The Preventive Effects And Mechanisms Of Lactoferrin On Alcoholic Liver Injury

BackgroundExcessive alcohol drinking-induced alcoholic liver injury(ALI)is a serious public health problem worldwide.The initiation and progression of ALI are associated with redox imbalance,lipid metabolism dysregulation and gut microbiota abnormality.Due to the absence of efficient pharmacological treatment at present,the ALI prevention by natural compounds has raised research attentions.Lactoferrin(Lf)is an iron-binding protein possessing many beneficial biological functions.Some studies have indicated the preventive potentials of Lf to non-alcoholic fatty liver disease(NAFLD).Although they are different,ALI has an overlapping pathophysiology of NAFLD.We speculated that Lf also can prevent ALI.However,the relevant studies are rare.Moreover,it is controversial for the digestion and absorption of Lf.All these problems need further exploration.ObjectiveThe project aimed to observe the preventive effects of bovine Lf on ethanol-induced cell injury in BRL-3A cells.Meanwhile,to determine whether oral Lf can be absorbed and produce biological activity in its original form.Based on the above studies,we focused on the preventive effects of Lf on ALI induced by different alcohol exposure patterns in male and female mice,and illustrate the potential mechanisms.Methods1.Cell experimentBased on BRL-3A cells,the treatment doses of ethanol and Lf were determined by CCK-8 assay.The cells were randomly divided into 4 groups and treated for 24 h:(1)control group(CON group): complete medium;(2)Lf treatment group(Lf group):complete medium containing 400 μg/ml Lf;(3)ethanol model group(Et OH group):complete medium containing 400 m M ethanol;(4)ethanol and Lf treatments group(Et OH+Lf group): complete medium containing 400 m M ethanol and 400 μg/ml Lf.Apoptosis was determined by flow cytometry,biomarkers were determined by kits,and protein expressions were determined by western blots.Besides,we used Nrf2 antagonist ML385 to explore how Nrf2 blocking affect the preventive effects of Lf on ethanol-induced cell injury via CCK-8 assay and lactate dehydrogenase(LDH)activity determination in cell culture supernatants.2.Studies on Lf digestion and absorptionLf was dissolved in artificial gastric juice,and incubated at 37℃ for 1 h and 4 h.Pepsin was inactivated by heating,p H was adjusted to 7.0,and Lf hydrolysate lyophilized powder was obtained after dialysis and freeze drying.SDS-Tris-PAGE and SDS-Tricine-PAGE were performed to test the protein molecular weight distributions of Lf hydrolysates.Mice were randomly divided into two groups for 3 days: one group was fed by AIN-93 G diet,while another group was fed by AIN-93 G diet containing0.8% Lf.In the third day,the feces of mice were collected,and half mice in each group were sacrificed,then serum and cecum contents were obtained.At day 4,Lf solution(10%,0.15 ml)was administered intragastrically to the mice in the second group every hour continuously for 3 times.After 30 min of last gavage,all mice were sacrificed,and their serum and cecum contents were collected.Lf detections were performed by ELISA kits3.Animal experimentsC57BL/6J mice were randomly divided into 4 groups and fed with different diets:(1)control group(CON)and(2)ethanol model group(Et OH): AIN-93 G diet;(3)low-dose Lf group(LLf): AIN-93 G diet with 0.4% casein replaced by bovine Lf;(4)high-dose Lf group(HLf): AIN-93 G diet with 4.0% casein replaced by bovine Lf.Modelling methods varied from experiment to experiment for the mice in last three groups.(1)The preventive effects of Lf on ALI in male miceALI was induced by giving 20% ethanol ad libitum for 8 weeks combined with four “binges”(3.6 g/kg·bw ethanol).(2)The preventive effects of Lf on acute ALI in female miceAcute ALI was induced by giving intragastrically ethanol(4.8 g/kg·bw)every 12 h for a continuous 3 times.