Objective This study aimed to explore the protective effect of procyanidin B2(PCB2)on acute liver injury induced by aflatoxin B1(AFB1)in rats.Methods Forty Sprague Dawley rats were randomly divided into control,AFB1,A...Objective This study aimed to explore the protective effect of procyanidin B2(PCB2)on acute liver injury induced by aflatoxin B1(AFB1)in rats.Methods Forty Sprague Dawley rats were randomly divided into control,AFB1,AFB1+PCB2,and PCB2 groups.The latter two groups were administrated PCB2 intragastrically(30 mg/kg body weight)for 7 d,whereas the control and AFB1 groups were given the same dose of double distilled water intragastrically.On the sixth day of treatment,the AFB1 and AFB1+PCB2 groups were intraperitoneally injected with AFB1(2 mg/kg).The control and PCB2 groups were intraperitoneally administered the same dose of dimethyl sulfoxide(DMSO).On the eighth day,all rats were euthanized:serum and liver tissue were isolated for further examination.Hepatic histological features were assessed by hematoxylin and eosin-stained sections.Weight,organ coefficient(liver,spleen,and kidney),liver function(serum alanine aminotransferase,aspartate aminotransferase,alkaline phosphatase,total bilirubin,and direct bilirubin),oxidative index(catalase,glutathione,superoxide dismutase,malondialdehyde,and 8-hydroxy-2′-deoxyguanosine),inflammation factor[hepatic interleukin-6(IL-6)m RNA expression and serum IL-6],and bcl-2/bax ratio were measured.Results AFB1 significantly caused hepatic histopathological damage,abnormal liver function,oxidative stress,inflammation,and bcl-2/bax ratio reduction compared with DMSO-treated controls.Our results indicate that PCB2 treatment can partially reverse the adverse liver conditions induced by AFB1.Conclusion Our findings indicate that PCB2 exhibits a protective effect on acute liver injury induced by AFB1.展开更多
Aflatoxin B1(AFB1)is one of the most common mycotoxins that threatens human health.As singlestranded oligonucleotides with high affinity and specificity,aptamers have incomparable effect on the targeted detection of A...Aflatoxin B1(AFB1)is one of the most common mycotoxins that threatens human health.As singlestranded oligonucleotides with high affinity and specificity,aptamers have incomparable effect on the targeted detection of AFB1.Herein,after 11 rounds of selection and analysis using a modified affinity chromatography-based SELEX strategy,the truncated 37 nt aptamer AF11-2 was successfully obtained.The aptamer shows good detection performance for AFB1,and can sensitively detect AFB1 in the range of 100-1000 nmol/L,with a detection limit of 42 nmol/L.In the detection of pretreated edible peanut oil samples,AF11-2 aptamer also showed a high recovery rate and good stability for AFB1,and achieved satisfactory results.In addition,AF11-2 aptamer can significantly enhance the fluorescence ability of AFB1,which is not available in traditional Afla17-2-3 aptamer.After molecular docking analysis,it was found that AF11-2 and Afla17-2-3 had different nucleotide binding sites for AFB1.Afla17-2-3 binds to the carbonyl O of AFB1,while AF11-2 binds to the pyrrolic O of AFB1,which may be the main reason that AF11-2 can enhance the fluorescence of AFB1.展开更多
Objective： To explore the impact of V5-epitope tag inserted in the commercial pcDNA5/FRT/V5-His TOPO expression vector on the metabolic activation of AFB1 by human CYP2A13. Methods ： A C-terminal 6 × Histag was...Objective： To explore the impact of V5-epitope tag inserted in the commercial pcDNA5/FRT/V5-His TOPO expression vector on the metabolic activation of AFB1 by human CYP2A13. Methods ： A C-terminal 6 × Histag was first introduced into CYP2A13 cDNA by PCR and subsequently transferred into the expressing vector pcDNA5/FRT. Another commercial pcDNA5/FRT/V5-His TOPO expression vector was used to develop the construct directly via PCR. Both of the constructs were then transfected into Flp-In CHO and allowed for the stable expression of CYP2A13. The mouse CYP2A5 and the vector alone were used as positive and negative control, respectively. The presence of CYP2A5 and CYP2A13 cDNA and their protein expression in the stable transfectant cells were deterrrfined by immunoblotting assay using a monoclonal antibody against 6 × Histag. The AFBl-induced cytotoxicity in these tranfected CHO cells were conducted by MTS assay and the IC50 of cell viability was used to compare the CYP enzyme metabolic activity in AFB1 metabolism among these cells. Results： In accordance with the Flp-In system working mechanism, all the transfectant cells presented same protein expression level. The CHO cells expressing CYP2A5 was more sensitive to AFB1 treatment than those cells expressing CYP2A13, there was about 30-fold ICs0 difference between the two cells （2.1 nmol/L vs 58 nmol/L）. Interestingly, CYP2A13 fused with V5-Histag had the lost of metabolic activity to AFB1 than that fused with Histag alone, the ICa, of the viability in CHO-2A13-His-V5 cells was about 20-fold less than CHO-2A13- His （〉 1 000 nmol/L vs 58 nmol/L）. However, there was no change between CYP2A5 fused with V5-Histag and Histag alone （2.4 nmol/L vs 2.1 nmol/L）. Conclusion： The results demonstrate that CYP2A13 fused with V5-epitope has a significant impact on its metabolic activation to AFB1, which indicated that it should be careful to select a new expressing vector for evaluating the enzyme activity in carcinogen metabolism.展开更多
