Chlorogenic Acid Alleviates Chronic Stress-induced Liver Injury In Rats Through The Gut-liver Axis

In the veterinary clinic,chronic stress is more common and can cause liver damage in animals and promote the development of liver diseases and aggravate their severity,which seriously endangers the growth and development of livestock and poultry and reduce the quality of livestock and poultry prod ucts.Recent studies have found that intestinal flora disorders are closely related to the development of liver disease.Therefore,based on the intestinal flora as the starting point,investigating the mechanism of action of chronic stress-induced liver injury and screening safe and effective preventive and curative drugs has become a research hotspot in the field of veterinary medicine and medical science.Chlorogenic acid(CGA)is one of the key active ingredients of Chinese traditional medicine honeysuckle,which has the characteristics of wide source,easy extraction,and low price.Current studies have proved that CGA has good protective effects on liver,intestine,kidney and other organs,and several recent studies have found that the regulation of intestinal flora by CGA is an important mechanism for its organ-protective effects.Therefore,this experiment used 16 S r DNA,untargeted metabolomics,and transcriptome sequencing technologies to detect the changes of flora in the intestine(ileum and colon),metabolite alterations in serum and liver gene transcription,respectively,linking intestinal flora,metabolism,and liver,thereby elucidating the specific mechanism of action of CGA in alleviating chronic stress-induced liver injury.To investigate the effect of CGA on chronic stress-induced liver injury and its intervention mechanism,36 male Wistar rats were first selected for the CGA dose screening test.36 rats were randomly divided into 6 groups,namely,CON group,CON +CAH group(150 mg/kg CGA),CRS group,CRS + CAL group(50 mg/kg CGA),CRS +CAM group(100 mg/kg CGA)and CRS+CAH group(150 mg/kg CGA).The stress procedure was 21 consecutive days of restraint stress(6 h /d).The changes in body weight and food intake of the rats were recor ded during the modeling period.At the end of the modeling period,the success of the model was verified by measuring behavioral changes and serum Corticosterone(CORT)levels.The protective effects of different doses of CGA on chronic stress-induced liver injury were clarified by detecting liver coefficients,liver structure,liver function,oxidative stress,apoptosis,and inflammatory factor levels in rats,and screening the optimal protective dose of CGA.Then another 18 male Wistar rats were used for the CGA protection mechanism investigation test.18 rats were randomly divided into 3 groups: CON group,CRS group,and CRS+CGA group(100 mg/kg CGA).Model validation was performed after restraint stress(6 h/d,21 d).The histopathological changes of intestine(ileum and colon),ultrastructural changes of intestine(ileum and colon),tight junction structure of intestine(ileum and colon),lipopolysaccharide(LPS)content in serum,intestinal flora(ileum and colon),serum metabolites,hepatic gene transc ription,and related gene expression were observed to elucidate the protective mechanism of CGA in alleviating chronic stress-induced liver injury.(1)The experimental results of the effect of different doses of CGA on liver injury caused by chronic stress in rats showed that:1)After chronic stress,the serum level of CORT in rats was significantly increased;behavioral tests revealed that the locomotor trajectories of rats were concentrated at the edge and small in scope,and the total distance,the res idence time in the central area,the number of crossing frames and the number of standing were significantly reduced,and the rats showed depression-like behavior.This indicated that the chronic stress model was successfully established.2)After chronic stress,the average daily weight gain,daily food intake,and liver coefficient of rats were reduced.There was no significant alleviation effect of the above indexes after low-dose CGA intervention.The above indexes were significantly increased after medium and high doses of CGA intervention.It showed that medium and high doses of CGA could significantly alleviate the influence of chronic stress on body weight,food intake and liver coefficient of rats.3)After chronic stress,the liver function indexes of rats,ALT and AST,increased significantly,and there was no significant change in ALT and AST after low-dose CGA intervention.The ALT and AST indexes were significantly reduced after medium and high dose CGA intervention.It showed that medium and hi gh doses of CGA could alleviate the liver dysfunction caused by chronic stress.4)After chronic stress,histopathological examination revealed irregularly aligned hepatic cords,swollen and lightly stained hepatocytes,granular degeneration,and cleaved and absent hepatocyte nuclei in rats liver.Low-dose CGA intervention in the liver did not alleviate the structural damage.In contrast,the liver structure of rats was more intact and the hepatocytes were less swollen after medium and high doses of CGA int ervention.The ultrastructural observations of liver showed that the structure of hepatocytes was disturbed after chronic stress,with swollen mitochondria and disappearance of cristae,dilated and swollen endoplasmic reticulum,more vacuoles in the cytopl asm,and wrinkled nuclei.After low-dose CGA intervention,the hepatocyte damage was not alleviated,while the structure of hepatocytes was relatively intact with clearer endoplasmic reticulum and mitochondrial structure after medium and high-dose CGA intervention.This indicated that the medium and high doses of CGA intervention can significantly repair the structural damage to liver caused by chronic stress.5)After chronic stress,the fluorescence intensity of ROS and level of MDA in rat liver were significantly increased,and the levels of SOD,GSH,and CAT were significantly decreased.Low-dose CGA intervention reduced MDA level and increased SOD,GSH,and CAT levels,but did not reduce ROS fluorescence intensity.Medium and high