Molecular mechanisms of hepatic stimulator substance gene knockout in promoting development and progression of nonalcoholic steatohepatitis / 中华传染病杂志

ABSTRACT

Objective To explore the molecular mechanisms of hepatic stimulator substance (HSS) gene knockout in promoting the development and progression of nonalcoholic steatohepatitis (NASH).Methods NASH model mice (n=20) with HSS wild-type (HSS+/+) or HSS gene knockout (HSS-/-) were constructed using modified choline-deficient diet (CD-diet),untreated C57BL6-HSS-/-and C57BL6-HSS+/+ mice (n=20) were considered as control.Ten mice of each group were killed at month 1 and 2,respectively.The levels of triglyceride (TG) and total cholesterol (TC) in liver were measured using ELISA method.Histopathology and collagen deposition in liver tissue were observed using HE staining and Masson staining,respectively.Lipid content in liver tissue was observed and calculated using oil red O staining.The levels of mRNA and proteins of peroxisome proliferators activated receptor gama coactivator 1 alpha (PGC-1α),mitochondrial transcription factor A (TFAM),transcription factor-E2 related factor α (Nrf2),[-loop,dynamin-related protein 1 (Drp1),mitochondrial fission 1 protein (Fis1),mitofusins 1 (Mfn1),autophagy related gene 3 (Atg3) in liver tissue were detected using Real-time PCR and Western blot,respectively.Content of malonaldehyde (MDA),cyclooxygenase Ⅳ (COX Ⅳ) and adenosine tirphosphate (ATP) were measured using kits,and the activity of respiratory chain complex Ⅴ and cytochrome C oxidase in liver tissue were measured using spectrophotometry.the comparison between groups was done by t test.Results The levels of HSS mRNA and protein in mice-HSS-/-were 0.154± 0.04 and 0.08± 0.01,respectively,which were both significantly lower than those in mice-HSS+/+ (0.952 ± 0.08 and 1.362±-0.130,respectively),and t he differences had statistical significance (t =10.244 and 10.375,respectively,both P＜0.05).One month and 2 months after NASH modeled,TC contents in mice-HSS-/ were (248.6±21.5) μmol/g and (217.4±18.0) μmol/g,respectively,which were both remarkably higher than those in mice-HSS+/+ [(153.5 ± 11.2) μmol/g and (140.8 ±7.5) μmol/g,respectively],and the differences had statistical significance (t=15.270 and 10.524,respectively,both P＜0.05).The results form HE staining,oil red O staining and Masson staining indicated that fat deposition,collage deposition and inflammation in liver tissues of mice-HSS-/-were severer than those in mice-HSS+/+.One month after NASH modeled,protein levels of Drp1,Fis1,Mfn1 and Atg3 in liver tissues of mice-HSS-/ were all significantly decreased compared with those in mice-HSS+/+,and the differences had statistical significance (t=10.705,24.072,9.892 and 17.540,respectively,all P＜ 0.05).Two months after NASH modeled,protein levels of Drp1,Fis1,Mfn1and Atg3 in liver tissues of mice-HSS-/ were all significantly decreased compared with those in mice-HSS+/+,and the differences had statistical significance (t=125.378,15.926,34.330 and 13.437,respectively,all P＜0.05).One month after NASH modeled,mRNA levels of Drp1,Fis1,Mfn1 and Atg3 in liver tissues of mice-HSS-/-were all significantly decreased compared with those in mice-HSS+/+,the differences had statistical significance (t=36.337,40.825,33.508 and 28.104,respectively,all P＜0.05).Two months after NASH modeled,mRNA levels of Drp1,Fis1,Mfn1 and Atg3 in liver tissues of mice-HSS-/-were all significantly decreased compared with those in mice-HSS+/+,and the differences had statistical significance (t=35.210,42.375,27.753 and 20.560,respectively,all P＜0.05).The protein levels of PGC-1α,TFAM,Nrf2 and D-loop in liver of C57BL6-HSS-/-group were lower than those in liver of C57BL6-HSS+/+ group,and the differences had statistical significance (one montht=20.548,31.036,19.445 and 10.974,respectively;two monthst=9.887,13.330,22.375 and 18.903,respectively,all P＜0.05).The mRNA levels of PGC-1α,TFAM,Nrf2 and D-loop in liver of C57BL6-HSS-/-group were all lower than those in C57BL6-HSS+/+ group,and the differences had statistical significance (one montht=9.087,12.582,21.451 and 7.774,respectively;two monthst=23.758,17.924,9.924 and 15.209,respectively,all P＜0.05).One month and 2 months after NASH modeled,the levels of ATP mRNA in liver of C57BL6-HSS / group were both significantly lower than those in C57BL6-HSS+/+,and the differences had statistical significance 0=43.775 and 28.375,respectively,both P＜0.05);the levels of COXⅣ mRNA in liver of C57BL6-HSS / group were 0.142 ± 0.06 and 0.068± 0.001,respectively,which were both significantly lower than those in C57BL6-HSS+/+ group (0.255± 0.08 and 0.172 ±0.06,respectively),and the differences had statistical significance (t=28.337 and 19.782,respectively,both P＜0.05);the levels of MDA mRNA in liver of C57BL6-HSS-/-group were 0.973 ±0.112 and 1.253±0.054,respectively,which were both significantly lower than those in C57BL6-HSS+/+ group (0.366±0.02 and 0.872±0.05,respectively),and the differences had statistical significance (t=8.357 and 6.582,respectively,both P＜0.05).Conclusion Deletion of HSS accelerates NASH progression via inhibiting mitochondrial fusion,which leads to dysfunction of mitochondrial respiratory chain and inhibition of fatty acid oxidation.