Dietary exposure to aflatoxin B1(AFB1)is harmful to the health and performance of domestic animals.The hepatic cytochrome P450s(CYPs),CYP1A1 and CYP2A6,are the primary enzymes responsible for the bioactivation of AFB1...Dietary exposure to aflatoxin B1(AFB1)is harmful to the health and performance of domestic animals.The hepatic cytochrome P450s(CYPs),CYP1A1 and CYP2A6,are the primary enzymes responsible for the bioactivation of AFB1to the highly toxic exo-AFB1-8,9-epoxide(AFBO)in chicks.However,the transcriptional regulation mechanism of these CYP genes in the liver of chicks in AFB1metabolism remains unknown.Dual-luciferase reporter assay,bioinformatics and site-directed mutation results indicated that specificity protein 1(SP1)and activator protein-1(AP-1)motifs were located in the core region-1,063/-948,-606/-541 of the CYP1A1 promoter as well as-636/-595,-503/-462,-147/-1 of the CYP2A6 promoter.Furthermore,overexpression and decoy oligodeoxynucleotide technologies demonstrated that SP1 and AP-1 were pivotal transcriptional activators regulating the promoter activity of CYP1A1 and CYP2A6.Moreover,bioactivation of AFB1to AFBO could be increased by upregulation of CYP1A1 and CYP2A6 expression,which was trans-activated owing to the upregulation of AP-1,rather than SP1,stimulated by AFB1-induced reactive oxygen species.Additionally,nano-selenium could reduce ROS,downregulate AP-1 expression and then decrease the expression of CYP1A1 and CYP2A6,thus alleviating the toxicity of AFB1.In conclusion,AP-1 and SP1 played important roles in the transactivation of CYP1A1 and CYP2A6 expression and further bioactivated AFB1to AFBO in chicken liver,which could provide novel targets for the remediation of aflatoxicosis in chicks.展开更多
Purpose: Aflatoxin B<sub>1</sub> is the most common mycotoxin in cereal crops;it is of stronger toxicity and has a carcinogenic effect. In recent years, a series of fluorescence sensors constructed on the ...Purpose: Aflatoxin B<sub>1</sub> is the most common mycotoxin in cereal crops;it is of stronger toxicity and has a carcinogenic effect. In recent years, a series of fluorescence sensors constructed on the basis of MoS<sub>2</sub>NS fluorescence quenching property have become a research hotspot. Therefore, we can construct a fast and simple analysis method with high specificity to detect AFB<sub>1</sub> by utilizing MoS<sub>2</sub>NS, which can be effectively applied to food safety monitoring and clinical diagnosis. Method: In the current research, a fluorescence biosensor is developed on the basis of a new type of two-dimensional nano-material namely MoS<sub>2</sub>NS applied for the detection of AFB<sub>1</sub>. The fluorescence of Apt@AFB<sub>1</sub> can be quickly quenched by MoS<sub>2</sub>NS through the fluorescence resonance energy transfer (FRET). When the target molecule AFB<sub>1</sub> exists, after the specificity binding between AFB<sub>1</sub> and aptamer, the Apt@AFB<sub>1</sub> loses its single stranded structure and is away from MoS<sub>2</sub>NS, and the fluorescence of Apt®AFB<sub>1</sub> cannot be quenched effectively. Such sensing signals can be used to achieve the sensitive detection of AFB<sub>1</sub>. Result: With this new method, under the optimized conditions, the AFB<sub>1</sub> is analyzed in the MoS<sub>2</sub>NS/Apt®AFB<sub>1</sub> sensing platform. Within the dynamic range of 0.2 - 25 ng/mL, the sensing platform expresses a good linear response to the level of AFB<sub>1</sub> with the R<sup>2</sup> = 0.9964 and LOD as 90 pg/mL. This method is applied to detect the actual serum samples and soybean milk with the recovery rate of 93.10% - 107.23% and 95.15% - 102.60% separately, and it can be used in the quantitative detection under the interference of other mycotoxins in a relatively accurate way. Conclusion: It is proved that this new detection method can be used as a potential biosensor platform for the detection of AFB<sub>1</sub>. This detection method features several advantages such as specificity, rapidness and low costs, which can meet the requirement of trace detection in clinical detection and food safety.展开更多
基金financially supported by National Natural Science Foundation of China[No.31360383]。
