ABSTRACT
ObjectiveTo investigate the influence of recombinant thioredoxin (TRX)on apoptosis of myocardium cell in viral myocarditis of mice.MethodsTwenty-four Balb/c mice,weighting 12 - 14 g,were randomly divided into 3 groups:the control group,the virus group and the protective group,8 mice in each group.The virus group and the protective group were injected with 0.1 ml 100TCID50 Coxackie virus B3 (CVB3)intraperitoneally,and the control group was injected equal volume of saline.Therewithal the protective group was injected with TRX(2 mg/kg) by tail vein,and the virus group was injected saline the same way.After 14 days all mice were killed and hearts were taken.Changes of myocardial histopathology was observed with optical microscope,cell apoptosis was checked by TUNEL technique,and the expression of apoptosis-related proteins (Bcl-2,caspase-3)in infiltrated cell of myocardium was determined by immunohistochemistry.Results(①)Lymphocyte infiltration and necrosis were observed in survivals of the virus group,sporadic coagulation necrosis and ballooning degeneration of cells were observed in the protective group,however no myocardial lesion was found in the control group.(②)TUNEL technique showed that the positive ratio of apoptosis in the virus group and the protective group[(90.23 ± 3.63)％,(20.02 ± 2.41)％] was significantly higher than that of the control group(0.00 ± 0.00,all P ＜ 0.05),the positive ratio of apoptosis in the protective group was significantly lower than that of the virus group (P ＜ 0.05 ).(③)Immunohistochemistry showed that the expression of protein Bcl-2(+,++,+++) in the virus group and the protective group was significantly higher than that of the control group (all P ＜ 0.05).The expression of protein Bcl-2 in the protective group was significantly higher than that of the virus group(P ＜ 0.05).The expression of caspase-3 (+,++) was significantly higher in the virus group and the protective group than the control group (all P ＜ 0.05).Compared with the virus group,the expression of caspase-3 in the protective group was significantly lower(P ＜ 0.05).ConclusionTRX could inhibit cardiomyocyte apoptosis in viral myocarditis mice and the inhibition is related to regulation of apoptosis-related protein expression.
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Objective To observe the protective function of recombinant human thioredoxin(TRX) on HeLa cell injury induced by Coxsackie virus 3m(CVB3m) and to study the inhibiting effect of TRX on viral replication. Methods We infected HeLa cells with 10TCID50 CVB3m and then protected these cells with TRX (2,5,10 mg/L). The protective group of TRX, viral group, control group of TRX, and normal control group were included. Six parallel wells were set up in each group. The cell growth was observed by methyl thiazolyl tetrazolium(MTT) and contrast phase microscope. Results The results of contrast phase microscope revealed that HeLa cells were arranged tightly and polygon in normal control group; untightly, became circle and abscission in viral group; HeLa cells morphous improved by increasing TRX concentration in TRX protective group(2,5,10mg/L). MTT results of the inhibitory ratio on cell growth of TRX(2,5,10 mg/L) control group(1.2%,2.9%,6.3%) were compared with normal control group(0), there was no significant difference(all P > 0.05); and while the inhibitory ratio on cell growth of TRX(2,5,10 mg/L) protective group(32.0%,28.0%,27.0%) was compared with virus infective group(51.7%), there was a significant difference (all P < 0.05). The inhibition study of viral replication showed that compared the inhibitory ratio on cell growth of TRX(2,5,10 mg/L) protective group(26.0%,27.0%, 10.9%) with virus infective group(60.0%), there was a significant difference(all P < 0.05). In the protective groups, there was a significant difference (all P < 0.05) between low dose groups(2,5 mg/L) and high dose groups( 10 mg/L). Conclusions The recombinant TRX(2,5,10 mg/L) may alleviate HeLa cell's injury induced by virus and the construct has no significant toxicity. TRX(2,5,10 mg/L) is effective in inhibiting virus CVB3m replication.
ABSTRACT
<p><b>BACKGROUND</b>Thioredoxin is one of the most important redox regulating proteins. Although thioredoxin has been shown to protect cells against different kinds of oxidative stress, the role of thioredoxin in myocardial ischemia and reperfusion injury has not been fully understood. This study was conducted to explore the protective role of human thioredoxin on myocardial ischemia and reperfusion injury and its potential mechanisms.</p><p><b>METHODS</b>Purified human thioredoxin was injected into adult Wistar rats, which were subjected to 30 minutes of myocardial ischemia followed by 2 or 24 hours of reperfusion. We detected 1) the infarct size; 2) the level of malondisldehyde (MDA) in serum; 3) the expression of caspase-9, and cytochrome c in/out of mitochondria by Western blotting; 4) apoptosis by terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) assay and caspase-3 and its protein by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting; 5) the expression of bcl-2 and bax in cardium by immunohistochemical (IHC) assay.</p><p><b>RESULTS</b>Human thioredoxin reduced myocardial ischemia/reperfusion injury as evidenced by significant decrease of myocardial infarct size (P < 0.01), notable reduction of myocyte apoptosis (P < 0.01), lower systemic oxidative stress level (P < 0.01) after reperfusion for 2 hours, and few inflammatory cell infiltration after reperfusion for 24 hours in rats. Furthermore, treatment with human thioredoxin significantly reduced the release of mitochondrial cytochrome C (P < 0.05), and inhibited the activity of caspase-9 (P < 0.05) and caspase-3 (P < 0.01 in mRNA and P < 0.05 at protein level). Meanwhile, human thioredoxin markedly increased bcl-2 expression (P < 0.05).</p><p><b>CONCLUSIONS</b>These results strongly suggest that human thioredoxin has cardioprotective effects on myocardial ischemia/reperfusion and its anti-apoptotic role may be mediated by modulating bcl-2 and the mitochondria-dependent apoptotic signaling pathway.</p>
Subject(s)
Animals , Humans , Rats , Apoptosis , Caspase 3 , Genetics , Mitochondria, Heart , Physiology , Mitochondrial Membrane Transport Proteins , Myocardial Reperfusion Injury , Oxidative Stress , Rats, Wistar , Thioredoxins , PharmacologyABSTRACT
Objective To evaluate protective effects of recombinant human thioredoxin(TRX) in myocardial injury of mice with viral myocarditis. Methods We established viral myocarditis models by intraperitoneal injection with 0.1 ml 100TCID50 Coxsackie virus 3m(CVB3m), along with tail vein injection of recombinant human TRX (2 mg/kg) for protection. The control group was given equivalent volume of normal saline. The mice were killed 7 days following the injections. Serum lactate dehydrogenase (LDH) activity was determined and myocardial injury was examined with light microscopy. Results The somm LDH activity in Coxsackie virns-infected mice [(3130.50±390.57)U/L] was higher than that of animals in the control group[ (1617.86±155.42)U/L] and that of TRX protection group[ (1959.43±540.75)U/L], the difference being statistically significant (P<0.05); there was no significant difference between TRX protection group and the control group(P 0.05). Light microscopy showed that five of the eight Coxsackie rims-infected mice had myocardial lesions, including focal myocardial necrosis and inflammatory infiltration. There was no myocardial injury in the TRX protection group. Conclusions Recombinant human TRX can lessen myocardial injuries induced by infection with CVB3m, and so can protect myocardium.