Abstract：Objective To investigate the effect of glutaryl-CoA dehydrogenase (GCDH) gene silencing and accumulation of lysine metabolites on the viability of hepatocytes. Methods BRL cells were divided into normal control group, negative control group, and GCDH silencing group. The shRNA lentiviral vector for silencing GCDH gene was constructed, and the BRL hepatocytes in the GCDH silencing group and the negative control group were infected with this lentivirus and negative control virus respectively, and then cultured in a medium containing 5 mmol/L lysine. Immunofluorescence assay was used to measure the infection efficiency of lentivirus. Western blot was used to measure the expression of GCDH protein. MTT assay was used to evaluate cell viability. Hoechest33342 staining was used to measure cell apoptosis. Western blot was used to measure the expression of Caspase-3, an index of cell apoptosis. Results The lentivirus constructed effectively silenced the GCDH gene in hepatocytes (P < 0.01). MTT assay and Hoechest 33342 staining showed no significant differences in cell viability and apoptosis between groups (P > 0.05). There was also no significant difference in the expression of Caspase-3 protein between groups (P > 0.05). Conclusions GCDH gene silencing and accumulation of lysine metabolites may not cause marked hepatocyte injury.
GAO Jin-Zhi,ZHANG Cai,YI Qin et al. Effect of glutaryl-CoA dehydrogenase gene silencing and high-concentration lysine on the viability of BRL hepatocytes[J]. CJCP, 2017, 19(9): 1014-1019.
Govender R, Mitha A, Mubaiwa L. A review of patients with glutaric aciduria type 1 at Inkosi Albert Luthuli Central Hospital, Durban, South Africa[J]. S Afr Med J, 2017, 107(3):201-204.
[2]
Tsai FC, Lee HJ, Wang AG, et al. Experiences during newborn screening for glutaric aciduria type 1:diagnosis, treatment, genotype, phenotype, and outcomes[J]. J Chin Med Assoc, 2017, 80(4):253-261.
[3]
Yang L, Yin H, Yang R, et al. Diagnosis, treatment and outcome of glutaric aciduria type I in Zhejiang Province, China[J]. Med Sci Monit, 2011, 17(7):55-59.
[4]
Jafari P, Braissant O, Bonafé L, et al. The unsolved puzzle of neuropathogenesis in glutaric aciduria type I[J]. Mol Genet Metab, 2011, 104(4):425-437.
[5]
Boy N, Mühlhausen C, Maier EM, et al. Proposed recommendations for diagnosing and managing individuals with glutaric aciduria type I:second revision[J]. J Inherit Metab Dis, 2017, 40(1):75-101.
[6]
Zinnanti WJ, Lazovic J, Wolpert EB, et al. A diet-induced mouse model for glutaric aciduria type I[J]. Brain, 2006, 129(Pt 4):899-910.
[7]
Seminotti B, da Rosa MS, Fernandes CG, et al. Induction of oxidative stress in brain of glutaryl-CoA dehydrogenase deficient mice by acute lysine administration[J]. Mol Genet Metab, 2012, 106(1):31-38.
[8]
Seminotti B, Ribeiro RT, Amaral AU, et al. Acute lysine overload provokes protein oxidative damage and reduction of antioxidant defenses in the brain of infant glutaryl-CoA dehydrogenase deficient mice:a role for oxidative stress in GA I neuropathology[J]. J Neurol Sci, 2014, 344(1-2):105-113.
[9]
Gao J, Zhang C, Fu X, et al. Effects of targeted suppression of glutaryl-CoA dehydrogenase by lentivirus-mediated shRNA and excessive intake of lysine on apoptosis in rat striatal neurons[J]. PLoS One, 2013, 8(5):e63084.
[10]
Braissant O, Jafari P, Remacle N, et al. Immunolocalization of glutaryl-CoA dehydrogenase (GCDH) in adult and embryonic rat brain and peripheral tissues[J]. Neuroscience, 2017, 343:355-363.
[11]
Kölker S, Valayannopoulos V, Burlina AB, et al. The phenotypic spectrum of organic acidurias and urea cycle disorders. Part 2:the evolving clinical phenotype[J]. J Inherit Metab Dis, 2015, 38(6):1059-1074.
[12]
Wang Q, Li X, Ding Y, et al. Clinical and mutational spectra of 23 Chinese patients with glutaric aciduria type 1[J]. Brain Dev, 2014, 36(9):813-822.
[13]
Sauer SW. Biochemistry and bioenergetics of glutaryl-CoA dehydrogenase deficiency[J]. J Inherit Metab Dis, 2007, 30(5):673-680.
[14]
Sauer SW, Opp S, Hoffmann GF, et al. Therapeutic modulation of cerebral L-lysine metabolism in a mouse model for glutaric aciduria type I[J]. Brain, 2011, 134(Pt 1):157-170.
