目的应用生物信息学方法探究猪重组NK-lysin(prNK-lysin)抑制肝癌细胞转移潜在的作用靶点和通路。方法将肝癌细胞设置为空白对照组,PBS处理组和prNK-lysin处理组,37℃作用6 h后,应用高效液相色谱串联质谱对肽段进行鉴定,按照Foldchange=...目的应用生物信息学方法探究猪重组NK-lysin(prNK-lysin)抑制肝癌细胞转移潜在的作用靶点和通路。方法将肝癌细胞设置为空白对照组,PBS处理组和prNK-lysin处理组,37℃作用6 h后,应用高效液相色谱串联质谱对肽段进行鉴定,按照Foldchange=1.2倍且P<0.05筛选出差异表达蛋白。基于GO、KEGG等数据库对差异表达蛋白进行GO功能分析和KEGG通路分析。运用RT-qPCR验证细胞中多肽-N-乙酰半乳糖胺基转移酶13(GALNT13)、跨膜蛋白51(TMEM51)和FKBP脯酰异构酶3(FKBP3)的mRNA相对表达量。Western blot验证FKBP3的蛋白表达量。结果蛋白组学表明,与空白对照组相比,prNK-lysin处理组中有1989个差异表达蛋白;与PBS处理组相比,prNK-lysin处理组中有2753个差异表达蛋白;PBS处理组和空白对照组相比,有15个差异表达蛋白。相对于PBS处理组和空白对照组,prNK-lysin处理组中共有1909个差异表达蛋白。GO和KEGG分析表明差异表达蛋白主要参与Viral process、translational initiation、RNAbinding等过程,主要富集于Ribosome、Protein process in endoplasmic reticulum、RNA transport等通路。RT-qPCR表明,与空白对照组相比,prNK-lysin处理组显著升高了细胞内GALNT13(1.54±0.06 vs 1.02±0.17,P<0.05)和TMEM51(1.27±0.07 vs 1.00±0.04,P<0.01)的mRNA相对表达量,显著降低了FKBP3(0.43±0.06 vs 1.02±0.24,P<0.05)的mRNA相对表达量。Western blotting表明prNK-lysin处理组与空白对照组相比显著降低了细胞内FKBP3(0.68±0.02 vs 1.02±0.03,P<0.001)的蛋白表达量。结论prNK-lysin处理后肝癌细胞SMMOL/LC-7721的蛋白质组与空白组相比发生显著变化,prNK-lysin作用于FKBP3蛋白,并能通过影响细胞内氧化磷酸化和糖酵解等通路发挥其抑制作用。展开更多
Diabetes-associated cognitive dysfunction has already been attracted considerable attention.Advanced glycation end products(AGEs)from daily diets are thought to be a vital contributor to the development of this diseas...Diabetes-associated cognitive dysfunction has already been attracted considerable attention.Advanced glycation end products(AGEs)from daily diets are thought to be a vital contributor to the development of this diseases.However,the effect of one of the best-characterized exogenous AGEs N^(ε)-(carboxymethyl)lysine(CML)on cognitive function is not fully reported.In the present study,diabetical Goto-Kakizaki(GK)rats were treated with free CML for 8-weeks.It was found that oral consumption of exogenous CML significantly aggravated diabetes-associated cognitive dysfunction in behavioral test.In details,exogenous CML increased levels of oxidative stress,promoted the activation of glial cells in the brain,up-regulated the release of inflammatory cytokines interleukin-6,inhibited the protein expression of the brain-derived neurotrophic factor and thus led to neuroinflammation.Furthermore,exogenous CML promoted the amyloidogenesis in the brain of GK rats,and inhibited the expression of GLUT4.Additionally,several tricarboxylic acid cycle and glutamate-glutamine/γ-aminobutyric acid cycle intermediates including pyruvate,succinic acid,glutamine,glutamate were significantly changed in brain of GK rats treated with exogenous free CML.In conclusion,exogenous free CML is a potentially noxious compounds led to aggravated diabetes-associated cognitive dysfunction which could be possibly explained by its effects on neuroinflammation,energy and neurotransmitter amino acid homeostasis.展开更多
胚胎着床是一个复杂的生理过程,除了拥有优质胚胎以外,还需要良好的子宫内膜容受性以及母胎之间的协调对话。NK2同源框蛋白-1(NK2 homeobox protein 1,NKX2-1)是一种含有同源结构域的转录因子。最近研究表明NKX2-1可以通过调控一系列细...胚胎着床是一个复杂的生理过程,除了拥有优质胚胎以外,还需要良好的子宫内膜容受性以及母胎之间的协调对话。NK2同源框蛋白-1(NK2 homeobox protein 1,NKX2-1)是一种含有同源结构域的转录因子。最近研究表明NKX2-1可以通过调控一系列细胞因子及黏附分子的作用影响胚胎着床过程,从而导致反复种植失败(recurrent implantation failure,RIF)的发生。本文就NKX2-1的结构、功能及其与反复种植失败之间的关系做一综述,并对今后研究方向作一展望,以期对RIF的发生机制和防治策略有更深入的了解。展开更多
