Metabolic Transformation Of Citrinin And Alterations To Injuries With Chronic Alcohol Co-exposure And Intervention Of Polygonatum Sibiricum

Monascus is widely used in the production of food,especially in the yellow wine industry.However,there is a potential risk of producing mycotoxin citrinin（CIT）during the Monascus metabolism.CIT is highly nephrotoxic,mutagenic,teratogenic,and carcinogenic,and threats the safety of Monascus products and the brewing industry.There is little research on CIT metabolism and transformation in the body,especially its impact on the gut-liver-kidney axis and gut-liver-brain axis.It is unclear whether it changes the metabolism of CIT and enhances the toxicity with chronic alcohol co-exposure.Therefore,it is necessary to study the effect of CIT metabolism and its toxicities with the involvement of chronic alcohol co-exposure.Meanwhile,it is important to find ways to reduce the injuries of combined effects,such as using polysaccharides and saponins from steamed Polygonatum sibiricum for intervention.This article focuses on the study of CIT metabolism using liver microsomes,cytochrome 450（P450）enzymes,and animal experiments.The study tracked the metabolites,investigated the mechanism of CIT injury with chronic alcohol co-exposure,and explored the intervention mechanism of polysaccharides and saponins for steamed Polygonatum sibiricum to the co-exposure.1.Four CIT metabolites were found in in vitro and in vivo.The transformation pathway of CIT was studied through liver microsomes,P450 enzymes,and molecular docking models,and two metabolites and their content were confirmed in miceLiver microsomes incubation was applied to study CIT metabolites.Combined with high-resolution time-of-flight mass spectrometry（TOF-MS）and Unifi software,four new metabolites were discovered,named 3-hydroxy-citrinin（M1）,4,5-ene-citrinin（M2）,5-hydroxymethyl-citrinin（M3）,and 7-hydroxy-citrinin（M4）.The four metabolites involved phase I metabolism,including hydrogenation,dehydrogenation,and oxygenation.In the liver microsomes,the content of the four metabolites rapidly increased within 30minutes and then increased very slowly.During the entire metabolic process,the content of M1was significantly higher than that of M2,M3,and M4（P<0.05）.By studying the metabolism of CIT with different P450 subtypes with molecular docking models,it was found that CYP3A4mainly participated in the generation of M1;CYP1A2 also promoted the generation of M4while generating M1;CYP2C9 and CYP2D6 could promote to the generation of M1 to M4,but to a less contribution to CYP3A4 and CYP1A2.Animal experiments showed that M1 and M4 were found in the intestine,liver,kidney,and hippocampus of mice,but M2 and M3 were not found in in vivo.The levels of CIT and the metabolites in the intestine were significantly higher than those in the liver,kidney,and hippocampus（P<0.05）.It displayed the low levels of CIT and its metabolites in the liver,kidney,and hippocampus,but the proportions of CIT were relatively high in these organs（P<0.05）.This indicates that there are differences in the types and levels of CIT and its metabolites in various tissues of the body.2.The effects of CIT metabolites were analyzed on the intestine,liver,kidney,and hippocampus in mice using gut microbiome and metabolomics analysis,and the alternations were investigated on CIT metabolism and the injuries to the gut-liver-kidney axis and gut-liver-brain axis with chronic alcohol exposureThe studies of CIT injury are mainly focused on the kidney,but it is paid little attention to CIT injuries on the intestines and other organs.Previous studies have found the existence of CIT and its metabolites in the intestines.In the animal experiments,CIT（5 mg/kg）caused significant damage to the intestines,decreased density of intestinal villi in mice,and downregulated levels of acetic acid and propionic acid.It also led to the decrease of Firmicutes and increase of Bacteroidetes in the phylum level,and reduced the abundance of Lactobacillus in the genus level.Under the influence of chronic alcohol exposure（5～35%）（co-exposure in short）,M1 significantly decreased and M4 slightly increased in the intestines（P<0.05）.It shows that co-exposure further exacerbated CIT injury to the intestines,manifested as aggravated shortening of intestinal villi,decreased levels of acetic acid and propionic acid,increased Bacteroidetes in the phylum level,exacerbated the decrease in Lactobacillus,and promoted the increase in Bacteroidetes,Prevotella,and Ruminococcus in the genus level,leading to intestinal microbiota disorder.CIT metabolism promoted significant liver and kidney damage,with no effect on thehippocampus.With co-exposure,both M1 and M4 in the liver were increased（P<0.05）,and the significant increase of M4 in the kidney and