Endoplasmic Reticulum Stress Is Involved In Carbon Tetrachloride-induced Acute And Chronic Liver Injury In Mice And Its Partly Mechanism Of Action

The liver is not only an important organ in the metabolism for human, but also atarget organ which is most vulnerable to the damage of numerous extraneous factors. Ashort-term expose of high dose xenobiotics induce acute liver injury, and long-termexpose of xenobiotics induced chronic liver injury. Liver fibrosis is a repair response tochronic liver injury. Liver fibrosis is a slow pathological process and a commonpathological change in chronic liver disease,which has become a worldwide healthproblem. The change of texture in the liver is abnormal hyperplasia and deposition ofliver extracellular matrix (ECM), which may cause severe liver fibrosis or cirrhosis andeven lead to liver failure if not treated in time. Nevertheless, the molecular mechanismsof acute and chronic liver injury remain poorly understood. Many anti-liver injury drugshave been developed in recent years, but the effects have not been affirmed yet. It is ofsignificance to find an ideal anti-fibrosis drug and understand the pathogenesis of liverfibrosis.The endoplasmic reticulum (ER) stress plays an important role in numerous liverdiseases. ER is the organelle responsible for protein folding and maturation andmaintaining Ca2+homeostasis. Accumulation of unfolded/misfolded proteins andalteration of Ca2+homeostasis in the ER lumen triggers ER stress. ER stress is aself-defence mechanism,but strong and long lasting ER stress leads to irreversible cellinjury. Many liver diseases such as viral hepatitis, alcohol liver disease, drug induced hepatitis, nonalcoholic fatty liver diseases are related to ER stress. A recent reportshowed that bile acids caused hepatic ER stress and Unfolded Protein Reaction (UPR)signaling activation in a model of cholestasis-induced hepatic fibrosis. Nevertheless,whether ER stress is involved in the pathogenesis of acute and chronic liver injury,especially for hepatic fibrogenesis, remain poorly understood.Carbon tetrachloride (CCl₄)-induced mouse models of acute and chronic liverinjury were for study of its molecular mechanism. The aim of this study was to observea single dose and long-term of CCl₄-induced hepatic ER stress and UPR signalingactivation, moreover, investigate the effects of Sodium4-phenylbutyrate (PBA) onCCl₄-induced hepatic fibrogenesis and elucidate its molecular mechanism in mice.1. The roles of ER Stress on a single dose of CCl₄-induced liver injury in mice1.1The effects of a single dose of CCl₄-induced liver injury in miceTo investigate the effects of a single dose of CCl₄-induced liver injury in mice,mice were sacrificed at0,2,6,12,24and72hours after a single intraperitonealinjection of CCl₄(0.30ml/kg BW). Results showed the absolute and relative liverweights were significantly increased from12h to72h after a single intraperitonealinjection of CCl₄. The lever of serum ALT was slightly increased, beginning as early as2h and remained significantly elevated up to24h after a single dose of CCl₄administration. CCl₄-induced hepatic histopathological damage are analyzed withhematoxylin and eosin (HE) staining. Numerous hepatocytes with necrosis wereobserved at6h after a single injection of CCl₄. Liver zonal necrosis was observed at12h and was significant at24h. Numberous inflammatory cells around the necrotic tissuewere observed in liver sections at72h. These results demonstrated a single dose ofCCl₄-induced liver injury in mice.1.2The effects of a single dose of CCl₄-induced ER Stress in mice To investigate the effects of a single dose of CCl₄-induced ER Stress in mice,western blotting and immunohistochemistry were for the determination of ER Stress.Asingle dose of CCl₄administration significantly increased the level of hepatic GRP78,an ER chaperone, used by western blotting and positive hepatocytes of GRP78determined by immunohistochemistry. In addition, a single dose of CCl₄administrationsignificantly increased the level of hepatic peIF2α and pIRE1α were significantlyincreased in CCl₄-treated mice.1.3The effects of a single dose of CCl₄-induced hepatic apoptosis in miceTo investigate the effects of a single