Pharmacokinetic Study Of Cinnamaldehyde And Its Submicrion Emulsion And Pharmacological Study Of LPS Induced Myocarditis

1 Background LPS is the main component of the cell wall of Gram-negative bacteria.The release of LPS in the blood will cause acute immune system reaction.The acute myocardial inflammation will cause decreasing cardiac contractility,increase of heart rate and the ejection fraction decreased.Severe myocardial inflammation can even lead to heart failure or death.Cinnamon is a traditional Chinese medicine commonly used in Chinese medicine prescriptions.The antibacterial and cardic protetive effect of Cinnamon attractive more and more attention around the word.As the composition of cinnamon is complex,it is difficult to clarify its mechanism and targets,which makes it hard to develop and utilize cinnamon.With the development of natural medicine research,the theory of using the main ingredient of the herbal medicine to replace the herb itself accepted by more and more researchers.Cinnamaldehyde is the main component of volatile oil of Cinnamon,also showed significant antibacterial and cardiac protection.We can clarify its mechanism in the body by using modern Chinese medicine pharmacology theory.Due to the poor water solubility of cinnamaldehyde and the unclear metabolic parameters in vivo.In order to make more efficient use of traditional Chinese medicine,this study firstly explored the pharmacokinetic parameters of cinnamaldehyde and its water-soluble emulsions,the protective effect on LPS-induced myocardial injury and its mechanism provided the experimental basis for the development of new drugs of cinnamaldehyde.2 Methods 2.1 Study on the pharmacokinetics parameters of cinnamaldehyde in rats: establishment of GC-MS method for the determination of cinnamaldehyde in rat blood and tissues.Column: DB-5MS(30 m × 0.25 mm,0.25 ?m;Agilent Technologies)Capillary Column to separate samples.The initial oven temperature was 50 ?C for 1 min and then increased to 160 ?C at 10 ?C/min for 1 min,then increased to 280 at 20 ?C/min and held at the final temp for 1 min.Helium was used as carrier gas at a constant flow rate of 1.0 m L/min.The transfer line and ion source temperatures were both at 250?C.Ionization was carried out in electron impact ionization(EI)mode at 70 e V.Detection was operated under selected ion monitoring(SIM)mode.Quantifying ion mass(131 m/z,105 m/z and 92 m/z)was selected for simultaneously investigating cinnamaldehyde and its metabolites.The established method was used to analyze the metabolism and distribution of cinnamaldehyde in vivo.2.2 Cinnamaldehyde submicron in plasma pharmacokinetics and tissue distribution in rats: In order to improve the water solubility and pharmacokinetic parameters of cinnamaldehyde,we used high pressure homogenization way to prepare a novel submicron cinnamaldehyde.The pharmacokinetic parameters of submicron emulsion cinnamaldehyde in vivo was determined by the method of mentioned in 2.1.and compared with the pharmacokinetic parameters of cinnamaldehyde.2.3 The protective effect of cinnamaldehyde on LPS-induced sepsis in rats: Rats were divided as 6 groups: control group,model group,vehicle group,low dose,middle dose and high dose cinnamaldehyde treatment group.(1)The same parts of the heart were embedded by paraffin and used for H&E staining.(2)The levels of TNF-?,IL-1? and IL-6 in the serum were measured by ELISA.(3)Fractional shortening(FS)was were recorded by using a VEVO 770 high-resolution imaging system.(4)The expression of LC3 and TLR4 protein in rat heart tissue was detected by immunohistochemistry and Western Blot.(4)The expression of LC3 and TLR4 protein in the heart was detected by Western Blot.(5)The MAPK pathways was detected by measuring the key proteins of JNK,ERK and p38 and its phosphorylation.This part was to explore the molecular mechanism of LPS-induced myocardial injury.2.4 Protective effect of cinnamaldehyde on LPS-induced cardiomyocyte injury: Myocardial cell was isolated and cultured for 36 h before use.Cells were separated as normal group,model group,low dose,middle dose and the high dose group(0.01,0.1,1 ?M).The following parameters were tested:(1)ROS production and related inflammatory protein in cardiomyocytes was evaluated by confocal microscopy and Western Blot analysis.