The Effect And Mechanism Of Alleviating Hyperuricemia By Lacticaseibacillus Rhamnosus NCUH061012 And Fermented Carrots

Recently,the incidence of hyperuricemia(HUA)has shown the trend of significant rise and younger,which has attracted prevalent attention,and HUA has emerged as a public health burden.Up to now,these drugs for treating HUA are often limited in clinical application,because of their side effects.Therefore,safe and effective strategies are urgently needed to be developed to prevent and treat HUA.In this study,lactic acid bacteria(LAB)with anti-HUA potential were obtained,and fermented carrots with anti-HUA potential was developed.Key differences between unfermented carrots as well as fermented carrots were determined,and their anti-HUA activity was explored in vitro.The effect of anti-HUA by LAB and its fermented carrots was analyzed in HUA mice.Moreover,the effect of fermented carrots in patients with HUA was analyzed through population experiments.Finally,the potential mechanism of alleviating HUA in mice by LAB and its fermented carrots was revealed by multiple perspectives in biochemical indicators,gut microbiota,fecal metabolome,and serum metabolome.(1)Lacticaseibacillus rhamnosus NCUH061012(LR012)with anti-HUA potential were obtained by a comprehensive and effective screening method.LR012,Limosilactobacillus fermentum NCUH003018,Limosilactobacillus reuteri NCUH064056 and Lactobacillus gasseri NCUH066006 were preliminarily selected from ninety strains of edible LAB,and showed the ability of efficient degradation of inosine and guanosine,inhibition of xanthine oxidase(XO)activity,the tolerance to acid and bile salt,adhesion to epithelium cells,antibacterial activity,and antibiotic susceptibility in vitro.HUA mice model was established,and the results showed that these four strains of LAB reduced the levels of serum uric acid(SUA),creatinine(CRE)and urea nitrogen(BUN).Among them,LR012 was the most effective in lowering the SUA levels in mice,which prompted the decrease of SUA by 36.88%compared with HUA model group.Moreover,the levels of CRE and BUN in the LR012 group were not significantly different from the normal control group.Therefore,the LR012 is considered as a probiotic with anti-HUA potential.(2)LR012 with anti-HUA potential was used to ferment carrots,which enhance the potential of anti-HUA activity for fermented carrots.LR012 consumed fructose that could promote the development of HUA,generated short-chain fatty acids(SCFAs)that was beneficial for host healthy,and significantly increased the levels of free amino acids as well as free polyphenols in carrots.LR012 significantly changed the metabolic profile of non-volatile substances,and there were sixty-nine differential metabolites between unfermented carrots and fermented carrots,such as purines,amino acids and polyphenols,that can significantly participate in the metabolic pathways including purine metabolism,and amino acid metabolism.LR012 significantly increased the ability of carrots to inhibit the uptake of uric acid in h URAT1-expressing oocytes,and significantly increased the activity to inhibit XO.In fermented carrots,chlorogenic acid was 100.23 μg/m L.The levels of caffeic acid,ferulic acid,p-coumaric acid,as well as gallic acid in fermented carrots increased to31.57 μg/m L,10.58 μg/m L,4.91 μg/m L and 3.41 μg/m L by 14.34%,37.14%,32.70% and 36.40% compared with unfermented carrots,respectively.And the levels of 16-hydroxypalmitic acid as well as 4-hydroxypyrazolo[3,4-d]pyrimidine was increased to 1.69 μg/m L and 1.40 μg/m L,respectively.These changes could be beneficial for promoting the anti-HUA effect of fermented carrots.(3)Animal experiments and population experiments were conducted to comprehensively investigate the anti-HUA effects of LR012 and its fermented carrots.The treatment of LR012,unfermented carrots and fermented carrots alleviated HUA in mice,among which fermented carrots showed the best.In mice,SUA levels in the fermented carrots group were 55.42% lower than that in the HUA model group,and were not significantly different from the normal control group.The treatment of fermented carrots significantly alleviated systemic inflammation and kidney injury induced by HUA.And The levels of tumor necrosis factor-α,interleukin 1β,CRE and BUN as well as renal coefficient were significantly lower than those in the HUA model group,and were not significant difference from the normal control group.Fermented carrots inhibited XO activity in the liver,reduced the levels of uric acid reabsorption transporters URAT1 and GLUT 9 in the kidney,increased the levels of uric acid excretion transporter OAT1 in the kidney as well as the levels of uric acid excretion transporter ABCG2 in the intestine to regulate uric acid production and excretion in HUA mice.Population experiments have shown that fermented carrots effectively reduced SUA levels in HUA patients.After 30 days of intervention by fermented carrots,the SUA levels decreased in 94.44% of HUA patients.The mean SUA levels of HUA patients were decreased from 486.25 μmol/L to 414.31 μmol/L.and the SUA levels in 55.56% of HUA patients were reduced to the normal range.(4)Integrative multiomics revealed the underlying mechanism of alleviating HUA by fermented carrots.Fermented carrots improved the ability of the gut microbiota to degrade uric acid,recovered the diversity of gut microbiota affected by HUA,reversed the decrease in Firmicutes and the increase in Bacteroidetes induced by HUA,reduced Bacteroides,enriched beneficial bacteria(like Lactobacillus,Candidatus＿Saccharimonas,Ruminiclostridium＿5,and Ruminococcus＿1),along with increasing SCFAs and decreasing lipopolysaccharide.The fecal untargeted metabolome indicated that fermented carrots reversed the abnormal up-or down-regulation of intestinal metabolites induced by HUA,such as indol-3-methanol,adenosine and creatine.The serum metabolome indicated that fermented carrots reversed the abnormal up-or down-regulation of serum metabolites induced by HUA,such as L-methionine,hercynine,5-aminoimidazole ribonucleotide,12-ketodeoxycholic acid,and L-histidine.Furthermore,these alterations in gut microbiota,intestinal metabolites and serum metabolites showed positive associations with improving systemic inflammation and kidney damage,regulating uric acid production and excretion,and reducing SUA levels.In conclusion,fermented carrots alleviated HUA by regulating gut microbiota,intestinal metabolites and serum metabolites.