dose of CCl₄-induced hepatic apoptosis inmice, TUNEL was for the determination of hepatic apoptosis. TUNEL+cells weresignificantly increased in liver of mice at24h after a single intraperitoneal injection ofCCl₄.1.4ER stress inhibitor alleviates CCl₄-induced hepatic apoptosis in miceTo investigate the effects of ER stress inhibitor, PBA, on a single dose ofCCl₄-induced hepatic apoptosis in mice, mice received three doses of PBA, two (150mg/kg) intraperitoneally injected12h and24h before CCl₄(0.30ml/kg BW), the thirdinjected12h after CCl₄. All mice were sacrificed at24h after CCl₄. TUNEL was for thedetermination of hepatic apoptosis. PBA significantly alleviated CCl₄-induced hepaticapoptosis in mice at24h after a single intraperitoneal injection of CCl₄.2. The roles of ER stress on CCl₄-induced liver fibrosis and its molecularmechanism2.1CCl₄administration induces liver fibrosis model in miceMice were intraperitoneally injected with CCl₄(0.15ml/kg BW, twice per week) incombination with PBA (150mg/kg, twice per day) for8weeks. Liver weight wassignificantly increased in mice administered with CCl₄. Results showed long-term CCl₄ administration significantly increased the levels of serum ALT, AST, ALP, TBIL andTBA. Hepatic hydroxyproline content was also significantly increased in miceadministered with CCl₄. Further analysis showed that the levels of serum weresignificantly increased in mice administered with CCl₄. CCl₄-induced hepatichistopathological damage are including numerous inflammatory cells around thenecrotic tissue were observed in liver sections from CCl₄-treated mice using H&Estaining. CCl₄-induced hepatic fibrosis was determined using Sirius red staining. Asexpected, an obvious bridging fibrosis was observed in liver of mice administered withCCl₄. These results demonstrated long-term CCl₄administration induce liver fibrosis inmice.2.2ER stress in CCl₄-induced liver fibrosis model in miceTo investigate long-term CCl₄-induced hepatic ER stress, the effects of CCl₄onhepatic ER stress were analyzed using western blotting. Results showed long-term CCl₄administration significantly increased the level of hepatic GRP78, an ER chaperone. Inaddition, long-term CCl₄administration significantly increased the level of ATF6protein in hepatic nuclear extracts. The levels of hepatic peIF2α and pIRE1α were alsosignificantly increased in CCl₄-treated mice. These results suggested long-term CCl₄induced ER stress.3PBA alleviates CCl₄-induced liver fibrosisTo investigate the effects of PBA, which could act as a chemical chaperone thatinhibits ER stress and the UPR signaling activation, on long-term CCl₄-induced liverfibrosis, mice were intraperitoneally injected with CCl₄(0.15ml/kg BW, twice per week)in combination with PBA (150mg/kg, twice per day) for8weeks. Interestingly, PBAsignificantly alleviated CCl₄-induced elevation of liver weight. The analysis of serumbiological parameters showed that PBA significantly alleviated CCl₄-induced elevationof serum DBIL and TBA levels. The effect of PBA on CCl₄-induced hepatic histopathological damage was determined by HE staining.The results showed that thenumber of inflammatory cells was significantly decreased in liver of mice administeredwith PBA plus CCl₄as compared with CCl₄alone. In addition, the grades of necrosiswere slightly lower in liver of mice administered with PBA plus CCl₄as compared withCCl₄alone. CCl₄-induced hepatic fibrosis was determined using Sirius red staining. Asexpected, PBA significantly alleviated CCl₄-induced hepatic fibrosis. Further analysisshowed that the area of hepatic fibrosis was significantly reduced in mice administeredwith PBA plus CCl₄as compared with CCl₄alone. The effects of PBA on hepatichydroxyproline, an indicator of hepatic fibrosis, were then analyzed. Result showedPBA significantly attenuated CCl₄-induced elevation of hepatic hydroxyproline content.These results showed PBA protects mice from CCl₄-induced liver fibrosis.The effects of PBA on CCl₄-induced hepatic α-SMA, a marker associated withHSC activation, were analyzed by immunohistochemistry and western blotting.Immunohistochemistry showed that the percentage of α-SMA-positive area, α-SMAwas mainly distributed in area of liver with