Hepatocyte-derived MANF Alleviates Hepatic Ischemia-reperfusion Injury Via Regulating Endoplasmic Reticulum Stress-induced Apoptosis In Mice

Ischemia/reperfusion?I/R?injury?IRI?of the liver graft has been demonstrated an important influence on the short-and long-term outcomes after liver transplantation.Though previous studies have figured out the prolonged IRI caused irreversible damage,due to the complex mechanisms underlying hepatic IRI,a completely valid approach had not been proposed yet.It is becoming clear that disruption of endoplasmic reticulum?ER?protein homeostasis,which also known as ER stress,has been involved in IRI.So,ER perturbations are treated as novel subcellular effectors involved in the ischemia-reperfusion injury.As an ER stress-inducible protein,mesencephalic astrocyte-derived neurotrophic factor?MANF?has been proven to be increased during ischemic brain injury.It has been found that MANF has neuroprotective effect on dopaminergic and nondopaminergic neurons.Besides,studies show that MANF also has a protective effect on podocytes,chondrocytes,pancreatic Beta cells and Retinal Ganglion cells by modulating the apoptosis and proliferation process.However,the role of MANF in liver ischemia reperfusion?I/R?injury has not been studied yet.Object:To explore how MANF exerts impact on the liver IRI and the underlying mechanismsMethods:In order to investigate the role of MANF in the process of liver ischemia-reperfusion,Hepatocyte-derived MANF knockout(MANF ^hep-/-)mice and their wild-type?WT?littermates were used in our research.Mice partial?70%?warm hepatic I/R model was established by vascular occlusion.MANF protein was injected into the tail vein before 1h occlusion.After reperfusion,the apoptosis and relative protein level were tested.AST,ALT,I/R injury area and Suzuki score were used to evaluate the extent of I/R injury.OGD test was performed on primary hepatocytes to simulate IRI in vitro.RNA sequence was used to detect the cellular signal pathway activation while MANF knockout.Immunohistochemistry and immunofluorescence were used to characterize the expression and location of relative protein.At last,we used CO-IP and mass spectrometry to verify the potential protein,which could bind to MANF.Results:1.Hepatic I/R increased MANF expressionA mice model of partial?70%?warm hepatic ischemia-reperfusion was firstly made in vivo.The successful of partial?70%?warm hepatic ischemia-reperfusion model was confirmed by the significant increased AST and ALT levels.MANF protein levels in liver tissues from wild-type?WT?mice subjected to hepatic I/R was validated by western blotting.A significantly increased MANF protein level was observed in liver tissue 3h following I/R compared with the sham operation samples.Further immunohistochemical analysis and HE staining also confirmed that MANF highly expressed around the I/R injury area.2.Hepatocyte-derived MANF knockout aggravated hepatic I/R injuryThe efficiency of MANF knockout were validated by western blotting and RT-PCR.It was found that MANF was almost knocked out in mice liver tissues.We performed HE staining and biochemical parameters determination.As a result,serum AST and ALT levels were significantly increased at 3 h and 6 h in MANF ^hep-/-,compared with WT mice.Consistent with this result,more severe liver tissue injury as revealed by HE staining was found in MANF ^hep-/-mice,compared with WT mice.3.Hepatocyte-derived MANF knockout aggravated I/R-induced hepatocyte apoptosis.To investigate whether MANF affect hepatocyte survival in hepatic I/R-injury,the primary hepatocytes were isolated from WT and MANF ^hep-/-mice and treated with OGD,and the colocalization of MANF and Annexin V was detected.It was interesting to find that the presence of Annexin V on the cell membrane is related to these cells expressing less,while those cells highly expressing MANF have less Annexin V on the cell membrane.TUNEL fluorescence assay also showed that MANF knockout significantly increased the number of apoptotic cells in the primarily cultured hepatocytes and liver tissues,compared with WT.Several major apoptosis-related markers were detected in the primarily cultured hepatocytes,including the antiapoptotic protein Bcl-2,proapoptotic protein BAX,and cleaved caspase-3.We found that a lower ratio of Bcl-2/BAX and a higher level of cleaved caspase-3 were observed in MANF ^hep-/-mice,compared with WT mice after I/R.4.Recombinant human MANF?rh MANF?protected I/R-liver injury via attenuating apoptosisTo confirm the protective effect of MANF on I/R-induce liver injury,we administrated rh