significance than that in normal group,SIN-1group(5.3441±0.1602，P<0.01) was significantly increased.3.2.2The relative expression level of mRNA for Tbx5was significantlyincreased in the hypoxia group (7.4372±0.554, P<0.01), hypoxia+FeTTPsgroup (1.4635±0.1315) was no overt significance than that in normalgroup,SIN-1group(6.0329±0.3361，P<0.01) was significantly increased.3.3The changes of the transcription levels of iNOS, Nkx2-5, Tbx5incardiomyocytes in hypoxic were stimulated with different concentrations ofTongxinluo treated (360μg/ml,540μg/ml,720μg/ml)3.3.1The relative expression level of mRNA for iNOS was no overtsignificance in the360μg/ml group (0.0271±0.0019),540μg/ml group(0.0133±0.0010, P<0.01),720μg/ml group (0.0112±0.0013, P<0.01) wassignificantly lower than that in control group (0.0263±0.0022) incardiomyocytes with TXL.3.3.2The relative expression level of mRNA for Nkx2-5was no overtsignificance in the360μg/ml group (0.0204±0.0019),540μg/ml group(0.0134±0.0019, P<0.05),720μg/ml group (0.0113±0.0017, P<0.01) wassignificantly lower than that in control group (0.0213±0.0013) incardiomyocytes with TXL.3.3.3The relative expression level of mRNA for Nkx2-5was no overtsignificance in the360μg/ml group (0.0310±0.0021),540μg/ml group(0.0307±0.0025),720μg/ml group (0.0158±0.0008, P<0.01) was significantlylower than that in control group (0.0333±0.0029) in cardiomyocytes with TXL.Conclusion:1High glucose or hypoxia can make the early transcription factorexpression of Nkx2-5, Tbx5in primary myocardial cells increase.2The changes of iNOS consistent with Nkx2-5, Tbx5coursed by highglucose or hypoxia, ONOO-could influenced the expression of Nkx2-5andTbx5, ONOO-might be an important mechanism which upregulate theexpression of the early transcription factor of Nkx2-5and Tbx5.3540μg/ml Tongxinluo has the strongest effect of inhibiting theexpression of iNOS, Nkx2-5, Tbx5of high glucose situmulated in primarymyocardial cells;720μg/ml Tongxinluo is the strongest inhibitory for theexpression of iNOS, Nkx2-5, Tbx5of hypoxia situmulated in primarymyocardial cells. |
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