In this study,Ulva prolifera protein was used for preparing angiotensin-I converting enzyme(ACE)-inhibitory peptide via virtual gastrointestinal digestion and in silico screening.Some parameters of the obtained peptid...In this study,Ulva prolifera protein was used for preparing angiotensin-I converting enzyme(ACE)-inhibitory peptide via virtual gastrointestinal digestion and in silico screening.Some parameters of the obtained peptide,such as inhibition kinetics,docking mechanism,stability,transport pathway,were explored by Lineweaver-Burk plots,molecular docking,in vitro stimulate gastrointestinal(GI)digestion and Caco-2 cells monolayer model,respectively.Then,a novel anti-ACE peptide LDF(IC_(50),(1.66±0.34)μmol/L)was screened and synthesized by chemical synthesis.It was a no-competitive inhibitor and its anti-ACE inhibitory effect mainly attributable to four Conventional Hydrogen Bonds and Zn701 interactions.It could keep activity during simulated GI digestion in vitro and was transported by peptide transporter PepT1 and passive-mediated mode.Besides,it could activate Endothelial nitric oxide synthase(eNOS)activity to promote the production of NO and reduce Endothelin-1(ET-1)secretion induced by AngiotensinⅡ(AngⅡ)in Human Umbilical Vein Endothelial Cells(HUVECs).Meanwhile,it could promote mice splenocytes proliferation in a concentration-dependent manner.Our study indicated that this peptide was a potential ingredient functioning on vasodilation and enhancing immunity.展开更多
目的观察去泛素化酶Abraxas兄弟蛋白(ABRO1)对李斯特菌(LM)感染的小鼠单核巨噬细胞J774A.1白细胞介素(IL)-1β释放的影响,并探讨相关机制。方法培养J774A.1细胞,分别感染有李斯特菌溶血素O(LLO)的野生型LM菌株(野生型组)、敲除LLO的hly...目的观察去泛素化酶Abraxas兄弟蛋白(ABRO1)对李斯特菌(LM)感染的小鼠单核巨噬细胞J774A.1白细胞介素(IL)-1β释放的影响,并探讨相关机制。方法培养J774A.1细胞,分别感染有李斯特菌溶血素O(LLO)的野生型LM菌株(野生型组)、敲除LLO的hly基因缺失(Δhly)LM菌株(基因缺失组)及Δhly株回补hly基因的LM菌株(回补株组),采用Western blotting法检测ABRO1蛋白,ELISA法检测细胞培养液上清中的IL-1β。将J774A.1细胞分为NI组(未感染LM菌株)、WT组(感染WT LM菌株)与Δhly组(感染Δhly LM菌株),各组分别转染NC si RNA、ABRO1 si RNA,采用ELISA法检测细胞培养液上清中的IL-1β,采用Western blotting法检测炎症小体相关分子Caspase-1、p20(Caspase-1活化形式)、IL-1β、p17。结果随着感染时间延长,野生型组、回补株组ABRO1表达、IL-1β水平逐渐增高,在感染120 min达到最高、均高于基因缺失组(P均<0.05);基因缺失组不同时点ABRO1表达、IL-1β水平无明显变化。WT组转染ABRO1 si RNA的细胞培养液上清中IL-1β水平及p20、p17表达低于转染NC si RNA的细胞(P均<0.05);WT组转染NC si RNA的细胞培养液上清中IL-1β水平及p20、p17表达高于NI组(P均<0.05);Δhly组转染NC si RNA的细胞培养液上清中IL-1β水平及p20、p17表达低于WT组(P均<0.05)。结论李斯特菌感染的J774A.1细胞中ABRO1表达增高,下调ABRO1表达后,J774A.1细胞中IL-1β释放减少;LLO可能通过激活炎症小体从而促进LM感染诱导的IL-1β释放。展开更多
BACKGROUND The self-assembly of solid organs from stem cells has the potential to greatly expand the applicability of regenerative medicine.Stem cells can self-organise into microsized organ units,partially modelling ...BACKGROUND The self-assembly of solid organs from stem cells has the potential to greatly expand the applicability of regenerative medicine.Stem cells can self-organise into microsized organ units,partially modelling tissue function and regeneration.Dental pulp organoids have been used to recapitulate the processes of tooth development and related diseases.However,the lack of vasculature limits the utility of dental pulp organoids.AIM To improve survival and aid in recovery after stem cell transplantation,we demonstrated the three-dimensional(3D)self-assembly of adult stem cell-human dental pulp stem cells(hDPSCs)and endothelial cells(ECs)into a novel type of spheroid-shaped dental pulp organoid in vitro under hypoxia and conditioned medium(CM).METHODS During culture,primary hDPSCs were induced to differentiate into ECs by exposing them to a hypoxic environment and CM.The hypoxic pretreated hDPSCs were then mixed with ECs at specific ratios and conditioned in a 3D environment to