Hepatocyte-derived MANF Attenuates Rifampicin-induced Cholestatic Hepatic Injury Via Inhibiting ATF4-CHOP Signal Activation

Rifampicin(RFP)has been known to be potentially hepatotoxic and often used as an inducer of cholestatic hepatic injury.Here we found that mesencephalic astrocyte-derived neurotrophic factor(MANF),an endoplasmic reticulum(ER)stress inducible protein,is a protector in RFP-induced liver injury.In cholestatic hepatic injury mice induced by RFP,the liver/body ratio and the levels of serum alanine aminotransferase(ALT),aspartate aminotransferase(AST),alkaline phosphatase(ALP),total bile acid(TBA),total bilirubin(TBIL),and direct bilirubin(DBIL)were significantly increased.Meanwhile,the protein and m RNA levels of MANF were remarkably elevated in the liver injury mice.In hepatocyte-specific MANF knockout(HKO)mice,an extra increase in the liver/body ratio and serum ALT,AST,ALP,TBA,TBIL,and DBIL levels was detected after treatment with RFP.In addition,recombinant human MANF(rhMANF)treatment efficiently reduced the liver/body ratio and serum ALT,AST,ALP,TBA,TBIL,and DBIL levels in RFP-induced liver injury mice.Furthermore,we found there is an increase in the number of the apoptotic cells,detected by TUNEL staining in the liver tissues of HKO mice.Meanwhile,the protein levels of C/EBP-homologous protein(CHOP),Ki67,and the proliferating cell nuclear antigen(PCNA),as well as the m RNA level of Ki67 were elevated after treated with RFP,and these parameters were increased more significantly in HKO mice than that in wild type(WT)controls in RFP-induced liver injury.The rhMANF treatment can rescue the cell apoptosis and reduce the protein and m RNA levels of CHOP,Ki67,and PCNA elevated by MANF deletion and RFP.In HKO mice,immunoglobulin heavy chain binding protein(BIP)and activating transcription factor 4(ATF4)were predominantly increased after treatment with RFP,which were reduced by rhMANF treatment.Therefore,we conclude that hepatocyte-derived MANF attenuates RFP-induced cholestatic hepatic injury via inhibiting ATF4-CHOP signal activation and subsequent cell apoptosis.1.MANF was up-regulated in the liver tissues of mice treated with RFP To know how RFP induced-liver injury affects MANF expression,we treated C57BL/6 wide type male mice with RFP(300 mg/kg)for intragastric injection every day.Two weeks later,the animals were sacrificed 6 h after the last RFP treatment.The vehicle controls were treated with CMC-Na in equal volume.The protein and m RNA levels of MANF were significantly increased in the liver tissues of mice after administered with RFP.We also found that the livers became yellow and the size of livers was enlarged after treated with RFP,compared with vehicle group.The effects of RFP on biochemical parameters were analyzed.We found that there was a remarkable increase in liver/body ratio,serum ALT,AST,ALP,TBA,TBIL,and DBIL levels.These data suggest that we have successfully induced the cholestatic liver injury with RFP,and MANF expression was induced in RFP-induced liver injury.2.Hepatocyte-specific MANF knockout aggravates RFP-induced liver injury.To know the role of MANF upregulation in RFP-induced liver injury,we prepared a mouse model in that MANF was specifically deleted in hepatocytes and this manipulation resulted in a significant reduction of MANF levels in the liver tissue,but there was no significant change in the size,morphology,structure,and function of liver.However,after treatment with RFP,there were a significant increase in liver/body ratio and serum ALT,AST,ALP,TBA,TBIL,and DBIL levels in HKO mice,compared with WT.The pathological change was more remarkable in HKO mice after treated with RFP.These data suggest that hepatocyte-specific MANF knockout aggravates RFP-induced liver injury.3.Rh MANF attenuates RFP-induced liver injury.To verify the protective effects of MANF on RFP-induced liver injury,rhMANF(5,10,and 20 μg),diluted in PBS,was injected via tail vein into animals 6 h after the last RFP treatment once a week for two weeks.After treated