Protective Effects Of Cinnamaldehyde In Liver Of Salmonella Gallinarum-challenged Young-chicks

BackgroundChicken salmonellosis,a bacterial disease caused by Salmonella,seriously endanger the chicken industry,posing large economic damage to poultry production,especially chicks in brooding stage.Chicken salmonellosis is characterized by depression,poor growth,diarrhoea,dehydration,hepatomegaly,friable liver consistency,yellow-greenish colour of the liver,and multifocal hepatic necrosis.Excessive use of antibiotics against Salmonella infection has resulted multi-drug resistant Salmonella strains which limits further antibiotic use against chicken salmonellosis.There is an urgent requirement for the development of the novel drugs to treat multi-drug resistant Salmonella infections.Natural compounds have been proved beneficial for the treatment of such bacterial infections.Beneficial roles of cinnamaldehyde due to its anti-bacterial,anti-inflammatory,and anti-oxidative properties have been elucidated.However,the ameliorating effect of cinnamaldehyde in liver damage due to S.Gallinarum infection to young chicks remained largely unknown.Therefore,the objective of this study was to investigate the protective effects of cinnamaldehyde on liver damage caused by intraperitoneal infection of S.Gallinarum to young chicks.The study is divided into following parts,1.Protective Effects of Cinnamaldehyde on the Inflammatory Response and hepatic injury to Salmonella Gallinarum-Challenged Young ChicksIn this section protective effects of cinnamaldehyde on Salmonella colonization in hepatic tissue（CFU）,serum level of hepatic enzymes（ALT/AST）,relative m RNA expression of inflammation related genes（IL-1β,IL-6,IL-12,NF-кB,TNF-α,My D-88,IL-8,IL-10 and i NOS）,Salmonella-induced hepatic morphological damage（using H&E staining and TEM techniques）were investigated.A total of 90 specific pathogen-free（SPF）day-old chicks were purchased and acclimatized for3 days.The flock was divided into three groups,each with 30 chicks,i.e.,control group（CON）,challenge group（SG）,and treatment group（SG+CA）.At 0 day post-infection（0 DPI）,PBS（intraperitoneally）in the CON group,0.3 m L of 3×10?⁷ CFU/m L S.Gallinarum solution in the SG and SG+CA groups（intraperitoneally）,and 150 mg/kg body weight cinnamaldehyde in the SG+CA group（orally）were administered.Cinnamaldehyde in SG+CA was administered till 3DPI.On 1,3,and 5 DPI blood samples were collected from 10 chicks（in each group）and slaughtered humanely on the respective days to collect liver samples.Our results indicated a significant（P<0.05）decrease in hepatic CFU by cinnamaldehyde treatment,for average hepatic colonization(Log₁₀ CFU/g)at 3 DPI were 4.08±0.06 for SG,and 2.97±0.1for SG+CA group.The results of serum ALT and AST concentrations showed the protective efficiency of cinnamaldehyde in minimizing the hepatocytic damage.At 3 DPI,the serum ALT and AST concentrations were from 29.51±1.7 IU/L（SG group）down to 19.36IU/L（SG+CA group）,and from 29.14±1.36 IU/L（SG group）down to 15.50±0.94 IU/L（SG+CA group）,respectively.Moreover,cinnamaldehyde significantly（P<0.05）decreased the relative m RNA expression of pro-inflammatory factors（IL-1β,IL-6,IL-12,NF-кB,TNF-α,and My D-88）and increased the relative m RNA expression of anti-inflammatory factors（IL-8,IL-10,and i NOS）in S.Gallinarum-challenged chicken hepatocytes,at 1,3,and 5 DPI,revealing its anti-inflammatory properties against Salmonella infection to chicken.Histopathological examination indicated that Salmonella infection resulted in haemorrhages,necrosis,and immune cell infiltration in hepatic tissue whereas cinnamaldehyde treatment group showed normal hepatocytic architecture.TEM findings also showed distorted nuclear membrane,intracellular vacuolation,damaged mitochondria and other organelles of Salmonella-challenged hepatocytes,whereas cinnamaldehyde treated group showed relatively normal hepatocytic morphology.Hence,these results showed the protective effects of cinnamaldehyde on the inflammatory response and injury to the hepatocytes of S.Gallinarum-challenged young chicks.2.Protective Effects of Cinnamaldehyde on the mitochondrial health and Energy