MANF?His-tagged?to MANF ^hep-/-mice by tail vein.We found that MANF injection significantly alleviated the I/R injury in MANF ^hep-/-livers.The presence of His-tagged rh MANF in liver tissues was detected by using immunohistochemistry assay with anti-His antibody after rh MANF administration.Similarly,the protective effect of rh MANF was observed from the reduction of AST and ALT levels.Meanwhile,rh MANF increased the ratio of Bcl-2/BAX and reduced the level of cleaved caspase-3 in the liver tissues of either WT or MANF ^hep-/-mice,compared with saline controls.5.MANF was involved in I/R-Induced ER stress and UPR signal activation.We observed an increase in the levels of BIP and CHOP,the indicators of ER stress,in the liver tissues at 6 h after reperfusion following 1 h ischemia,suggesting that I/R induces ER stress.We also found Hepatocyte-derived MANF knockout up-regulated BIP and CHOP expression,which was more significant after I/R in liver tissues.These results suggest that MANF deficiency in hepatocytes induces ER stress.Accordingly,the levels of p-IRE1?,ATF4,and ATF6 were significantly increased in the liver tissues of MANF ^hep-/-mice,compared with WT mice after I/R.This finding was further confirmed in the primarily cultured hepatocytes exposed to OGD simulation in vitro.In agreement with the in vivo study,the levels of p-IRE1?,ATF4,and ATF6 and BIP were upregulated in the hepatocytes of MANF ^hep-/-mice within 24 h after OGD,compared with WT mice.These results indicate Hepatocyte-derived MANF knockout activates UPR signal pathways,especially ATF4/CHOP pathway,with or without liver I/R injury.To know whether MANF rescues the influence of MANF knockout,the purified rh MANF protein was used to the MANF ^hep-/-mice.As expected,the elevated levels of BIP,CHOP,p-IRE1?,ATF6,and ATF4 in both WT and MANF ^hep-/-mice suffering from I/R injury were reduced after rh MANF injection,compared with the saline controls.Taken together,MANF is involved in I/R-Induced ER stress and UPR signal activation.6.Hepatocyte-derived MANF knockout activated JNK/c-JUN/CHOP pathway in mice.The primarily cultured hepatocytes from 3 mice in each group(WT and MANF ^hep-/-mice)were used to for RNA sequencing.A total of 652 differential genes were detected,with 448 genes upregulated and 204 genes downregulated.A differential gene KEGG database revealed the differences between WT and MANF ^hep-/-hepatocytes.It was found that the differential genes were much highly expressed in two pathways:protein processing in the ER and MAPK signaling pathway.The important regulatory factors Hsp A5?BIP?,Ddit3?CHOP?,and JUN were highly expressed in the hepatocytes with MANF knockout,while JNUB,JUND and FOS expression were not significantly changed.This result suggests that JNK/c-Jun activation may play a critical role in MANF deficiency hepatocytes suffering from I/R-triggered UPR.To further confirm the activation of JNK,c-Jun,and CHOP,the levels of p-JNK,c-Jun,and CHOP were measured by western blotting and Immunohistochemistry.The increased levels of p-JNK,c-Jun,and CHOP were found in MANF ^hep-/-mice,compared with WT mice either with or without I/R injury.This result suggests that MANF knockout activates JNK/c-Jun/CHOP pathway in hepatocytes,which is involved in I/R-induced liver injury.7.MANF interacted with GRP75 and affected its bioactivityTo further explore the relationship between MANF and UPR pathway,we used mass spectrometry to detect the composition of the protein solution obtained from CO-IP experiment.We found that MANF could bind to GRP75,the result was further proved by the immunofluorescence and western blotting.The co-localization of MANF and GRP75was also found in the liver tissue after I/R,which indicated the interaction between MANF and GRP75 during hepatic I/R injury.We observed an increase level of GRP75,in the liver tissues after reperfusion,suggesting that GRP75 was involved in the process of I/R injury like BIP.After I/R injury,the expression level of GRP 75 in the liver tissues of MANF ^hep-/-mice was significantly increased compared with WT liver tissues.Moreover,CLPP,a marker for mitochondrial unfolded protein response?mt UPR?,was also significantly up-regulated after MANF knockout,indicating that MANF knockout can affect the bioactivity of GRP 75,which might further aggravate the endoplasmic reticulum and mitochondrial UPR?Conclusion:In summary,our study has identified MANF as a crucial modulator in I/R-triggered liver injury by inhibiting ER stress-mediated apoptosis specifically via JNK/C-Jun/CHOP pathway,which may shed light on the potential therapy.