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
Viral protein R(Vpr) plays an important role in the replication and pathogenesis of Human immunodeficiency virus type 1(HIV-1). Some of the various functions attributed to Vpr, including the induction of G2/M cell cyc...Viral protein R(Vpr) plays an important role in the replication and pathogenesis of Human immunodeficiency virus type 1(HIV-1). Some of the various functions attributed to Vpr, including the induction of G2/M cell cycle arrest, activating the NF-κB pathway, and promoting viral reverse transcription, might be interrelated. To test this hypothesis, a panel of Vpr mutants were investigated for their ability to induce G2/M arrest and to activate the NF-κB pathway. The results showed that the Vpr mutants that failed to activate NF-κB also lost the activity to induce G2/M arrest, which suggests that inducing G2/M arrest via Vpr depends at least partially on the activation of NF-κB. This latter possibility is supported by data showing that knocking down the key factors in the NF-κB pathway – p65, Rel B, IKKα, or IKKβ– partially rescued the G2/M arrest induced by Vpr.Our results suggest that the NF-κB pathway is probably involved in Vpr-induced G2/M cell cycle arrest.展开更多
Conjunctival melanoma(CM) is a rare and fatal malignant eye tumor. In this study, we deciphered a novel anti-CM mechanism of a natural tetracyclic compound named as cucurbitacin B(CuB). We found that CuB remarkably in...Conjunctival melanoma(CM) is a rare and fatal malignant eye tumor. In this study, we deciphered a novel anti-CM mechanism of a natural tetracyclic compound named as cucurbitacin B(CuB). We found that CuB remarkably inhibited the proliferation of CM cells including CM-AS16,CRMM1, CRMM2 and CM2005.1, without toxicity to normal cells. CuB can also induce CM cells G2/M cell cycle arrest. RNA-seq screening identified KIF20A, a key downstream effector of FOXM1 pathway, was abolished by CuB treatment. Further target identification by activity-based protein profiling chemoproteomic approach revealed that GRP78 is a potential target of CuB. Several lines of evidence demonstrated that CuB interacted with GRP78 and bound with a Kdvalue of0.11 μmol/L. Furthermore, ATPase activity evaluation showed that CuB suppressed GRP78 both in human recombinant GRP78 protein and cellular lysates. Knockdown of the GRP78 gene significantly induced the downregulation of FOXM1 and related pathway proteins including KIF20A, underlying an interesting therapeutic perspective. Finally, CuB significantly inhibited tumor progression in NCG mice without causing obvious side effects in vivo. Taken together, our current work proved that GRP78-FOXM1-KIF20A as a promising pathway for CM therapy, and the traditional medicine CuB as a candidate drug to hinder this pathway.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supported by grants from the Chinese Ministry of Health (2012ZX10001006)the National Natural Science Foundation of China (81271812 and 31370182)+1 种基金111 Project (B08011)the Postgraduate Scholarship Program of the China Scholarship Council
文摘Viral protein R(Vpr) plays an important role in the replication and pathogenesis of Human immunodeficiency virus type 1(HIV-1). Some of the various functions attributed to Vpr, including the induction of G2/M cell cycle arrest, activating the NF-κB pathway, and promoting viral reverse transcription, might be interrelated. To test this hypothesis, a panel of Vpr mutants were investigated for their ability to induce G2/M arrest and to activate the NF-κB pathway. The results showed that the Vpr mutants that failed to activate NF-κB also lost the activity to induce G2/M arrest, which suggests that inducing G2/M arrest via Vpr depends at least partially on the activation of NF-κB. This latter possibility is supported by data showing that knocking down the key factors in the NF-κB pathway – p65, Rel B, IKKα, or IKKβ– partially rescued the G2/M arrest induced by Vpr.Our results suggest that the NF-κB pathway is probably involved in Vpr-induced G2/M cell cycle arrest.
基金supported by the National Mega-project for Innovative Drugs of China(2019ZX09721001-004-003)the National Natural Science Foundation of China(82003603 and 81872747)+1 种基金the Innovative Research Team of High-level Local Universities in Shanghai,the National Special Fund for State Key Laboratory of Bioreactor Engineering(2060204,China)Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism(2021 Sci&Tech 03-28,China).
文摘Conjunctival melanoma(CM) is a rare and fatal malignant eye tumor. In this study, we deciphered a novel anti-CM mechanism of a natural tetracyclic compound named as cucurbitacin B(CuB). We found that CuB remarkably inhibited the proliferation of CM cells including CM-AS16,CRMM1, CRMM2 and CM2005.1, without toxicity to normal cells. CuB can also induce CM cells G2/M cell cycle arrest. RNA-seq screening identified KIF20A, a key downstream effector of FOXM1 pathway, was abolished by CuB treatment. Further target identification by activity-based protein profiling chemoproteomic approach revealed that GRP78 is a potential target of CuB. Several lines of evidence demonstrated that CuB interacted with GRP78 and bound with a Kdvalue of0.11 μmol/L. Furthermore, ATPase activity evaluation showed that CuB suppressed GRP78 both in human recombinant GRP78 protein and cellular lysates. Knockdown of the GRP78 gene significantly induced the downregulation of FOXM1 and related pathway proteins including KIF20A, underlying an interesting therapeutic perspective. Finally, CuB significantly inhibited tumor progression in NCG mice without causing obvious side effects in vivo. Taken together, our current work proved that GRP78-FOXM1-KIF20A as a promising pathway for CM therapy, and the traditional medicine CuB as a candidate drug to hinder this pathway.