produce prevascularized dental pulp organoids.The biological characteristics of the organoids were analysed,and the regulatory pathways associated with angiogenesis were studied.RESULTS The combination of these two agents resulted in prevascularized human dental pulp organoids(Vorganoids)that more closely resembled dental pulp tissue in terms of morphology and function.Single-cell RNA sequencing of dental pulp tissue and RNA sequencing of Vorganoids were integrated to analyse key regulatory pathways associated with angiogenesis.The biomarkers forkhead box protein O1 and fibroblast growth factor 2 were identified to be involved in the regulation of Vorganoids.CONCLUSION In this innovative study,we effectively established an in vitro model of Vorganoids and used it to elucidate new mechanisms of angiogenesis during regeneration,facilitating the development of clinical treatment strategies.展开更多
Human μ-opioid receptor （HμOR） with a tag of six consecutive histidines at its carboxyl terminus had been expressed in recombinant baculovirus infected Sf9 insect cells.The maximal binding capacity for the [3H] di...Human μ-opioid receptor （HμOR） with a tag of six consecutive histidines at its carboxyl terminus had been expressed in recombinant baculovirus infected Sf9 insect cells.The maximal binding capacity for the [3H] diprenorphine and [3H]ohmefentanyl （Ohm） were 9.1± 0.7 and 6.52±0.23 nmol/g protein, respectively. The [3H] diprenorphine or [3H] Ohm binding to the receptor expressed in Sf9 cells was strongly inhibited by μ-selective agonists [D-Ala2], N-methylPhe4, glyol5]enkephalin （DAGO）, Ohm, and morphine, but neither by δ nor by K selective agonist. Na+ （100 mM） and GTP （50 μM） could reduce HμOR agonists etorphine and Ohm affinity binding to the overexpressed HμOR. μ-selective agonists DAGO and Ohm effectively stimulated [35S]GTPγS binding (EC50 = 2.7nM and 6.9 nM) and inhibited forskolin- stimulated cAMP accumulation (IC50 = 0.9 nM and 0.3 nM). The agonist-dependent effects could be blocked by opioid antagonist naloxone or by pretreatment of cells with pertussis toxin （PTX）. These results demonstrated that HμOR overexpressed in Sf9 insect cells functionally coupled to endogenous Gi/o proteins.展开更多
Superparamagnetic poly（styrene）-co-poly（2-acrylanmido-2-methyl propanesulfonic acid） （PSt-co-PAMPS） and poly（methylmethacrylate）-co-poly（glycidyl methacrylate） （PMMA-co-PGMA） microspheres with mean size of...Superparamagnetic poly（styrene）-co-poly（2-acrylanmido-2-methyl propanesulfonic acid） （PSt-co-PAMPS） and poly（methylmethacrylate）-co-poly（glycidyl methacrylate） （PMMA-co-PGMA） microspheres with mean size of 170 nm were prepared by emulsion polymerization in the presence of oleic acid-coated Fe3O4 nanoparticles. The structures, morphologies, diameter and diameter distribution of the as-prepared microspheres were identified by Fourier transform infrared spectroscopy （FT-IR） and transmission electron microscopy （TEM）. The saturation magnetizations of PSt-co-PAMPS and PMMA-co-PGMA microspheres are 21.94 and 25.07 emu/g, respectively. The as-synthesized magnetic microspheres were used for immobilization of Bovine serum albumin （BSA） by physical interaction and covalent interaction respectively. The equilibrium amount of BSA immobilized onto PMMA-co-PGMA microspheres was 86.48 mg/g microspheres in 90 min, while on PSt-co-PAMPS microspheres was 59.62 mg/g microspheres in 120 min.展开更多