hippocampus（P<0.05）.CIT metabolism exacerbated liver and kidney injury and caused hippocampal injury with co-exposure,specifically promoting the increase of ALT and AST levels in the liver,the increase of CRE and BUN concentrations in the kidney,and the decrease of Ach levels and increase of GABA levels in the hippocampus.SOD,GSH levels in the liver,kidney,and hippocampus were significantly decreased,while TNF-αand Caspase-3 levels were significantly increased.Metabolomic analysis revealed that serine,aspartic acid,proline,tyrosine,succinicsemialdehyde,indole,and uric acid were the main metabolites that affected the liver;phosphocholine,glucose 6-phosphate,inosinic acid,and indoleacetic acid were the main metabolites that affected the kidney;and orotic acid,dimethyl sulfone and citric acid metabolism were the main metabolites that affected the hippocampus.Furthermore,tryptophan metabolism and purine metabolism were found to be common metabolic pathways between the liver and kidney,while sulfur metabolism,alanine,aspartic acid,and glutamic acid metabolism were common metabolic pathways between the liver and brain.The correlation analysis found that CIT and M1 were positively correlated with Lactobacillus,negatively correlated with M4,while other genera were the opposite correlations.There were significant correlations between increased CIT metabolites and differential metabolites in the liver,kidney,and brain.It revealed the enhanced positive correlation of increased CIT metabolites with the tryptophan metabolism and the alanine,aspartate and glutamate metabolism pathways,and negative correlation with the purine metabolism and sulfur metabolism pathways.In addition,orotic acid and dimethyl sulfone could be considered as marker metabolites of hippocampal damage with the co-exposure.These results indicated that gut microbiota disorder was more severe under co-exposure and induced intestinal barrier dysfunction.A large amount of CIT crossed the intestine into the body and induced metabolism.Under co-exposure,the increase in CIT metabolites disrupted the metabolism of the liver,kidney,and hippocampus,causing metabolic disorders in the gut-liver-brain axis and gut-liver-kidney axis.3.Through screening of functional ingredients beneficial to the intestines,the newpolysaccharides and saponins were extracted and isolated from steamed Polygonatum sibiricum,and their structures were analyzed and identifiedFermentation in vitro showed that steamed Polygonatum sibiricum polysaccharides promoted increases of acetic acid and propionic acid.It promoted the proliferation of beneficial bacteria such as Parabacteroides and Bifidobacterium,and inhibited the proliferation of harmful bacteria such as Shigella and Enterococcus.Steamed Polygonatum sibiricum saponins promoted the increase of acetic acid and propionic acid,inhibited the proliferation of harmful bacteria such as Shigella,and promoted the proliferation of beneficial bacteria such as Enterococcus,Roseburia,Weissella,and Faecalibacterium.This indicated that steamed Polygonatum sibiricum polysaccharides and saponins had beneficial effects on gut microbiota.Further isolation,purification,and in vitro fermentation led to the discovery of an active small molecule polysaccharide with a molecular weight of 1800,composed of 11 sugar residues,including fructose and glucose,which is inconsistent with existing reports on Polygonatum sibiricum polysaccharides.NMR analysis of the polysaccharide’s spectrum confirmed that glucose was connected to the starting position of fructose at the 2nd position in anαconfiguration,and that the positioning of fructose was connected with 2→3 and 2→5 bonds,with the specific connection beingα-glc1→2Fru[2→3 Fru]₆[2→5 Fru]₄,which was determined as Polygonatum sibiricum polysaccharides.At the same time,liquid chromatography analysis identified the two main saponins,sibiricosides A and B,which were determined to be Polygonatum sibiricum saponins.4.Intervention effect of Polygonatum sibiricum polysaccharide on the CIT metabolismand the gut-liver-kidney axis injury with the co-exposureThe intervention of Polygonatum sibiricum polysaccharide on the intestinal injury was manifested as an increase in the levels of CIT and M1 in the intestine（P<0.05）,and a decrease in M4（P<0.05）.The decrease in intestinal villi was inhibited,and the levels of acetic acid and propionic acid were increased.The intervention promoted the increase of Firmicutes and suppressed the proliferation of Bacteroidetes and the ratio of Bacteroidetes/Firmicutes.It also promoted the recovery of Lactobacillus and inhibitd the proliferation of Bacteroides,Prevotella,and Ruminococcus.Polygonatum sibiricum polysaccharide also had an intervention effect on liver and kidney injury caused by co-exposure,manifested as a decrease