Cinnamaldehyde Derivative Against Viral Myocarditis Caused By CVB3

Viral myocarditis (viral myocarditis,VMC) is a violation of the heart caused by myocardial virus real or localized or diffuse interstitial disease, clinical frequent disease in children and young adults in the incidence rate showed the trend of rising year by year . VMC virus intrusion is a major cause of induced, among which group of Coxsackie B virus type 3 coxsackievirus B3,CVB₃) is most common, which can lead to cardiac disease and death. TLR4 on the cell membrane of myocardial identify viruses or virus components of myocardial cells after injury (both belong to PAMP), activation of TLR4 via TLR4-NF-κB pathway to further activate the nuclear transcription factor NF-κB, which can cause a range of the inflammatory response,including inflammatory cytokine(sTNF, IL-1, IL-6, INF_-γ, etc.) release, cell adhesion molecules, recruitment of inflammatory cells. Therefore, in the treatment of viral myocarditis, today there is no cure, study how CVB₃ in vivo inhibition of the drug is of great significance, providing a new way of thinking. Pharmacology studies have shown that cinnamicaldehyde (Cinnamaldehyde,CA)with anti-fungal, anti-virus, anti-tumor effects , 2008,Youn HS et al CA further confirmed in vitro LPS induced TLR4-NF-κB signaling pathway significantly inhibited. Early experiments show CA through the TLR4-NF-κB pathway on viral myocarditis has obvious therapeutic effect, but CA is chemically unstable in the body,so to CA as a leading compound synthesized three derivatives,namelyα-bromo-4-ch lorocinnamaldehyde,α-bromo-4- methylcinnamaldehyde,4-chlorocinnamaldehyde. Three derivatives comparison and research TLR4-NF-κB signal transduction inhibition, to identify the most sensitive structure of the most effective drugs.ObjectiveEvaluation to the synthesis of CA as a leading compound in the treatment of deriveatives on VMC to explore the effective dose and mechanism of action, provide the basis data for the development of new drugs.MethodPart 1 Synthesis cinnamaldehyde derivatives1. Method By using CA as the lead compound, we synthesize a series of derivatives through aldol condensation and addition reaction. Including: (1) we study the effect of the changed substituent and substitutions site on biological activity by doing the donating electron group and withdrawing electron group substitution to the five sites on the benzene ring of CA. (2) we design the dual-functional molecular by the interaction between CA and acyclovir, ganciclovir respectively.(3) We study the derivatives synthesis of CA and their activities, further more, we analyze the derivatives by applying the FTIR, NMR and hydrogen spectrum. 2. Results and Conclusions: By analyzing the physical and chemical properties of the various derivatives, we found the stable property of theα-bromo,p- chlorocinnamaldehyde andα-bromo,p-methylcinnamaldehyde system, which can be used to study the anti-CVB₃ effect.Part 2 In vitro experiments1. Cytotoxicity assays ofα-bromo-4-chlorocinnamaldehyde,α-bromo-4- meth ylcinn- amaldehyde, 4-chlorocinnamaldehyde1.1 Methods Primary cultured myocardial cells were incubated in the culture at 24-well plates. Myocardial cells were exposed toα-bromo-4-chlorocinnamaldehyde,α-bromo-4-methylcinnamaldehyde,4-chlorocinnamaldehyde at 0-1000μM for 72h. Myocytes cultured in the absence of drug treatment were used as control group. Compared cytopathic effect (Cytopathic effect,CPE) among groups. The MTT reduction assay was used to assess cell viability(n=3).1.2 Results Our data shows that the TC₅₀ ofα-bromo-4-chlorocinnamaldehyde,α-bromo-4-methylcinn-amaldehyde,4-chlorocinnamaldehyde is 2151.28μg/mL,1475.32μg/mL,22460.32μg/mL.1.3 Conclusion The characteristic of toxicity of drugs on the myocardial cells were slower cell beating, cell type flat shrink, falling from the bottom wall 96, or even death. 