(3)The preventive effects of Lf on chronic ALI in female miceChronic ALI was induced by giving 20% ethanol ad libitum for 12 weeks.Serum transaminase levels were tested by commercial kits;hepatic morphological structures were assessed by HE staining;protein expressions were determined by western blots;m RNA expressions were detected by q RT-PCR;and gut microbiota were evaluated by 16 S r RNA sequencing for feces samples.Results1.Cell experimentIn BRL-3A cells,Lf treatment reduced ethanol-induced death and apoptosis,especially early apoptosis,meanwhile,Lf treatment alleviated LDH excessive release.Ethanol induced CYP2E1 overexpression but did not affect ADH1 protein level,while the expressions of the two proteins were not affected by Lf treatment.Compared with Et OH group,the expression levels of fatty acid β-oxidation key enzymes were higher in Et OH+Lf group.Lf treatment reversed the reduction of nuclear Nrf2 induced by ethanol without affecting cytoplasm Nrf2 level.Besides,Lf can restored the decreased antioxidant enzyme activities caused by ethanol.However,block of Nrf2 nuclear translocation eliminated the protective effects of Lf.2.Studies on Lf digestion and absorptionAll intact bovine Lf molecules were hydrolyzed after 1 h of artificial gastric juice simulated digestion.Further prolonged processing time had few effects on the protein molecular weight distributions of Lf hydrolysates,and they were mainly concentrated 5to 20 k Da.Under normal Lf supplement,no intact Lf molecules were detected in serum,cecum contents and feces of mice.However,acute ultra-large dose of Lf intake could made intact Lf molecules appear in cecum contents,whereas only trace amount of Lf was detected in serum.These suggested that oral Lf cannot be absorbed into blood and produce biological effects in its original form unless extreme conditions.3.Animal experiments(1)The preventive effects of Lf on ALI in male miceLf treatment significantly reduced the mortality rate and hepatic malondialdehyde contents.Compared with HLf treatment,LLf treatment had more potent effects on the reduction in serum aspartate aminotransferase level and the improvements of hepatic pathological morphology.Lf promoted ALDH2 protein expression and inhibited CYP2E1 overexpression,resulting in the reduced hepatic superoxide and inflammation levels,which ultimately led to the hepatic injury alleviation.However,HLf increased ACC and FAS protein levels in liver,suggesting that Lf excessive intake may weaken its beneficial effects.Besides,LLf treatment enhanced the relative abundances of Akkermansia and Lactobacillus.(2)The preventive effects of Lf on acute ALI in female miceHLf but not LLf could restore the increases of liver weight,hepatic triglyceride(TG)content and serum transaminase level.Meanwhile,HLf treatment had a more obvious alleviating effects on ethanol-induced hepatic morphological changes than LLf treatment.Compared with Et OH group,CYPE21 protein expression,ADH activity and ALDH activity were not affected by Lf treatment.The ethanol-induced hepatic reactive oxygen species level increase was not ameliorated by Lf.Metabolomics and bioinformatics analysis results suggested an important role of redox-stress response capacity(RRC).Western blots showed that HLf promoted AKT and AMPK activations and upregulated Nrf2 and LC3-II expressions,which was associated with RRC improvement.(3)The preventive effects of Lf on chronic ALI in female miceHLf treatment could significantly decrease hepatic TG contents and improved hepatic morphological structures.HLf suppressed CYPE21 overexpression and up-regulated ADH1 protein expression.In liver,HLf treatment promoted AKT and AMPK activation,as well as upregulated Nrf2 to increase hepatic antioxidative enzyme activities.AMPK activation was also beneficial to lipid metabolism regulation,manifested as the inhibitions of ACC and FAS,and the upregulations of HSL and CPT1 A.Meanwhile,compared with Et OH group,Akkermansia relative abundance was significantly increased in LLf group,but decreased in HLf group.As a whole,HLf had no obvious beneficial effects on gut microbiota.Conclusions1.Lf alleviates ethanol-induced cell injury via promoting Nrf2 nuclear translocation in BRL-3A cells.2.Oral Lf can not be absorbed into blood in its original form,but it still has the preventive effects on ALI in vivo.3.Althought the specific preventive mechanisms of Lf for ALI are affected by gender and alcohol exposure patterns,all of them are associated with hepatic redox homeostasis maintaining.Besides,the regulations of alcohol metabolism and gut microbiota by Lf may also play a role in the process.The optimum Lf dose may differed by sex.