BACKGROUND Advanced glycation end products(AGEs)are diabetic metabolic toxic products that cannot be ignored.Nε-(carboxymethyl)lysine(CML),a component of AGEs,could increase macrophage lipid uptake,promote foam cell ...BACKGROUND Advanced glycation end products(AGEs)are diabetic metabolic toxic products that cannot be ignored.Nε-(carboxymethyl)lysine(CML),a component of AGEs,could increase macrophage lipid uptake,promote foam cell formation,and thereby accelerate atherosclerosis.The receptor for AGEs(RAGE)and cluster of differentiation 36(CD36)were the receptors of CML.However,it is still unknown whether RAGE and CD36 play key roles in CML-promoted lipid uptake.AIM Our study aimed to explore the role of RAGE and CD36 in CML-induced macrophage lipid uptake.METHODS In this study,we examined the effect of CML on lipid uptake by Raw264.7 macrophages.After adding 10 mmol/L CML,the lipid accumulation in macrophages was confirmed by oil red O staining.Expression changes of CD36 and RAGE were detected with immunoblotting and quantitative real-time polymerase chain reaction.The interaction between CML with CD36 and RAGE was verified by immunoprecipitation.We synthesized a novel N-succinimidyl-4-18Ffluorobenzoate-CML radioactive probe.Radioactive receptor-ligand binding assays were performed to test the binding affinity between CML with CD36 and RAGE.The effects of blocking CD36 or RAGE on CML-promoting lipid uptake were also detected.RESULTS The study revealed that CML significantly promoted lipid uptake by macrophages.Immunoprecipitation and radioactive receptor-ligand binding assays indicated that CML could specifically bind to both CD36 and RAGE.CML had a higher affinity for CD36 than RAGE.ARG82,ASN71,and THR70 were the potential interacting amino acids that CD36 binds to CML Anti-CD36 and anti-RAGE could block the uptake of CML by macrophages.The lipid uptake promotion effect of CML was significantly attenuated after blocking CD36 or RAGE.CONCLUSION Our results suggest that the binding of CML with CD36 and RAGE promotes macrophage lipid uptake.展开更多
文摘目的应用生物信息学方法探究猪重组NK-lysin(prNK-lysin)抑制肝癌细胞转移潜在的作用靶点和通路。方法将肝癌细胞设置为空白对照组,PBS处理组和prNK-lysin处理组,37℃作用6 h后,应用高效液相色谱串联质谱对肽段进行鉴定,按照Foldchange=1.2倍且P<0.05筛选出差异表达蛋白。基于GO、KEGG等数据库对差异表达蛋白进行GO功能分析和KEGG通路分析。运用RT-qPCR验证细胞中多肽-N-乙酰半乳糖胺基转移酶13(GALNT13)、跨膜蛋白51(TMEM51)和FKBP脯酰异构酶3(FKBP3)的mRNA相对表达量。Western blot验证FKBP3的蛋白表达量。结果蛋白组学表明,与空白对照组相比,prNK-lysin处理组中有1989个差异表达蛋白;与PBS处理组相比,prNK-lysin处理组中有2753个差异表达蛋白;PBS处理组和空白对照组相比,有15个差异表达蛋白。相对于PBS处理组和空白对照组,prNK-lysin处理组中共有1909个差异表达蛋白。GO和KEGG分析表明差异表达蛋白主要参与Viral process、translational initiation、RNAbinding等过程,主要富集于Ribosome、Protein process in endoplasmic reticulum、RNA transport等通路。RT-qPCR表明,与空白对照组相比,prNK-lysin处理组显著升高了细胞内GALNT13(1.54±0.06 vs 1.02±0.17,P<0.05)和TMEM51(1.27±0.07 vs 1.00±0.04,P<0.01)的mRNA相对表达量,显著降低了FKBP3(0.43±0.06 vs 1.02±0.24,P<0.05)的mRNA相对表达量。Western blotting表明prNK-lysin处理组与空白对照组相比显著降低了细胞内FKBP3(0.68±0.02 vs 1.02±0.03,P<0.001)的蛋白表达量。结论prNK-lysin处理后肝癌细胞SMMOL/LC-7721的蛋白质组与空白组相比发生显著变化,prNK-lysin作用于FKBP3蛋白,并能通过影响细胞内氧化磷酸化和糖酵解等通路发挥其抑制作用。
基金supported by the National Natural Science Foundation of China(32302258,32172317)Changsha Municipal Natural Science Foundation(kq2202223).
