Programming Effect Of Fatty Liver In Adult Offspring Rats Induced By Exposure To Retrorsine During Lactation

Objective:Pyrrolizidine alkaloids(PAs)are a natural component with hepatotoxicity,widely distributed in 3%of flowering plants in the world.Chinese herbal medicines or foods contained with PAs are still widely consumed in China.After maternal ingestion,milk will be contaminated and infants will also be exposed to PAs.Early cases suggest that exposure to retrorsine(RTS)during lactation can lead to fatty liver in young offspring,but whether fatty liver lasts until adulthood is unclear.In this project,RTS was used as a representative drug for PAs to explore the programming effects of RTS exposure during lactation on fatty liver in adult offspring rats.Methods:During postnatal week(PW)1 to 3,mother Wistar rats were given 5 or 20 mg/kg·d of RTS by intragastric administration(RTS5 and RTS20 groups),the control group was given the same volume of vehicle.Milk was collected in the last second day of PW3,and blood and liver of parts of the offspring were collected on the first day of PW4.Hematoxylin-eosin(H&E)and oil red O staining were used to observe liver histopathological changes.UHPLC-QE Orbitrap/MS method was used for metabolomics analysis of milk components.LC-MS/MS method was used to detect the content of RTS prototype drugs and metabolites in milk,blood and liver.Biochemical kits were used to detect serum liver functional indexes and lipid levels in PW3 and PW12 offspring rats.Real-time quantitative PCR was used to detect the expression changes of genes related to fatty acid uptake,synthesis,β-oxidation and efflux in livers of PW3 and PW12 offspring rats,as well as genes related to the activation of pregnane X receptor(PXR).Chromatin immunoprecipitation assay(ChIP)was used to detect histone acetylation and methylation in the PXR promoter region.Further,L02 cells were treated with different concentrations(10,100 and 1000 nM)of RTS for 72 h to detect the lipid level of the cells and observe the effect of RTS on the expression of PXR and lipid metabolism related genes.Results:①Compared with the control group,RTS caused a significant decrease in body weight,body weight gain rate and liver index of PW3 offspring(P<0.01,P<0.05).Pups in the RTS20 group died from 15 to 36 days after birth.Female in RTS5 group showed obvious catch-up growth(P<0.05);②The milk composition in the treatment group changed,mainly manifested by a decrease in lipid content.Compared with the mother,there is a large amount of RTS in milk(P<0.01),but the content of pyrrole protein adducts(PPAs)is much less(P<0.01).The content of serum RTS and liver PPAs in offspring are less than mother(P<0.01),the content of PPAs in males offspring liver are more than that in females(P<0.01)③There is a significant vacuolar degeneration in the hepatic cells in the treatment group of the PW3 offspring,with a dose-dependent manner,and oil red O staining is positive.In the RTS5 group,PW12 offspring hepatocytes still showed steatosis;④Compared with the control group,activities of alanine aminotransferase(ALT),aspartate aminotransferase(AST)and alkaline phosphatase(AKP)in PW3 RTS offspring were increased(P<0.01),the levels of total cholesterol(TCHO)and low density lipoprotein chesterol(LDL-C)were increased(P<0.01),while the level of low density lipoprotein cholesterol(HDL-C)was decreased(P<0.01),and the ratio of LDL-C/DL-C was increased(P<0.01),Serum total bile acids(TB A)and triglyceride(TG)levels was also increased(P<0.01),and contents of liver TG and free fatty acid(FFA)were significantly increased(P<0.01,P<0.05).compared with the control group,the activities of ALT,AST and AKP as well as levels of serum TCHO and TBA were not significantly changed in PW12 RTS offspring.Serum TG levels in males were increased(P<0.05).Levels of liver TG and FFA were still increased significantly(P<0.01,P<0.05);⑤Compared with the control group,the expression of fatty acid transport protein 2(FATP2),fatty acid transport protein 5(FATP5),fatty acid-binding protein 1(FABP1),fatty acid synthase(FASN),acetyl CoA carboxylase α(Acetyl CoA carboxylase α,ACCα),carnitine palmitoyltransferase lα(Carnitine palmitoyltransferase 1α,CPT1α),peroxisome prol iferator-activated receptor α(PPARα)and microsomal triglyceride transfer protein(MTTP)were decreased,while expression of fatty acid translocase/cluster of differentiation(FAT/CD36)and peroxisome proliferator-activated receptor γ(PPARγ)(P<0.01,P<0.05)were increased by RTS treatment in PW3 liver(P<0.01,P<0.05),with a dose-dependent manner.Changes of the above genes in RTS5 group continued to PW12;⑥Expression of PXR gene of the PW3 and PW12 offspring were increased in RTS treatment group(P<0.01,P<0.05).The expression of cytochrome P450 3A(cytochrome P450 3A,CYP3A),which is a downstream gene regulated by PXR,was also increased(P<0.05).Expression of farnesoid X receptor(FXR)in PW3 offspring decreased(P<0.01),but showed no change in PW12;⑦histone 3 lysine 9 acetylation(H3K9ac)level of PXR promoter was increased in PW12 RTS offspring liver(P<0.01,P<0.05),while other histone acetylation or methylation sites in PXR promoter region did not change significantly;⑧after RTS treatment for 72 h,The content of TG and FFA in LO2 cells showed a concentration-dependent increase(P<0.05,P<0.01),and the gene expressions of FATP2,FATP5,FASN,CPT1α,PPARα,MTTP,CD36 and PPARγshowed consistent changes with those in animals experiment(P<0.01,P<0.05).Conclusion:RTS exposure during lactation led to fatty liver in PW3 offspring,and PXR activation by RTS may play a role.On one hand,PXR activation may increase the expression of downstream gene PPARγ and increase fatty acid uptake;on the other hand,it may reduce the expression of PPARα and reduce fatty acid β-oxidation,and eventually leads to steatosis.In addition,the increase of H3K9ac level in the PXR promoter region may be involved in the continued increase of PXR expression,therefore,continuously increasing liver fatty acid intake and reducing fatty acid β-oxidation occur,which may ultimately lead to the programming effect of fatty liver in adult offspring.