0-1000μMα-bromo-4-chlorocinnamaldehyde ,α-bromo-4- methylcinnamaldehyde induced an overt concentration-dependent decrease in cell viability.The loss in cell viability was not significant at relatively low concentrations(0-10μM). 4-chlorocinnamaldehyde failed to affect the cell viability of myocardial cells even at the highest concentration(1000μM).2 Anti-CVB₃ activity test ofα-bromo-4-chlorocinnamaldehyde,α-bromo-4- meth- ylcinn-amaldehyde, 4-chlorocinnamaldehyde In vitro2.1 Methods Myocardial cells were exposed to the virus (100×TCID₅₀, 0.1mL/well)for 1 hour,washed with PBS three times,and incubated with a serial dilution ofα-bromo-4-chlorocinnamaldehyde,α-bromo-4-methylcinnamaldehyde,4-chlorocinnamaldehyde (0.1-1000μM).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. The effect of Cinnamaldehyde derivatives on the mRNA expression of some important cytokines,such as TNF-α. IL-1β.IL-6 was measured in cardiac muscle cells by ELISA.2.2 Results With the increase of concentration,the typical CPE ,such as cell turned to be round and small, particle increased and falling off,were decreased. The most effective dose ofα-bromo-4-chlorocinnamaldehyde ,α-bromo-4-methylcinnamaldehyde both were 10μmol/mL and the Cell viability was 61% and 65% respectively.The IC50 value of cinnamaldehyde derivatives on CVB₃ were 178.94μg/mL,173.35μg/mL,6045.25μg/mL and the TI were 12.02 , 8.51 , 3.71 , respectively. Three CA derivatives can inhibit the CVB₃mRNA replication and TNF-α.L-1β.IL-6 secretion to some extent.2.3 Conclusion This observation indicated that 10–1000μM 4-chlorocinnamaldehyde had potent anti-CVB₃ activity in reducing viral titers of primary cultured myocardial cells. Viral titers of myocardial cells in the infected group weresignificantly decreased in vitro byα-bromo-4-chlorocinnamaldehyde,α-bromo-4-methylcinnamaldehyde at 10-1000μM in a concentration-depend- ent manner, but also produced significant toxicity on host cells. After a series of in vitro experiments has found thatα-bromo -chlorocinnamicaldehyde has a high efficiency and low toxicity characteristics on treatment of viral myocarditis.Part 3 In vivo experiments1. The treatment of different doses ofα-bromo-chlorocinnamicaldehyde on VMC mice.1.1 Methods A total of 70 BALB/c male mice, aged 4 weeks, were included in this study. Of the 70 mice, 60 were inoculated intraperitoneally with CVB3 (100×TCID₅₀). 50 of the inoculated mice were given cinnamaldehyde (30mg/kg)α-bromo-4-chlorocinnamaldehyde (54mg/kg,18mg/kg,6mg/kg) and interferon (10mg/kg)for 7 days. Another 20 inoculated mice, the CVB-infected controls , were treated with 0.9% NaCl solution daily intraperitoneally. For the normal controls, mice without inoculation (n=6) were given 0.9% NaCl solution daily intraperitoneally. On day 7, the mice were killed.Mouse hearts were randomly collected; one third of each organ was fixed in 10% buffered formalin for tissue-staining studies,one third was frozen for Western blot analysis, and the final third was for virus titer test.1.2 Results High dose ofα-bromo-4-chlorocinnamaldehyde can significantly inhibit the TLR4 and NF-κB protein level (P <0.01), and can significantly reduce the viral titers of cardiac muscle (P <0.01).1.3 Conclusion CA as a precursor to the synthesis of derivatives have a therapeutic effect on the VMC, among these high dose ofα-bromo-chloro cinnamic aldehyde has most obvious effect , even than the cinnamic aldehyde.Experimental results show that the three synthetic derivatives inhibit the TLR4-NF-κB signaling pathway to play a therapeutic role in VMC, but its mechanism needs further study.