(2)To observe the autophagosomes in cardiomyocytes by transmission electron microscopy and laser confocal microscopy.(3)Apoptosis after LPS stimulation was tested by TUNEL.(4)The effect of cinnamaldehyde on TLR4-NOX4 signaling pathway was detected by Western Blot and si RNA.(5)Using molecular docking to fit the target of cinnamaldehyde on cardiomyocytes and explore its mechanism.3 Results 3.1 The pharmacokinetics and tissue distribution of cinnamaldehyde in rats:The method for determination of cinnamaldehyde by GC-MS was established in this part.The linear range of cinnamaldehyde in plasma was 20~2000 ng / m L(r2?0.999);For all samples,LOQ of the method were above 20 ng/m L,and only the acquired data above 20 ng/m L were available for statistical analysis.The intra-day and inter-day precision variations were less than 10.4% and 12.2%.Using the above method,the half-life of cinnamaldehyde in plasma was 6.7 ± 1.5 h and 3.26 ± 2.9 h after oral and intravenous injection.The linear range of cinnamaldehyde measured in all tissue samples were 20~2000 ng / m L(r2?0.999).The intra-day and inter-day precision variations were less than 11.3%.Experiments found that cinnamaldehyde can penetrate the blood-brain barrier and in the spleen concentration was higher than heart,liver,lung,kidney and brain.3.2 Submicron emulsion cinnamaldehyde in rats plasma pharmacokinetics and tissue distribution characteristics: Cinnamaldehyde was mixed with soybean oil,lecithin and glycerol making submicoemulsion liquid.The mean particle size,zeta potential and entrapment efficiency of submicron microemulsion were 130 ± 5.92 nm,-25.7 ± 6.00 m V and 99.5 ± 0.25%.After intravenous injection of 20 mg / kg cinnamaldehyde submicron emulsion,the maximum concentration of cinnamaldehyde in plasma was increased from 547 ± 142 ng/m L to 1052 ± 184 ng/m L.Tissue distribution characteristics has changed,the concentration in liver,kidney and spleen improved,no significant increase found in other organizations.3.3 Cinnamaldehyde on LPS-induced heart damage in rats: Cinnamaldehyde can significantly improve the cardiac function after LPS stimulation.The results showed that the levels of TNF-?,IL-1? and IL-6 in serum were sharply increased after LPS stimulation.The results of echocardiography showed that the FS value of heart decreased after LPS stimulation.Immunohistochemistry and Western Blot showed that inflammatory and autophagy-related proteins,such as TLR4 and LC3 were elevated.The MAPK pathway,JNK,ERK and p38 were phosphorylated in large quantities.In the pre-treatment of cinnamaldehyde,we found that the above phenomenon were ameliorated in different degrees.3.4 The protective effect of cinnamaldehyde on lipopolysaccharide-induced cardiomyocyte injury: Cinnamaldehyde regulates cardiomyocyte autophagy by inhibiting TLR4-NOX4 signaling pathway to protect cardiomyocytes.Compared with the model group,cinnamaldehyde significantly reduced the content of ROS in cardiomyocytes and down regulated the inflammation related protein JNK.In the observation of transmission electron and confocal microscopy,the number of autophagosomes in the model group was significantly increased,and the autophagosomes was reduced after cinnamaldehyde treatment.TLR4 si RNA and NOX4 si RNA demonstrated that cinnamaldehyde played a role in the regulation of autophagy and apoptosis by inhibiting TLR4-NOX4 signaling pathway.4 ConclusionsIn this study,the pharmacokinetics and other methods were used to simulate the metabolism and division of cinnamaldehyde in vivo.The dosage form of cinnamaldehyde was further developed for by using the submicron emulsion system.And the changes of pharmacokinetics and tissue distribution were discussed.Based on the previous study on the protective effect of cinnamaldehyde,the effect of cinnamaldehyde on the myocardium in vivo and in vitro was discussed.The new form of cinnamon has been presented to domestic and foreign counterparts for cinnamon step by step development and utilization laid the foundation.