| Viral myocarditis (VMC)which is frequently linical occurring disease severely threatens people's health. However, there is still no effective drug to treat this disease.Coxsackievirus B3 (CVB3) infection is believed to be a principle trigger leading to human myocarditis. Following infection, cell lysis and islet degeneration occurs, and such infections in vivo might have long-term impacts. The endogenous opiate system is activated byβ-endogenous opioid peptide (β-EP) in murine VMC because of CVB3, which might affect the neuroimmunoical modulating effects by nuclear factor kappa B(NF-κB). TLR4 is also activated by CVB3 persistence effect. These change could cause a serial of inflammatory response by TLR4-NF-κB signal trasduction passway ,induce further cellular apomorphosis and necrosis in myocardium, which results backward heart failure(CCF)in mice with VMC. Therefore, Inhibitors of CVB3 duplicating,β-EP level and TLR4 - NF-κB signal trasduction passway not only possess important significance on illuminating of pathology turnover of VMC,but also establish empirical foundation on effective drug exploitation of VMC.Cinnamaldehyde(CA)is an active compound isolated from of the stem bark of Cimmamomum cassia, a traditional Chinese medicinal herb,which has the main constituent of cinnamon volatile oil (>75%), is anα,β-unsaturated carbonyl derivative with a mono-substituted benzene ring. The pharmacological research shows that, in the human body, CA has an effect of anti-bacteria, anti-virus, and has effect on central nervous system(CNS) and immunologic system. Although cinnamaldehyde has been demonstrated to suppress the growth of influenza A/PR/8 virus(K. Hayashi.,2007),little information is available regarding its effects on CVB3-induced viral myocarditis. It has been further demonstrated that cinnamaldehyde suppresses LPS-induced activation of TLR4 - NF-κB in a dose-dependent manner(K. Hayashi.,2008). These results strongly implicate an immuno-modulatory role for CA.Our protophase work has been demonstrated that CA has a treatment effect on VMC mice. Therapeutic efficacy of CA is better than radix astragali parenteral solution and ribavirin, however, its acting mechanism did not clear. CA, a reactive aldehyde rapidly oxidized to cinnamic acid(CD), is unstable in blood. The primary urinary metabolites for CA include glycine or glucuronic acid conjugates of benzoic acid that form as a result ofβ-oxidation of CD. We don't know wether CA possesses any anti-CVB3 or myocardial protective effects in vivo and in vitro in a manner similar to CD. In order to provide experimental data for developing an application of CA, we select CD as a control group to carry out pharmacology study in vitro and in vivo.ObjectiveTo Investigate mechanism of action of CA and CD, firstly,we explore the effect of anti-CVB3 in vivo and vitro of CA and CD ;secondly,we explore the effects of CA and CD onβ-EP,кopiate recepto(rкOR)and TLR4-NF-κB signal trasduction pathway in VMC miceMethods and resultsPrimary part:Study in vitro1 Cytotoxicity assays of CA and CD1.1 Methods To establish the maximal non-cytotoxic dose of chemicals on a myocardial cell, primary cultured myocardial cells (1×105 cells/well) were cultured in two 24-well plates, each well having a final volume of 1 mL containing 20% fetal bovine serum. Myocardial cells were exposed to CA and CD at 0, 0.001, 0.01,0.1, 1, 10, 100, and 1000μM for 72 hours, after which cell viability was determined. The MTT reduction assay was used to assess cell viability. Myocytes cultured in the absence of drug treatment were used as control group.Data were presented as mean±S.D. of at least three independent cultures (experiments).1.2 Results Our data showed that an LogIC50 value of CA in inhibiting myocardial cells viability was-4.125. As shown in MTT reduction assay, 0.1-1000μM CA induced an overt concentration-dependent decrease in cell viability. The loss in cell viability was not significant at relatively low concentrations. Cell viability was reduced to approximately 70%, 54%, 13%and 0% in cells treated for 72 hours with 0.1, 1, 10 and100μM of CA, respectively. CD failed to affect the cell viability of myocardial cells even at the highest concentration (1000μM). Cell viability of 1000μM CD was approximately 97% which were significantly elevateder than CA group(P<0.01).1.3 Conclusion CD even at the highest concentration (10 mM) and did not affect the cell viability in Myocardial cells,while CA has a low order of toxicity through dermal route. The unsaturated aldehydes may product cytotoxicity of CA.2 Anti- CVB3 activity test of CA and CD In vitro2.1 Methods Myocardial cells were exposed to the virus (100×TCID50, 0.1 mL/well) for 1 hour, washed with PBS three times, and incubated with a serial dilution of CA and CD (0.001-100μM). Myocytes with viral infection were used as the CVB3 control. Myocytes cultured in the absence of viral infection and drug treatment were used as controls. Following a 72-hour culture, viral titers were measured on HeLa cells. Results of viral titers were expressed as ? lgTCID50 (n = 3).2.2 Results Viral titers of myocardial cells in the infected group were significantly decreased in vitro by CD at 10 - 1000μM in a concentration-dependent manner, viability of myocardial cells were significantly elevateder than CVB group and CA group (p <0.01, n = 3 each).Viral titers of myocardial cells in the CA group at 100-1000μM were lower than CVB group (P < 0.05), however,the viability of myocardial cells in CA group at 100-1000μM has no statistically significant difference with CVB group.2.3 Conclusion This observation indicated that 10–1000μM CD had potent anti- CVB3 activity in reducing viral titers of primary cultured myocardial cells .CA had anti- CVB3 activity at 100-1000μM ,however, little or no viability of myocardial cells was observed at the same concentration.3 To investigate mechanism of anti-CVB3 action of CA and CD In vitro3.1 Methods Myocytes (1×105 cells/well) were cultured in each well of a 24-well plate with a final volume of 1 mL including 20% fetal bovine serum. Myocardial cells were exposed to the virus (100 TCID50, 0.1 mLper well) at-1, 0, 1 h and washed with PBS three times, and then incubated with or without the treatment of 100μL CA, and CD (100μM). After culture for 72 h, Mechanism of action of chemicals was assessed by different experimental procedures: survey and record maximal viral CPE, then virus titers were measured on HeLa cells; MTT test,virus neutralizing antibody experiment,immunofluorescence test and electron microscope test were carried out.3.2 Results Viral titers and of myocardial cells in the infected group were significantly decreased in vitro by CD at for -1, 0, 1 h CVB3 treatment (p <0.05 -0.01, n = 3 each).,and has higher viability of myocardial cells than CVB and CA groups(p <0.01). Mechanism of action of CD may inactivate directly and inhibit CVB3 duplicating in cells ,however it could not produce prophylactic protection action on myocytes because there are little deviation between -1, 0, 1 h CVB3 treatmen(tP>0.05). Viral titers of myocardial cells in the CA group at 0h CVB3 treatment were lower than CVBgroup (P < 0.05), however,the viability of myocardial cells in CA group has no statistically significant difference with CVB group.3.3 Conclusion CA could inactivate CVB3 directly in vitro with cytotoxicity in myocardial cell by CVB3 infected. CD may inactivate directly and inhibit CVB3 duplicating in cells, however it could not produce prophylactic protection action on myocardial cell by CVB3 infected..Second part:Study inn vivo experiments1 To observe the change ofкOR expression in myocardium of VMC mice.1.1 Methods 50 BALB/c mice weighing 20-25 g were used for all experiments. The mice were randomly divided according to experiment protocol and inoculated intraperitoneally with CVB3 (100×TCID50)0.1mL. Mice without inoculation (n = 16) were given i.p.0.9% NaCl solution daily and used as normal controls. Mice were euthanized, and hearts were collected on Days 5,7 ,14, 21 and28. RT-PCR technique and WESTERN BLOT technique were used to investigate the change of expression ofκOR mRNA protein in rat at different time point during VMC with CVB3 infected.1.2 Results On the 5,7,14,21 day after viral infection, expression ofкOR mRNA was dramatically increased in myocardium of VMC mice(P<0.01). On the 7,14,21 day after viral infection, expression ofкOR protein in myocardium of VMC mice was dramatically increased(P<0.05).1.3 Conclusion The up-regulation ofκOR gene and protein were observed at the different time during VMC. it is helpful for to know theκOR to act the pathology effect during VMC.2 To investigate mechanism of action of CA and CD therapy VMC mice2.1 Methods A total of 165 BALB/c male mice at 4 weeks of age were included in this study. Of the 165 mice, 150 were inoculated intraperitoneally with CVB3 (100×TCID50). 120 of the inoculated mice were given CA(i.p. 30 mg/kg, n = 30) and CD (i.p. 30 mg/kg, n = 30) for 21 days and U50,488H (i.p. 30 mg/kg, n = 30) and NXL(Naloxone , i.p.2mg/kg, n = 30) were used as controls group, respectively. Another 30 inoculated mice were treated with i.p. 0.9% NaCl solution daily and were used as CVB3 group. Mice without inoculation (n = 16) were given i.p.0.9% NaCl solution daily and used as normal controls. Mice were euthanized(n = 8), and hearts were collected on Days 10 (n = 8)and 21. On Day 10 and 21, mouse hearts were randomly collected. the HW/BW ratio was detect.One part was fixed in 10% buffered formalin for tissue staining studies, others was frozen for RT-PCR and WESTERN BLOT analysis, and the blood third was homogenized to detectβ-EP. These mice (n = 8 for 10 day) were excluded from the survival analysis. On Day 21, the HW/BW ratio, survival ratio, and pathological examination were evaluated.2.2 Results The 21-day survival rate in the CA group (47.6%, n = 21)and U50,488H group (52.4%,n = 21) was significantly greater than that in the CVB group (14.2%, n = 21; p < 0.05). On Days 10 and 21 after inoculation,the expression ofκORmRAN,TLR4mRAN,κOR protein and TLR4 protein was dramatically increased in the CVB group, while treatment with CA and U50,488H inhibited these protein expression. The Hw/Bw ratio and histopathologic score were significantly reduced in the CA and U50,488H treatment groups compared with that of the CVB group (p <0.05), On Days 21 after inoculation,theβ-EP level was significantly reduced in the CA and U50,488H treatment groups compared with that of the CVB group (p <0.05). The 21-day survival rate in the NXL group was significantly greater than that in the CVB group (57.1%, n = 21); on Days 10 and 21 after inoculation,theβ-EP level was significantly reduced in NXL group. On Days 21 after inoculation , histopathologic score were significantly reduced in NXL group and CD group(p <0.05). On Days 10 and 21 after inoculation,the expression of CVB3mRNA was significantly reduced in CA group and CD group(p <0.01). CA possesses similar antiviral activity with CD via i.p. administration. It means that CA has therapeutic effect on viral myocarditis by its metabolites- CD which is mainly responsible for the antiviral activities.2.3 Conclusion The mechanism of action of CA on VMC:1) CA has therapeutic effect on viral myocarditis by its metabolites- CD which is mainly responsible for the antiviral activities;2)CA could reduce inflammatory response by TLR4-NF-κB signal trasduction passway in heart of VMC mice;3)CA possesses similar antiviral activity with U50,488H via onκOR,however,wether or not they has inhibit effect on arrhythmia of VMC mice is unknown.It needs further study.To the best of our knowledge,this is the first report on anti- CVB3 viral activity of CA and CD in vivo. This work suggests that CA may be a new source for the therapic drug used in the initial stage of viral myocarditis.
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