Throughout the globe,diabetes mellitus（DM） is increasing in incidence with limited therapies presently available to prevent or resolve the significant complications of this disorder.DM impacts multiple organs and af...Throughout the globe,diabetes mellitus（DM） is increasing in incidence with limited therapies presently available to prevent or resolve the significant complications of this disorder.DM impacts multiple organs and affects all components of the central and peripheral nervous systems that can range from dementia to diabetic neuropathy.The mechanistic target of rapamycin（m TOR） is a promising agent for the development of novel regenerative strategies for the treatment of DM.m TOR and its related signaling pathways impact multiple metabolic parameters that include cellular metabolic homeostasis,insulin resistance,insulin secretion,stem cell proliferation and differentiation,pancreatic β-cell function,and programmed cell death with apoptosis and autophagy.m TOR is central element for the protein complexes m TOR Complex 1（m TORC1） and m TOR Complex 2（m TORC2） and is a critical component for a number of signaling pathways that involve phosphoinositide 3-kinase（PI 3-K）,protein kinase B（Akt）,AMP activated protein kinase（AMPK）,silent mating type information regulation 2 homolog 1（Saccharomyces cerevisiae）（SIRT1）,Wnt1 inducible signaling pathway protein 1（WISP1）,and growth factors.As a result,m TOR represents an exciting target to offer new clinical avenues for the treatment of DM and the complications of this disease.Future studies directed to elucidate the delicate balance m TOR holds over cellular metabolism and the impact of its broad signaling pathways should foster the translation of these targets into effective clinical regimens for DM.展开更多
BACKGROUND： Acute organophosphate poisoning can cause injuries of multiple visceras; especially,central nervous system injury can increase risk factors of patients with severe acute organophosphate poisoning. An appl...BACKGROUND： Acute organophosphate poisoning can cause injuries of multiple visceras; especially,central nervous system injury can increase risk factors of patients with severe acute organophosphate poisoning. An application of modem image may increase diagnostic rate of brain injury in an earlier period and provide evidences for clinical treatment.OBJECTIVE： To reveal imaging manifestations, pathological characteristics and multi-ways injured mechanism of brain injury due to acute organophosphate poisoning.DESIGN： Contrast observational study.SETTING： Department of Medical Image, the Second Hospital of Hebei Medical University.MATERIALS： The experiment was carried out in the Department of Nerve Molecule Imaging Medicine and Laboratory of Neurology, the Second Hospital of Hebei Medical University from August 2003 to February 2004. A total of 30 healthy cats weighing 2.8 - 3.5 g and of both genders were selected from Animal Experimental Center of Hebei Medical University.METHODS： Thirty healthy cats were randomly divided into control group （n =5） and intoxication group （n=25）. Cats in the control group were subcutaneously injected with 0.3 mL/kg saline at four points; while, cats in the intoxication group were subcutaneously injected with 400 g/L 0.3 mL/kg O,O-dimethyl-S-（methoxycarbonylmethyl） thiophosphate at four points. Two minutes after intoxication, cats received muscular injection with 0.5 mg/kg atropine sulfate, and then, brain tissues were collected from parietal lobe, basal ganglia, hippocampus, cerebellum and brain stem were observed at 3, 6, 24 hours, 3 and 7 days after intoxication respectively under optic microscope and electron microscope and expressions of acetylcholinesterase （AChE）, choline acetyltransferase （ChAT）, glial fibrillary acidic protein （GFAP）,glutamic acid （Glu） and γ-amino butyric acid after immunohistochemical