OBJECTIVE To investigate whether aldo-keto reductases(AKRs)can act as a nitrore⁃ductase(NR)and bioactivate aristolochic acidⅠ(AA-Ⅰ)to produce AA-Ⅰ-DNA adducts.METHODS①Human-induced hepatocytes(hiHeps)and human bla...OBJECTIVE To investigate whether aldo-keto reductases(AKRs)can act as a nitrore⁃ductase(NR)and bioactivate aristolochic acidⅠ(AA-Ⅰ)to produce AA-Ⅰ-DNA adducts.METHODS①Human-induced hepatocytes(hiHeps)and human bladder RT4 cells were used as tool cells and treated with AA-Ⅰ0,0.5,1.0 and 2μmol·L^(-1)for 24 h.Cell viability was detected using the CCK-8 method,and the half maximal inhibition concentration(IC_(50))was calculated using the CCK-8 method and the level of DNA adduct production was calculated.②hiHeps and RT4 cells were treated with AKR inhibitor luteotin(0,5,10 and 25μmol·L^(-1))+AA-Ⅰ0.2 and 1.0μmol·L^(-1)for 24 h,respectively,and the levels of DNA adducts were detected by a liquid chromatography-tandem mass spectrometer(LC-MS/MS).③hiHeps cells were incubated with 80 nmol·L^(-1)small interfering RNAs(si-AKRs)for 48 h and treated with AA-Ⅰ1.0μmol·L^(-1)for 24 h.Real-time qualitative PCR(RT-qPCR)method was used to detect the mRNA expression of AKRs gene and LC-MS/MS technology was used to investigate the effect of specific AKR gene knockdown on DNA adduct levels.④500 nmol·L^(-1)human AKR recombinant proteins AKR1A1 and AA-Ⅰwere incubated in vitro under anaerobic conditions and the formation of AA-Ⅰ-DNA adducts was detected.RESULTS①The IC_(50)of AA-Ⅰto hiHeps and RT4 cells was 1.9 and 0.42μmol·L^(-1),respec⁃tively.The level of DNA adduct production of the two cell lines was significantly different(P<0.01).②Luteolin≥5μmol·L^(-1)significantly inhibited the production of AA-Ⅰ-DNA adducts in both cells(P<0.05),and there was a concentration-dependent effect in hiHeps cells(P<0.01,R=0.84).③In the AKR family,the knockdown of AKR1A1 gene up to 80%inhibited the generation of AA-Ⅰ-DNA adducts by 30%-40%.④The AA-Ⅰ-DNA adducts were detected in the incubation of recombinant protein AKR1A1 and AA-Ⅰunder anaerobic conditions in vitro,approximately 1 adduct per 107 nucleotides.CONCLU⁃SION AKR1A1 is involved in AA-Ⅰbioactivation,providing a reference for elucidation of the carcino⁃genic mechanism of AA-Ⅰ.展开更多
Transgenic mice ubiquitously overexpressing murine γ aminobutyric acid transporter subtype Ⅰ were created. Unexpectedly, these mice markedly exhibited heritable obesity, which features significantly increased body w...Transgenic mice ubiquitously overexpressing murine γ aminobutyric acid transporter subtype Ⅰ were created. Unexpectedly, these mice markedly exhibited heritable obesity, which features significantly increased body weight and fat deposition. Behavioral examination revealed that transgeinc mice have slightly reduced spontaneous locomotive capacity and altered feeding pattern. Tills preliminary finding indicates that the inappropriate level of γ-aminobutyric acid transporters may be directly or indirectly involved in the pathogenic mechanism underlying certain types of obesity.展开更多
文摘OBJECTIVE To investigate whether aldo-keto reductases(AKRs)can act as a nitrore⁃ductase(NR)and bioactivate aristolochic acidⅠ(AA-Ⅰ)to produce AA-Ⅰ-DNA adducts.METHODS①Human-induced hepatocytes(hiHeps)and human bladder RT4 cells were used as tool cells and treated with AA-Ⅰ0,0.5,1.0 and 2μmol·L^(-1)for 24 h.Cell viability was detected using the CCK-8 method,and the half maximal inhibition concentration(IC_(50))was calculated using the CCK-8 method and the level of DNA adduct production was calculated.②hiHeps and RT4 cells were treated with AKR inhibitor luteotin(0,5,10 and 25μmol·L^(-1))+AA-Ⅰ0.2 and 1.0μmol·L^(-1)for 24 h,respectively,and the levels of DNA adducts were detected by a liquid chromatography-tandem mass spectrometer(LC-MS/MS).③hiHeps cells were incubated with 80 nmol·L^(-1)small interfering RNAs(si-AKRs)for 48 h and treated with AA-Ⅰ1.0μmol·L^(-1)for 24 h.Real-time qualitative PCR(RT-qPCR)method was used to detect the mRNA expression of AKRs gene and LC-MS/MS technology was used to investigate the effect of specific AKR gene knockdown on DNA adduct levels.④500 nmol·L^(-1)human AKR recombinant proteins AKR1A1 and AA-Ⅰwere incubated in vitro under anaerobic conditions and the formation of AA-Ⅰ-DNA adducts was detected.RESULTS①The IC_(50)of AA-Ⅰto hiHeps and RT4 cells was 1.9 and 0.42μmol·L^(-1),respec⁃tively.The level of DNA adduct production of the two cell lines was significantly different(P<0.01).②Luteolin≥5μmol·L^(-1)significantly inhibited the production of AA-Ⅰ-DNA adducts in both cells(P<0.05),and there was a concentration-dependent effect in hiHeps cells(P<0.01,R=0.84).③In the AKR family,the knockdown of AKR1A1 gene up to 80%inhibited the generation of AA-Ⅰ-DNA adducts by 30%-40%.④The AA-Ⅰ-DNA adducts were detected in the incubation of recombinant protein AKR1A1 and AA-Ⅰunder anaerobic conditions in vitro,approximately 1 adduct per 107 nucleotides.CONCLU⁃SION AKR1A1 is involved in AA-Ⅰbioactivation,providing a reference for elucidation of the carcino⁃genic mechanism of AA-Ⅰ.
文摘Transgenic mice ubiquitously overexpressing murine γ aminobutyric acid transporter subtype Ⅰ were created. Unexpectedly, these mice markedly exhibited heritable obesity, which features significantly increased body weight and fat deposition. Behavioral examination revealed that transgeinc mice have slightly reduced spontaneous locomotive capacity and altered feeding pattern. Tills preliminary finding indicates that the inappropriate level of γ-aminobutyric acid transporters may be directly or indirectly involved in the pathogenic mechanism underlying certain types of obesity.