bridging fibrosis, was significantlydecreased in liver of mice administered with PBA plus CCl₄as compared with CCl₄alone. Correspondingly, PBA significantly reduced CCl₄-induced expression of hepaticα-SMA protein. The effects of PBA on CCl₄-induced expression of TGF-β1wereanalyzed by real-time RT-PCR. As expected, long-term CCl₄administrationsignificantly increased the levels of hepatic TGF-β1mRNAs. Interestingly, PBAsignificantly attenuated CCl₄-induced upregulation of hepatic TGF-β1mRNAs. Theseresults suggested the protection of PBA on CCl₄-induced liver fibrosis was associatedwith the inhibition of PBA on CCl₄-induced the activation of HSCs.The effects of PBA on CCl₄-induced expression of hepatic Mmp2and Mmp9wereanalyzed by real-time RT-PCR. Long-term CCl₄administration significantlyupregulated the expression of hepatic Mmp2and Mmp9. PBA significantly attenuated CCl₄-induced upregulation of hepatic Mmp2mRNA. The levels of hepatic Timp1andTimp2mRNA were significantly increased in mice administered with CCl₄. PBAsignificantly attenuated CCl₄-induced upregulation of hepatic Timp1mRNA. ActivatedHSCs produce type I collagen molecules. Thus, the effects of PBA on CCl₄-inducedhepatic mRNA accumulation of collagen1α1(Col1a1) and Col1a2were then analyzed.As expected, the levels of hepatic Col1a1and Col1a2mRNAs were obviouslyincreased in mice administered with CCl₄. PBA significantly attenuated CCl₄-inducedelevation of hepatic Col1a1and Col1a2mRNAs.To investigate the effects of PBA on long-term CCl₄-induced hepatic ER stress, thelevel of hepatic GRP78, eIF2α and IRE1α phosphorylation and nuclear ATF6proteinwere analyzed by western blotting. Interestingly, PBA significantly attenuatedCCl₄-induced elevation of hepatic GRP78and nuclear ATF6protein. In addition, PBAsignificantly inhibited CCl₄-induced hepatic eIF2α and IRE1α phosphorylation. Theseresults suggested PBA alleviated ER stress and UPR signaling activation.4The molecular mechanism of ER stress on CCl₄-induced liver fibrosis in mice4.1PBA alleviates CCl₄-induced activation of hepatic NF-κB signaling pathwayThe effects of PBA on CCl₄-induced hepatic NF-κB activation are analyzed bywestern blotting. Interestingly, PBA significantly inhibited CCl₄-induced nucleartranslocation of NF-κB p65. The effects of PBA on CCl₄-induced expression ofinflammatory cytokines were analyzed by real-time RT-PCR. As expected, long-termCCl₄administration significantly increased the levels of hepatic TNF-α mRNAs.Interestingly, PBA significantly attenuated CCl₄-induced upregulation of hepatic TNF-αmRNAs. These results suggested the protection of PBA on CCl₄-induced liver fibrosiswas mediated by the inhibition of PBA on CCl₄-induced hepatic NF-κB activation andinflammation. 4.2PBA alleviates CCl₄-induced activation of hepatic MAPK signaling pathwayThe effects of PBA on CCl₄-induced hepatic MAPK signaling were then analyzedby western blotting. The levels of hepatic pERK and pJNK were significantly increasedin mice administered with CCl₄. PBA significantly attenuated CCl₄-induced hepaticERK and JNK phosphorylation. Unexpectedly, the level of hepatic pp38wassignificantly decreased in mice administered with CCl₄. PBA had no effect on the levelof hepatic pp38. These results suggested the protection of PBA on CCl₄-induced liverfibrosis was associated with the inhibition of PBA on CCl₄-induced hepatic ERK andJNK phosphorylation.In summary, the present study demonstrates that a single dose of CCl₄administration induce liver injury, hepatic ER stress and UPR signaling activation withhepatic apoptosis. Long-term CCl₄administration also induced hepatic ER stress andUPR signaling activation, which may play an important role on CCl₄-induced HSCactivation and subsequent hepatic fibrosis. PBA, an ER chemical chaperone, inhibitsCCl₄-induced hepatic ER stress and UPR signaling activation. In addition, PBAalleviates CCl₄-induced inflammation through inhibiting activation of NF-κB and JNK,ERK signaling. Importantly, PBA effectively protects against CCl₄-induced HSCactivation and hepatic fibrosis. Therefore, ER stress is involved in CCl₄-induced acuteand chronic liver injury, and liver fibrosis partly mediated through activation of NF-κBand MAPK signaling.