文摘Objective This study aimed to explore the protective effect of procyanidin B2(PCB2)on acute liver injury induced by aflatoxin B1(AFB1)in rats.Methods Forty Sprague Dawley rats were randomly divided into control,AFB1,AFB1+PCB2,and PCB2 groups.The latter two groups were administrated PCB2 intragastrically(30 mg/kg body weight)for 7 d,whereas the control and AFB1 groups were given the same dose of double distilled water intragastrically.On the sixth day of treatment,the AFB1 and AFB1+PCB2 groups were intraperitoneally injected with AFB1(2 mg/kg).The control and PCB2 groups were intraperitoneally administered the same dose of dimethyl sulfoxide(DMSO).On the eighth day,all rats were euthanized:serum and liver tissue were isolated for further examination.Hepatic histological features were assessed by hematoxylin and eosin-stained sections.Weight,organ coefficient(liver,spleen,and kidney),liver function(serum alanine aminotransferase,aspartate aminotransferase,alkaline phosphatase,total bilirubin,and direct bilirubin),oxidative index(catalase,glutathione,superoxide dismutase,malondialdehyde,and 8-hydroxy-2′-deoxyguanosine),inflammation factor[hepatic interleukin-6(IL-6)m RNA expression and serum IL-6],and bcl-2/bax ratio were measured.Results AFB1 significantly caused hepatic histopathological damage,abnormal liver function,oxidative stress,inflammation,and bcl-2/bax ratio reduction compared with DMSO-treated controls.Our results indicate that PCB2 treatment can partially reverse the adverse liver conditions induced by AFB1.Conclusion Our findings indicate that PCB2 exhibits a protective effect on acute liver injury induced by AFB1.
基金supported by the National Natural Science Foundation of China(Nos.32071392,21775160 and 31900999)the Natural Science Foundation of Jiangsu Province(No.BE2020766)the Science Foundation of Jiangxi Province(No.20192ACB21033)。
文摘Aflatoxin B1(AFB1)is one of the most common mycotoxins that threatens human health.As singlestranded oligonucleotides with high affinity and specificity,aptamers have incomparable effect on the targeted detection of AFB1.Herein,after 11 rounds of selection and analysis using a modified affinity chromatography-based SELEX strategy,the truncated 37 nt aptamer AF11-2 was successfully obtained.The aptamer shows good detection performance for AFB1,and can sensitively detect AFB1 in the range of 100-1000 nmol/L,with a detection limit of 42 nmol/L.In the detection of pretreated edible peanut oil samples,AF11-2 aptamer also showed a high recovery rate and good stability for AFB1,and achieved satisfactory results.In addition,AF11-2 aptamer can significantly enhance the fluorescence ability of AFB1,which is not available in traditional Afla17-2-3 aptamer.After molecular docking analysis,it was found that AF11-2 and Afla17-2-3 had different nucleotide binding sites for AFB1.Afla17-2-3 binds to the carbonyl O of AFB1,while AF11-2 binds to the pyrrolic O of AFB1,which may be the main reason that AF11-2 can enhance the fluorescence of AFB1.
文摘Objective： To explore the impact of V5-epitope tag inserted in the commercial pcDNA5/FRT/V5-His TOPO expression vector on the metabolic activation of AFB1 by human CYP2A13. Methods ： A C-terminal 6 × Histag was first introduced into CYP2A13 cDNA by PCR and subsequently transferred into the expressing vector pcDNA5/FRT. Another commercial pcDNA5/FRT/V5-His TOPO expression vector was used to develop the construct directly via PCR. Both of the constructs were then transfected into Flp-In CHO and allowed for the stable expression of CYP2A13. The mouse CYP2A5 and the vector alone were used as positive and negative control, respectively. The presence of CYP2A5 and CYP2A13 cDNA and their protein expression in the stable transfectant cells were deterrrfined by immunoblotting assay using a monoclonal antibody against 6 × Histag. The AFBl-induced cytotoxicity in these tranfected CHO cells were conducted by MTS assay and the IC50 of cell viability was used to compare the CYP enzyme metabolic activity in AFB1 metabolism among these cells. Results： In accordance with the Flp-In system working mechanism, all the transfectant cells presented same protein expression level. The CHO cells expressing CYP2A5 was more sensitive to AFB1 treatment than those cells expressing CYP2A13, there was about 30-fold ICs0 difference between the two cells （2.1 nmol/L vs 58 nmol/L）. Interestingly, CYP2A13 fused with V5-Histag had the lost of metabolic activity to AFB1 than that fused with Histag alone, the ICa, of the viability in CHO-2A13-His-V5 cells was about 20-fold less than CHO-2A13- His （〉 1 000 nmol/L vs 58 nmol/L）. However, there was no change between CYP2A5 fused with V5-Histag and Histag alone （2.4 nmol/L vs 2.1 nmol/L）. Conclusion： The results demonstrate that CYP2A13 fused with V5-epitope has a significant impact on its metabolic activation to AFB1, which indicated that it should be careful to select a new expressing vector for evaluating the enzyme activity in carcinogen metabolism.
