Effect Of Endoplasmic Reticulum Stress In Chronic Intermittent Rats Of Liver Dysfunction

Objective: To study the role of endoplasmic reticulum stress in chronic intermittent hypoxia-induced liver injury in rats.Methods: A total of 18 male Sprague-Dawley rats(specific-pathogen-free)weighing 280-300 g were randomly assigned to three groups: a normal control group(NC),a CIH groups(CIH)and a CIH plus N-acetylcysteine supplement group(CIHN).In the CIHN group,rats received 1 g/kg body weight fresh N-acetylcysteine solution via gavage 30 min before exposure to CIH.The CIH and CIHN groups were exposed to intermittent hypoxia,while the control group received the same frequency of pulse air from 9 a.m.to 5 p.m.for 8 consecutive weeks.After 8 weeks,all animals were fasted for 12 h,and then blood samples were collected through cardiac puncture under 10% chloral hydrate(300 mg/ kg).The collected blood was stored at-80 °C after centrifugation.The liver was removed after cardiac perfusion and stored at-80 °C.The biochemical index was determined by automatic biochemical analyzer.The contents of MDA in the liver were determined by TBA method.The activity of T-SOD in the liver was determined by hydroxylamine method.The content of GSH in the liver was determined by spectrophotometry.The expression of GRP78,p-PERK,p-IRE1,ATF6 CHOP and Caspase12 protein were detected by Western blot.The expression of GRP78 mRNA was detected by RT-qPCR.HE staining was used to observe the pathological changes of liver.Immunohistochemical staining was used to observe the expression of GRP78,CHOP and Caspase12.TUNEL / DAPI double staining was used to detect hepatocyte apoptosis.Result: 1.Compared with NC group,the content of MDA in CIH group was significantly increased(P <0.01),T-SOD activity was significantly decreased(P <0.01)and GSH content was significantly decreased(P <0.01);Compared with CIH group,(P <0.01),NAC intervention could completely inhibit the increase of MDA content,partially inhibit the decrease of T-SOD activity and decrease the content of GSH.2.Compared with NC group,the levels of serum ALT and AST in CIH group were significantly increased(p <0.01;p <0.05),TC content was significantly decreased(p <0.05).Compared with CIH group,NAC intervention could inhibit the increase of ALT and AST(p <0.01;p <0.05),but had no obvious inhibitory effect on TC content(p> 0.05).Statistical analysis showed that NAC intervention only partially inhibited the changes in ALT and AST levels.3.Compared with NC group,the expression of GRP78,p-PERK,p-IRE1,CHOP and Caspase12 protein in CIH group were significantly up-regulated,while the expression of activated ATF6 protein was down-regulated.Compared with CIH group,the expression of GRP78,p-PERK,p-IRE1,CHOP and Caspase12 was up-regulated,but no significant inhibition was observed on the down-regulation of activated ATF6 expression.4.Compared with NC group,the expression of GRP78 mRNA in CIH group was significantly higher than that in CIH group(p<0.01)..Compared with CIH group,NAC intervention could significantly inhibit the upregulation of GRP78 mRNA expression(p<0.05)..Statistical analysis showed that NAC intervention only partially inhibited the upregulation of GRP78 mRNA levels.5.Compared with NC group,the level of serum GRP78 protein in CIH group were significantly increased(p<0.01).Compared with CIH group,the NAC intervention significantly increased the level of serum GRP78 protein(p <0.01).6.The liver sections from the normoxic group exhibited a normal structure,while those from the CIH groups showed moderate hepatic swelling along with cytoplasmic rarefaction in the hepatic lobule(Fig 3C).Moreover,the CIH rats had massive lymphocyte infiltration in the portal area(Fig 3D).Treatment with NAC reduced the severity of the moderate hepatocellular swelling and decreased inflammation in the rats exposed to CIH(Fig 3E and F).7.Immunohistochemical staining showed that GRP78 and Caspase12 were mainly expressed in cytoplasm,and CHOP was mainly expressed in cytoplasm.Compared with NC group,the expression of GRP78,CHOP and Caspase12 in CIH group was significantly up-regulated,the brown area was significantly increased and the brown color was significantly enhanced.Compared with CIH group,NAC intervention significantly inhibited the upregulation of GRP78,CHOP and Caspase12 expression,significantly reduced the brown area.Compared with CIH group,the scores of GRP78,CHOP and Caspase12 were significantly increased in CIH group(p<0.01;p<0.01;p<0.01).Compared with CIH group,NAC intervention significantly reduced the score of GRP78,CHOP and Caspase12(p<0.01;p<0.05;p<0.05).Statistical analysis showed that NAC intervention partially inhibited the up-regulation of GRP78 and Caspase12 protein expression,and completely inhibited the up-regulation of CHOP protein expression.8.TUNEL / DAPI staining showed that the apoptotic index(AI)of CIH group was significantly higher than that of NC group(p <0.01),while NAC intervention could alleviate apoptosis(p <0.01).In addition,the correlation analysis showed that the apoptotic index of liver cells was positively correlated with the expression of GRP78 mRNA(P <0.01).Conclusion: Specifically,increased reactive oxygen species(ROS)activates endoplasmic reticulum(ER)stress and ER stress-related apoptosis are potential underlying mechanisms of CIH-related liver dysfunction.NAC can relieve the ER stress by removing the ROS,thereby reducing the apoptosis of liver and improving the liver function.