staining.MAIN OUTCOME MEASURES： Results of MRI examinations; histological changes under optic microscope and electron microscope; expressions of AChE, ChAT, GFAP, Glu and γ -amino butyric acid after immunohistochemical staining.MAIN OUTCOME MEASURES： Results of MRI examinations; histological changes under optic microscope and electron microscope; expressions of AChE, ChAT, GFAP, Glu and γ -amino butyric acid after immunohistochemical staining.RESULTS： All 30 healthy cats were involved in the final analysis. ① Imaging and pathological observation： Image manifestations of brain injury induced by acute organophosphate poisoning showed as cerebral edema and symmetry signal abnormality of bilateral basal ganglia; while, pathological manifestations also showed as cerebral edema. ② Observation of immunohistochemical staining： As compared with the control group, after organophosphate poisoning, area of AChE immune-positive cells was decreased obviously （P〈0.01）, but area of ChAT immune-positive cells was not changed （P〉0.05）; in addition, positive cells of GFAP were increased remarkably （P〈0.01）, positive cells of γ -amino butyric acid in cerebral cortex were increased obviously （P〈0.05）, but numbers of positive cells of Glu were not changed （P〈0.05）.CONCLUSION： Multi-ways injured mechanism invovled in acute organophosphate poisoning. An application of modern image can increase diagnostic rate of brain injury in an earlier period and provide evidences for clinical treatment.展开更多
文摘In this study,Ulva prolifera protein was used for preparing angiotensin-I converting enzyme(ACE)-inhibitory peptide via virtual gastrointestinal digestion and in silico screening.Some parameters of the obtained peptide,such as inhibition kinetics,docking mechanism,stability,transport pathway,were explored by Lineweaver-Burk plots,molecular docking,in vitro stimulate gastrointestinal(GI)digestion and Caco-2 cells monolayer model,respectively.Then,a novel anti-ACE peptide LDF(IC_(50),(1.66±0.34)μmol/L)was screened and synthesized by chemical synthesis.It was a no-competitive inhibitor and its anti-ACE inhibitory effect mainly attributable to four Conventional Hydrogen Bonds and Zn701 interactions.It could keep activity during simulated GI digestion in vitro and was transported by peptide transporter PepT1 and passive-mediated mode.Besides,it could activate Endothelial nitric oxide synthase(eNOS)activity to promote the production of NO and reduce Endothelin-1(ET-1)secretion induced by AngiotensinⅡ(AngⅡ)in Human Umbilical Vein Endothelial Cells(HUVECs).Meanwhile,it could promote mice splenocytes proliferation in a concentration-dependent manner.Our study indicated that this peptide was a potential ingredient functioning on vasodilation and enhancing immunity.
文摘目的观察去泛素化酶Abraxas兄弟蛋白(ABRO1)对李斯特菌(LM)感染的小鼠单核巨噬细胞J774A.1白细胞介素(IL)-1β释放的影响,并探讨相关机制。方法培养J774A.1细胞,分别感染有李斯特菌溶血素O(LLO)的野生型LM菌株(野生型组)、敲除LLO的hly基因缺失(Δhly)LM菌株(基因缺失组)及Δhly株回补hly基因的LM菌株(回补株组),采用Western blotting法检测ABRO1蛋白,ELISA法检测细胞培养液上清中的IL-1β。将J774A.1细胞分为NI组(未感染LM菌株)、WT组(感染WT LM菌株)与Δhly组(感染Δhly LM菌株),各组分别转染NC si RNA、ABRO1 si RNA,采用ELISA法检测细胞培养液上清中的IL-1β,采用Western blotting法检测炎症小体相关分子Caspase-1、p20(Caspase-1活化形式)、IL-1β、p17。结果随着感染时间延长,野生型组、回补株组ABRO1表达、IL-1β水平逐渐增高,在感染120 min达到最高、均高于基因缺失组(P均<0.05);基因缺失组不同时点ABRO1表达、IL-1β水平无明显变化。WT组转染ABRO1 si RNA的细胞培养液上清中IL-1β水平及p20、p17表达低于转染NC si RNA的细胞(P均<0.05);WT组转染NC si RNA的细胞培养液上清中IL-1β水平及p20、p17表达高于NI组(P均<0.05);Δhly组转染NC si RNA的细胞培养液上清中IL-1β水平及p20、p17表达低于WT组(P均<0.05)。结论李斯特菌感染的J774A.1细胞中ABRO1表达增高,下调ABRO1表达后,J774A.1细胞中IL-1β释放减少;LLO可能通过激活炎症小体从而促进LM感染诱导的IL-1β释放。
基金Supported by the Science and Technology Programme of Guangzhou City,No.202201020341.