基金partly supported by the Chinese Natural Science Foundation Projects(31772636,32072775,32272915)the National Key Research and Development Program of China(2023YFD1301003)+1 种基金the Fundamental Research Funds for the Central Universities(2662023DKPY002)the Top-notch Young Talent Supporting Program to LHS,a gift from Beijing Deyuanshun Biotechnology Co.,Ltd。
文摘Dietary exposure to aflatoxin B1(AFB1)is harmful to the health and performance of domestic animals.The hepatic cytochrome P450s(CYPs),CYP1A1 and CYP2A6,are the primary enzymes responsible for the bioactivation of AFB1to the highly toxic exo-AFB1-8,9-epoxide(AFBO)in chicks.However,the transcriptional regulation mechanism of these CYP genes in the liver of chicks in AFB1metabolism remains unknown.Dual-luciferase reporter assay,bioinformatics and site-directed mutation results indicated that specificity protein 1(SP1)and activator protein-1(AP-1)motifs were located in the core region-1,063/-948,-606/-541 of the CYP1A1 promoter as well as-636/-595,-503/-462,-147/-1 of the CYP2A6 promoter.Furthermore,overexpression and decoy oligodeoxynucleotide technologies demonstrated that SP1 and AP-1 were pivotal transcriptional activators regulating the promoter activity of CYP1A1 and CYP2A6.Moreover,bioactivation of AFB1to AFBO could be increased by upregulation of CYP1A1 and CYP2A6 expression,which was trans-activated owing to the upregulation of AP-1,rather than SP1,stimulated by AFB1-induced reactive oxygen species.Additionally,nano-selenium could reduce ROS,downregulate AP-1 expression and then decrease the expression of CYP1A1 and CYP2A6,thus alleviating the toxicity of AFB1.In conclusion,AP-1 and SP1 played important roles in the transactivation of CYP1A1 and CYP2A6 expression and further bioactivated AFB1to AFBO in chicken liver,which could provide novel targets for the remediation of aflatoxicosis in chicks.
文摘Purpose: Aflatoxin B<sub>1</sub> is the most common mycotoxin in cereal crops;it is of stronger toxicity and has a carcinogenic effect. In recent years, a series of fluorescence sensors constructed on the basis of MoS<sub>2</sub>NS fluorescence quenching property have become a research hotspot. Therefore, we can construct a fast and simple analysis method with high specificity to detect AFB<sub>1</sub> by utilizing MoS<sub>2</sub>NS, which can be effectively applied to food safety monitoring and clinical diagnosis. Method: In the current research, a fluorescence biosensor is developed on the basis of a new type of two-dimensional nano-material namely MoS<sub>2</sub>NS applied for the detection of AFB<sub>1</sub>. The fluorescence of Apt@AFB<sub>1</sub> can be quickly quenched by MoS<sub>2</sub>NS through the fluorescence resonance energy transfer (FRET). When the target molecule AFB<sub>1</sub> exists, after the specificity binding between AFB<sub>1</sub> and aptamer, the Apt@AFB<sub>1</sub> loses its single stranded structure and is away from MoS<sub>2</sub>NS, and the fluorescence of Apt®AFB<sub>1</sub> cannot be quenched effectively. Such sensing signals can be used to achieve the sensitive detection of AFB<sub>1</sub>. Result: With this new method, under the optimized conditions, the AFB<sub>1</sub> is analyzed in the MoS<sub>2</sub>NS/Apt®AFB<sub>1</sub> sensing platform. Within the dynamic range of 0.2 - 25 ng/mL, the sensing platform expresses a good linear response to the level of AFB<sub>1</sub> with the R<sup>2</sup> = 0.9964 and LOD as 90 pg/mL. This method is applied to detect the actual serum samples and soybean milk with the recovery rate of 93.10% - 107.23% and 95.15% - 102.60% separately, and it can be used in the quantitative detection under the interference of other mycotoxins in a relatively accurate way. Conclusion: It is proved that this new detection method can be used as a potential biosensor platform for the detection of AFB<sub>1</sub>. This detection method features several advantages such as specificity, rapidness and low costs, which can meet the requirement of trace detection in clinical detection and food safety.