文摘Diabetes-associated cognitive dysfunction has already been attracted considerable attention.Advanced glycation end products(AGEs)from daily diets are thought to be a vital contributor to the development of this diseases.However,the effect of one of the best-characterized exogenous AGEs N^(ε)-(carboxymethyl)lysine(CML)on cognitive function is not fully reported.In the present study,diabetical Goto-Kakizaki(GK)rats were treated with free CML for 8-weeks.It was found that oral consumption of exogenous CML significantly aggravated diabetes-associated cognitive dysfunction in behavioral test.In details,exogenous CML increased levels of oxidative stress,promoted the activation of glial cells in the brain,up-regulated the release of inflammatory cytokines interleukin-6,inhibited the protein expression of the brain-derived neurotrophic factor and thus led to neuroinflammation.Furthermore,exogenous CML promoted the amyloidogenesis in the brain of GK rats,and inhibited the expression of GLUT4.Additionally,several tricarboxylic acid cycle and glutamate-glutamine/γ-aminobutyric acid cycle intermediates including pyruvate,succinic acid,glutamine,glutamate were significantly changed in brain of GK rats treated with exogenous free CML.In conclusion,exogenous free CML is a potentially noxious compounds led to aggravated diabetes-associated cognitive dysfunction which could be possibly explained by its effects on neuroinflammation,energy and neurotransmitter amino acid homeostasis.
基金Supported by The National Natural Science Foundation of China,No.82070455Natural Science Foundation of Jiangsu Province,No.BK20201225Medical Innovation Team Project of Jiangsu Province,No.CXTDA2017010。
文摘BACKGROUND Advanced glycation end products(AGEs)are diabetic metabolic toxic products that cannot be ignored.Nε-(carboxymethyl)lysine(CML),a component of AGEs,could increase macrophage lipid uptake,promote foam cell formation,and thereby accelerate atherosclerosis.The receptor for AGEs(RAGE)and cluster of differentiation 36(CD36)were the receptors of CML.However,it is still unknown whether RAGE and CD36 play key roles in CML-promoted lipid uptake.AIM Our study aimed to explore the role of RAGE and CD36 in CML-induced macrophage lipid uptake.METHODS In this study,we examined the effect of CML on lipid uptake by Raw264.7 macrophages.After adding 10 mmol/L CML,the lipid accumulation in macrophages was confirmed by oil red O staining.Expression changes of CD36 and RAGE were detected with immunoblotting and quantitative real-time polymerase chain reaction.The interaction between CML with CD36 and RAGE was verified by immunoprecipitation.We synthesized a novel N-succinimidyl-4-18Ffluorobenzoate-CML radioactive probe.Radioactive receptor-ligand binding assays were performed to test the binding affinity between CML with CD36 and RAGE.The effects of blocking CD36 or RAGE on CML-promoting lipid uptake were also detected.RESULTS The study revealed that CML significantly promoted lipid uptake by macrophages.Immunoprecipitation and radioactive receptor-ligand binding assays indicated that CML could specifically bind to both CD36 and RAGE.CML had a higher affinity for CD36 than RAGE.ARG82,ASN71,and THR70 were the potential interacting amino acids that CD36 binds to CML Anti-CD36 and anti-RAGE could block the uptake of CML by macrophages.The lipid uptake promotion effect of CML was significantly attenuated after blocking CD36 or RAGE.CONCLUSION Our results suggest that the binding of CML with CD36 and RAGE promotes macrophage lipid uptake.