Metabolism of Hepatocytes of Salmonella Gallinarum-Challenged Young ChicksInfections result into reactive oxygen species（ROS）production.ROS production leads to decrease in mitochondrial membrane potential eventually causing permeabilization and apoptosis.In this section,effect of cinnamaldehyde on S.Gallinarum-challenged chicken hepatocytic ROS production,Ψm,cytochrome c release,caspase 3 content,and ATP content in liver were investigated.Our finding demonstrated that S.Gallinarum-challenged hepatocytic ROS were significantly（P<0.05）decreased by cinnamaldehyde treatment at 1,3,and 5 DPI.Mitochondrial membrane potential and cytochrome c release were significantly（P<0.05）decreased by Salmonella infection while cinnamaldehyde treatment group exhibited approximately normal mitochondrial membrane potential and cytochrome c levels at 1,3,and5 DPI.At 3 DPI,caspase 3 content（ng/ml）were from 48.65±0.3（SG group）down to 36.35±0.9（SG+CA group）clearly indicating the beneficial effect of cinnamaldehyde.Moreover,Salmonella-induced increased hepatocytic ATP concentration was significantly（P<0.05）decreased by cinnamaldehyde treatment at 1,3,and 5 DPI.In conclusion these results showed the protective effects of cinnamaldehyde on the mitochondrial health and energy metabolism of hepatocytes of Salmonella Gallinarum-challenged young chicks.3.Transcriptome analysis of Salmonella Gallinarum challenged chicken hepatocytes treated with cinnamaldehydeTo get a deeper insight into the molecular mechanisms of cinnamaldehyde and induced gene expression changes at m RNA level,we performed transcriptome analysis of chicken hepatocytes infected with S.Gallinarum using RNA sequencing Illumina Platform（Hiseq xten/Nova seq 6000 Sequencing）.High-quality data with an adequate sequencing depth was generated by high-throughput RNA sequencing.GO function analysis and KEGG pathway analysis were combined to explore the mechanism of cinnamaldehyde action on S.Gallinarum induced metabolic changes in the hepatocytes of the chickens.We observed that,at 5 DPI,3genes,ENSGALG00000027188（SREBP1）,ENSGALG00000030034（G6Pase）,and ENSGALG00000007636（PEPCK1）of the AMPK pathway,which were down-regulated in the control＿vs＿challenge group,were up-regulated in the challenge＿vs＿treatment group.Moreover,2 genes,ENSGALG00000016456（Lipin-1）and ENSGALG00000041708（Wnt4）,which were down-regulated in the control＿vs＿challenge group,were found up-regulated in the challenge＿vs＿treatment group in the m TOR pathway.Up-regulated genes of the AMPK pathway were involved in the gluconeogenesis and fatty acid biosynthesis.At the same time,up-regulated genes of the m TOR pathway were involved in lipid biosynthesis.Conclusively,transcriptome analysis showed that Salmonella infection to chicken hepatocytes down regulate the genes involved in gluconeogenesis,fatty acid,and protein synthesis,while cinnamaldehyde treatment promotes gluconeogenesis,fatty acid and protein synthesis.ConclusionConclusively,current study revealed the protective effects of cinnamaldehyde on oxidative stress,inflammatory response,and apoptosis of the hepatocytes of S.Gallinarum-challenged young chicks.S.Gallinarum induces inflammation and apoptosis via NF-Kβ/caspase-3dependent pathway in the hepatocytes of young chicks.In addition,S.Gallinarum infection may also result in metabolic changes via modulating the AMPK-m TOR pathway.However,cinnamaldehyde treatment ameliorated inflammation and apoptosis caused by Salmonella infection by suppressing NF-Kβ/caspase-3 pathway.Transcriptome analysis revealed that cinnamaldehyde treatment reverted the metabolic changes caused by S.Gallinarum infection via the AMPK-m TOR pathway.We concluded that cinnamaldehyde has anti-bacterial,anti-inflammatory,anti-oxidative,and anti-apoptotic properties against S.Gallinarum infection to chicken hepatocytes and it can be a candidate drug for the treatment of Salmonella infections in poultry production.However,in future,long-term experimental studies and gene knock-out systems can be used to validate the genes involved in the mechanism of action of cinnamaldehyde in the mitigation of liver injury caused by S.Gallinarum infection to chicken.