with rhMANF,the liver/body ratio and serum ALT,AST,ALP,TBA,TBIL,and DBIL levels were decreased,compared with the PBS controls.The most effective dose of MANF was 20 μg per mouse.At this dose,there was a significant decrease in liver ratio and serum ALT,AST,ALP,TBA,TBIL,and DBIL levels,compared with the PBS controls.HE staining showed that rhMANF ameliorated the pathological changes caused by RFP.These data suggest that rhMANF prevents from RFP-induced liver injury.4.MANF deficiency promotes RFP-induced cell apoptosis To investigate whether MANF affects RFP-induced apoptosis,TUNEL staining was used,and the pro-apoptotic protein CHOP was also detected.The percentage of TUNEL-positive cells was significantly increased in the liver of mice treated with RFP.There was an extra increase in the number of TUNEL-positive cells in HKO mice after treated with RFP,compared with WT mice.The protein level of CHOP was also significantly elevated in HKO mice,compared with WT mice after treated with RFP.In consistence with TUNEL staining results,the number of CHOP-positive cells,especially the nucleus-positive cells were significantly increased in HKO mice,compared with WT mice after administered with RFP.These results suggest that MANF knockout promotes RFP-induced apoptosis.5.Rh MANF rescues the apoptosis induced by MANF deletion in RFP-induced liver injury.To investigate whether MANF rescues hepatic cells from the apoptosis induced by MANF deletion,the HKO mice were injected with different doses of rhMANF protein after RFP treatment,control mice were injected with equal volume PBS.The number of TUNEL-positive cells was significantly decreased in rhMANF supplementation mice compared with the PBS controls after treated with RFP.Meanwhile,the protein and m RNA levels of CHOP were significantly reduced after rhMANF supplementation.These results suggest that MANF prevents hepatic cells from RFP-induced apoptosis.6.MANF deficiency promotes RFP-induced proliferation.To observe the effect of MANF on RFP-induced proliferation,the proliferative markers Ki67 and PCNA were used.The numbers of Ki67-positive and PCNA-positive cells were significantly increased in the liver tissues of mice after treated with RFP.There was an extra increase of Ki67-positive and PCNA-positive cells in HKO mice,compared with WT mice.Meanwhile,Ki67 m RNA was also elevated in HKO mice,compared with WT mice.Western blot assay also showed the protein level of PCNA were upregulated in HKO mice,compared with WT mice.7.Rh MANF inhibits the proliferation induced by MANF deletion in RFP-induced liver injury.To investigate whether MANF rescues the effect of MANF deficiency on cell proliferation,the HKO mice were administrated with different doses of rhMANF.The levels of Ki67 and PCNA were significantly decreased after treatment with rhMANF.These results suggest that MANF protects against the alteration in pathological proliferation and apoptosis in liver.8.MANF inhibits ATF4 expression in RFP-induced liver injury.Our results also showed RFP increased the expressions of BIP and ATF4,as well as CHOP,but had no significant effect on ATF6 and XBP1.MANF deletion exerted more effect on ATF4 expression in RFP-induced liver injury,compared with WT mice.In consistence with this finding,rhMANF supplementation protected against RFP-induced ATF4 upregulation in HKO mice.To further explore the regulatory effect of MANF on ATF4 transcription,we constructed three truncates locating in the promoter region of ATF4 to luciferase reporter.Hep G2 cells were co-transfected the truncates with FLAG-MANF.The reporter activity was detected 24 h after transfection.It was found that MANF significantly inhibited the reporter activity of the three truncates,even the smallest fragment(-250 to 0 bp).These findings suggest that MANF may regulate ATF4 transcription.According to the above research results,this paper draws the following conclusions: Hepatocyte-derived MANF attenuates rifampicin-induced cholestatic hepatic injury via inhibiting ATF4-CHOP signal activation.