dose CGA interventions significantly reduced ROS fluorescence intensity,and MDA level and increased SOD,GSH,and CAT levels.It showed that the antioxidant effect of medium and high doses of CGA was better and could alleviate the liver oxidative stress damage caused by chroni c stress.6)After chronic stress,the protein expression levels of liver inflammatory factors(IL-6 and TNF-α)and the expression of the apoptotic protein Cleaved caspase-3 were significantly increased in rats.Low-dose CGA intervention had no significant effect on the above three indices.Medium and high doses of CGA significantly decreased the levels of IL-6,TNF-α,and Cleaved caspase-3.It showed that medium and high doses of CGA could alleviate liver inflammation and apoptosis(2)Investigation of the mechanism of CGA to alleviate liver injury in rats caused by chronic stress:1)The results of intestinal histopathology showed that the ileum was severely damaged after chronic stress,with intestinal epithelial cells detached and local inflammatory cell infiltration;the colon was less damaged,with only a small amount of inflammatory cell infiltration;the ileum and colonic villi were neatly arranged after CGA intervention,with less inflammatory cell infiltration.Ultrastructural observations showed that the epithelial cells of the ileum and colon were structurally disturbed after chronic stress,with swollen mitochondria,the disappearance of cristae,and wrinkled nuclei.The intercellular tight junction(TJ)structure is disrupted.After CGA intervention,the structure of ileal and colonic epithelial cells was relatively intact,most of the mitochondria were normal,and the nuclei were not abnormal.The intercellular TJ structure was clear and relatively continuous.The results of immunohistochemical s taining and RTq PCR assay for TJ key proteins showed that levels of key ileal and colonic TJ proteins(Claudin3,Occludin and ZO-1)were significantly reduced after chronic stress and significantly increased after CGA intervention.The results of serum LPS level assay showed that CGA was able to reduce the elevated LPS level in serum caused by chronic stress.It can be concluded that CGA can alleviate intestinal epithelial cell damage and promote TJ protein expression,thus repairing the mechanical barrier of the intestine.(2)The results of 16 S r DNA assay of ileal flora showed that Firmicutes was the dominant phylum in ileal intestine and Lactobacillus was the dominant genus.The abundance of Lactobacillus was significantly reduced after chronic stress.After CGA intervention,the abundance of Firmicutes increased,the ratio of Firmicutes/Bacteriodets(F/B)increased significantly,and the abundance of Lactobacillus increased.This indicated that CGA could adjust the intestinal flora and increase the abundance of Lactobacillus.(3)The results of 16 S r DNA assay of colonic flora showed that Firmicutes and Bacteriodets were the dominant phylum in the colonic intestine,and the genera of Lachnospiraceae＿NK4A136＿group,the Muribaculaceae＿unclassified,and Lactobacillus were the dominant genera.After chronic stress,the abundance of Actinobacteria decreased,the F/B ratio increased significantly,and the abundance of genera such as Lachnospiraceae＿NK4A136＿group increased.While the abundance of Roseburia increased after CGA intervention.It showed that CGA could adjust the intestinal flora and reduce the abundance of Lachnospiraceae＿NK4A136＿group.(4)The results of the serum untargeted metabolomics assay showed that chronic stress upregulated 304 metabolites and downregulated 648 metabolites;the CGA intervention upregulated 193 metabolites and downregulated 81 metabolites.A total of 56 potential differential biomarkers were identified after an analytical screening of differential metabolites,mainly involving tryptophan metabolism,nicotinate,and nicotinamide metabolism,and bile secretion.The levels of metabolites such as deoxycholic acid(DCA)increased and those of indole and 3-Hydroxyanthranilic acid(3-HAA)decreased after chronic stress,and CGA intervened to reverse the changes.This indicate d that CGA could regulate the metabolism of the body during chronic stress and play a protective role.(5)The results of liver transcriptome assay showed that 710 genes were up-regulated and 855 genes were down-regulated in liver gene expression after chronic stress,and 440 genes were up-regulated and 107 genes were down-regulated in liver gene expression after CGA intervention.The enrichment analysis revealed tha t retinol metabolism,PPAR signaling pathway,arachidonic acid metabolism,drug metabolism-cytochrome pathway,etc.were significantly altered.CYP450-related genes were also found to be present in several pathways,and the results were largely consistent with transcriptome sequencing after RT-q PCR.Chronic stress promoted the expression of most genes in CYP1 A,CYP2 B,CYP3 A,and CYP4 A families,while CGA intervention reduced their expression levels.This indicates that CGA can regulate the expression of key CYP450 family genes and alleviate liver injury.In summary,chronic stress can cause intestinal barrier damage,on the one hand,the intestinal mechanical barrier is damaged,and intestinal permeability is enhanced,on the other hand,the intestinal flora is disturbed and the metabolism of flora is changed,which causes pathogenic bacteria and their metabolites to enter the blood circulation,thus causing liver damage.When CG A was applied at different doses to intervene with chronic stress,it was found that the protective e effect could be achieved at a dose of 100 mg/kg,and the effect was the same as that of 150 mg/kg,which could improve the structural and functional damage of liver caused by chronic stress and alleviate the oxidative stress,apoptosis and inflammatory damage of liver.The mechanism of the protective effect is that CGA can repair intestinal barrier,promote TJ-related protein expression,restore intestinal flora homeostasis,regulate bile acid and tryptophan metabolism,reduce DCA and increase indole and 3-HAA metabolite levels,and reduce CYP450 family key protein expression,thus alleviating liver injury.