Metabolic Rewiring Of Kynurenine Pathway During Hepatic Ischemia-Reperfusion Injury Exacerbates Liver Damage By Impairing NAD Homeostasis

Hepatic ischemia-reperfusion(IR)injury remains a common problem with a lack of effective strategies and efficient pharmacological targets.The current pathological model of hepatic IR injury includes a two-stage process of ischemia-induced cellular injury and reperfusion-induced inflammatory response.This concept has led to a focus on the development of drugs with direct anti-inflammatory and anti-apoptotic properties,but the benefits achieved so far in the clinic have been unsatisfactory.In the present work,using a non-targeted metabolomic screen,we found that hepatic 3-hydroxy-2-aminobenzoic acid(3-HAA)and quinolinic acid(QA)were decreased and L-kynurenine(KYN)and kynurenine quinolinic acid(KYNA)were upregulated after hepatic IR in mice,and that kynurenine aminotransferase 2(Kyat2)functions at a key branch point of the kynurenine pathway(KP)in Ischemia is significantly upregulated in hepatocytes,shifting the tryptophan metabolic pathway from 3HAA and QA to KYNA synthesis,whereas elevated Kyat2 expression is mainly regulated by p-eIF2α.downregulation of QA synthesis blocks the ab initio biosynthesis of NAD,making hepatocytes dependent on the salvage pathway to maintain NAD levels and cellular antioxidants,and knockdown of Kyat2 effectively restores NAD levels and reduce lipid peroxidation damage in the liver.The nicotinamide phosphoribosyltransferase(NAMPT)inhibitor FK866 blocked the post-IR salvage pathway,which exacerbated hepatic oxidative damage and enhanced IR sensitivity,whereas the lipid peroxidation inhibitor Lip-1 inhibited oxidative damage,whereas the Naprt inhibitor 2-HNA blocked the post-IR Press-Hindle pathway with no significant effect on acute liver injury.Notably,in a mouse model,NAD production was effectively promoted by administration of nicotinamide mononucleotide(NMN)after IR and attenuated IR-induced oxidative stress,inflammation and cell death.In conclusion,our findings reveal that the ab initio synthesis of NAD is inhibited by metabolic reprogramming of KP during the hepatic IR process and provide evidence that upregulation of NAD synthesis is a promising therapeutic approach to rescue IR-induced liver injury.Purpose of the study1.The change of metabolite content in the tryptophan metabolic pathway after IR was determined by animal experiments and metabolome determination.2.Elucidate the causes and molecular mechanisms of metabolite content changes.3.To explore the effect of NAD homeostasis on liver injury and find small molecule substances that can effectively save liver ischemic damage,so as to provide effective treatment strategies for clinical practice.Study results1.After metabolome detection,the tryptophan metabolism of the mouse liver in the ischemic stage was metabolically tilted to the kynurenine metabolic branch,the content of 3-HAA and quinolinic acid decreased,and the content of kynurenine and canine urinary quinoline acid increased.2.Kyat2 expression is elevated in the ischemic stage of key enzymes Afinid and Kyat2 in the ischemic stage,there are no obvious changes in other genes in the pathway,Kyat2 is also expressed in samples of patients with liver resection,there is no significant change in the mRNA level of Kyat2 in ischemic liver,the upregulation of its protein level is parallel to the increase of p-Eif2α,and the inhibition of p-Eif2α by ISRIB weakens the upregulation of Kyat2 in ischemic liver.This paper indicates that p-eIF2α-mediated translation control modulates the expression of Hepatic IR-induced Kyat2 to a certain extent.After 3.IR,the level of NAD/NADH in the experimental group was reduced compared with the control group,and the targeted metabolome showed an increase in the expression level of lipid peroxidation-related genes,an upregulation of Ptgs2 expression at the RNA and protein levels,and enhanced IHC staining for EO6,MDA,4-HNE and other indicators.Compared with the NC group,the E06,MDA,4HNE,CD45 and Ly6b staining,liver enzyme levels,RNA expression of inflammatory factors in the siKyat2 group were reduced,the liver state became better,and the degree of damage was reduced.4.After ischemia and intraperitoneal injection of Nampt inhibitor FK866,the survival time of mice in the IR+FK866 group was significantly lower and dose-dependent than that of the IR+Vehicle group,and the NAD/NADH content was lower,and the liver status of the mice was observed after 24h of perfusion and liver enzymes were determined and stained by HE,CD45,E06,4HNE and inflammatory factor mRNA,and the results showed that the liver damage in the IR+FK866 group was higher than that in the IR+Vehicle group.After inhibiting Naprt,there is no significant effect.5.After ischemia and intraperitoneal injection of FK866+Lip-1,compared with FK866+Vehilce group,the survival of mice in FK866+Lip-1 group was prolonged,liver damage was reduced,liver status became better,liver enzyme levels decreased,HE,E06,4HNE,CD45 staining was weakened,and the expression of inflammatory factors decreased.6.After supplementing NMN,the liver state of mice improved,liver enzyme levels decreased,NAD/NADH content recovered,HE staining showed that liver necrotic areas were reduced,IHC staining and inflammatory factor qPCR results showed that the mice had reduced oxidative stress damage,reduced inflammatory infiltration,and NMN played a significant role in salvage.