in CIT,M1,and M4 levels,inhibition of the increase of liver ALT and AST,inhibition of the increase of kidney CRE and BUN,recovery of SOD and GSH levels in the liver and kidney,and inhibition of TNF-α,and an increase in Caspase-3.However,the polysaccharide had no effect on hippocampal injury.Metabolomics analysis found that Polygonatum sibiricum polysaccharide intervened in liver damage through the metabolism of tyramine,leucine,β-alanine,and indole-3-acetic acid;it intervened in kidney damage through the metabolism of riboflavin,hydroxyproline,and 6-phosphoamino glucose.The correlation analysis showed that CIT and M1 are positively correlated with Lactobacillus,and negatively correlated with M4,and other bacteria were the opposite correlations.There was a significant correlation between the decrease of CIT metabolites and differential metabolites in the liver and kidney.The decrease of CIT metabolites was significantly positively correlated with tryptophan,aspartic acid and tyrosine related metabolic pathways.Therefore,polysaccharide intervention could improve intestinal microbiota disorders,fix the barrier function against CIT,and inhibit the increase of M4.It also decreased the levels of CIT,M1,and M4 to alleviate liver and kidney injury,and improved the metabolic disorders of the intestinal-liver-kidney axis caused by co-exposure through the tryptophan,aspartic acid,and tyrosine metabolism pathways.5.Administration of Polygonatum sibiricum saponins promoted the intervention of CIT metabolism,as well as the injury to the gut-liver-kidney axis and the gut-liver-brain axis under the co-exposurePolygonatum sibiricum saponins intervened in the intestinal injury of co-exposure,manifested as an increase in the levels of CIT and M1 in the intestine（P<0.05）,a significant decrease in M4（P<0.05）,inhibition of the reduction of intestinal villi,an increase in levels of acetic acid and propionic acid.Saponins promoted the increase in Firmicutes,inhibition of Bacteroidetes,and the ratio of Bacteroidetes/Firmicutes.It also promoted the recovery of Lactobacillus,and inhibition of the proliferation of Bacteroides,Prevotella and Ruminococcus.Polygonatum sibiricum saponins also had the intervention effect on liver,kidney,and hippocampal injury,manifested as the decreased levels of CIT,M1,and M4,the inhibition of the liver ALT and AST levels,the inhibition of kidney CRE and BUN contents,the promotion of the ACh levels in the hippocampus,the inhibition of the GABA level.Saponins also showed the recovery of SOD,GSH levels in the liver,kidney,and hippocampus,and the inhibition of TNF-α,Caspase-3 increases.Metabolomic analysis showed that Polygonatum sibiricum saponins intervened in co-exposure-induced liver damage by affecting the metabolism of tyrosine,tyramine,and indoleacetic acid,and intervened in kidney injury through affecting the metabolism of riboflavin,phosphatidylcholine,glucose-6-phosphate,and cysteine.Polygonatum sibiricum saponins also intervened in hippocampal damage through affecting the metabolism of acrylamide,inosine,and lactic acid.Correlation analysis found that M1 was positively correlated with Lactobacillus and negatively correlated with M4,with other genera showing the opposite.The decrease of CIT metabolites was significantly correlated with differential metabolites in the liver,kidney,and brain.The decrease of CIT metabolites was significantly positively correlated with tryptophan,aspartic acid and tyrosine related metabolic pathways.These indicated that Polygonatum sibiricum saponins improved liver,kidney,and hippocampal damage by regulating the decrease of CIT and its metabolites,and improved the metabolic disorders of the intestine-liver-kidney and intestine-liver-brain axes caused by co-exposure through the tryptophan,aspartate,and tyrosine metabolic pathways.In summary,this study found new metabolic products of CIT and their changes in in vitro and in vivo,which were related to the metabolism of P450 subtypes.Chronic alcohol and CIT co-exopsure promoted the metabolism of CIT and exacerbated the damage to the intestine,liver,kidney,and brain as well as the metabolic disorder of the gut-liver-kidney axis and the gut-liver-brain axis.The purified and evaluated active ingredients of Polygonatum sibiricum such as polysaccharides and saponins,had beneficial effects on the intestinal flora and intervened in the injuries to the intestine,liver,and kidney caused by cp-exposure,improving the metabolic disorders of the gut-liver-kidney axis.Furthermore,saponins of Polygonatum sibiricum alleviated hippocampal damage and metabolic disorder of the gut-liver-brain axis.This study had laid a theoretical foundation for future research on the toxicological mechanism and intervention of mycotoxin CIT in red yeast rice and other fermented foods.