文摘BACKGROUND The self-assembly of solid organs from stem cells has the potential to greatly expand the applicability of regenerative medicine.Stem cells can self-organise into microsized organ units,partially modelling tissue function and regeneration.Dental pulp organoids have been used to recapitulate the processes of tooth development and related diseases.However,the lack of vasculature limits the utility of dental pulp organoids.AIM To improve survival and aid in recovery after stem cell transplantation,we demonstrated the three-dimensional(3D)self-assembly of adult stem cell-human dental pulp stem cells(hDPSCs)and endothelial cells(ECs)into a novel type of spheroid-shaped dental pulp organoid in vitro under hypoxia and conditioned medium(CM).METHODS During culture,primary hDPSCs were induced to differentiate into ECs by exposing them to a hypoxic environment and CM.The hypoxic pretreated hDPSCs were then mixed with ECs at specific ratios and conditioned in a 3D environment to produce prevascularized dental pulp organoids.The biological characteristics of the organoids were analysed,and the regulatory pathways associated with angiogenesis were studied.RESULTS The combination of these two agents resulted in prevascularized human dental pulp organoids(Vorganoids)that more closely resembled dental pulp tissue in terms of morphology and function.Single-cell RNA sequencing of dental pulp tissue and RNA sequencing of Vorganoids were integrated to analyse key regulatory pathways associated with angiogenesis.The biomarkers forkhead box protein O1 and fibroblast growth factor 2 were identified to be involved in the regulation of Vorganoids.CONCLUSION In this innovative study,we effectively established an in vitro model of Vorganoids and used it to elucidate new mechanisms of angiogenesis during regeneration,facilitating the development of clinical treatment strategies.
文摘Human μ-opioid receptor （HμOR） with a tag of six consecutive histidines at its carboxyl terminus had been expressed in recombinant baculovirus infected Sf9 insect cells.The maximal binding capacity for the [3H] diprenorphine and [3H]ohmefentanyl （Ohm） were 9.1± 0.7 and 6.52±0.23 nmol/g protein, respectively. The [3H] diprenorphine or [3H] Ohm binding to the receptor expressed in Sf9 cells was strongly inhibited by μ-selective agonists [D-Ala2], N-methylPhe4, glyol5]enkephalin （DAGO）, Ohm, and morphine, but neither by δ nor by K selective agonist. Na+ （100 mM） and GTP （50 μM） could reduce HμOR agonists etorphine and Ohm affinity binding to the overexpressed HμOR. μ-selective agonists DAGO and Ohm effectively stimulated [35S]GTPγS binding (EC50 = 2.7nM and 6.9 nM) and inhibited forskolin- stimulated cAMP accumulation (IC50 = 0.9 nM and 0.3 nM). The agonist-dependent effects could be blocked by opioid antagonist naloxone or by pretreatment of cells with pertussis toxin （PTX）. These results demonstrated that HμOR overexpressed in Sf9 insect cells functionally coupled to endogenous Gi/o proteins.
基金Funded by the National Natural Science Foundation of China (20876070)Technology Innovation Team of Universities Funded Project of Jiangsu Province (2007-5)
文摘Superparamagnetic poly（styrene）-co-poly（2-acrylanmido-2-methyl propanesulfonic acid） （PSt-co-PAMPS） and poly（methylmethacrylate）-co-poly（glycidyl methacrylate） （PMMA-co-PGMA） microspheres with mean size of 170 nm were prepared by emulsion polymerization in the presence of oleic acid-coated Fe3O4 nanoparticles. The structures, morphologies, diameter and diameter distribution of the as-prepared microspheres were identified by Fourier transform infrared spectroscopy （FT-IR） and transmission electron microscopy （TEM）. The saturation magnetizations of PSt-co-PAMPS and PMMA-co-PGMA microspheres are 21.94 and 25.07 emu/g, respectively. The as-synthesized magnetic microspheres were used for immobilization of Bovine serum albumin （BSA） by physical interaction and covalent interaction respectively. The equilibrium amount of BSA immobilized onto PMMA-co-PGMA microspheres was 86.48 mg/g microspheres in 90 min, while on PSt-co-PAMPS microspheres was 59.62 mg/g microspheres in 120 min.
基金supported by American Diabetes Association,American Heart Association,NIH NIEHS,NIH NIA,NIH NINDS,and NIH ARRA
文摘Throughout the globe,diabetes mellitus（DM） is increasing in incidence with limited therapies presently available to prevent or resolve the significant complications of this disorder.DM impacts multiple organs and affects all components of the central and peripheral nervous systems that can range from dementia to diabetic neuropathy.The mechanistic target of rapamycin（m TOR） is a promising agent for the development of novel regenerative strategies for the treatment of DM.m TOR and its related signaling pathways impact multiple metabolic parameters that include cellular metabolic homeostasis,insulin resistance,insulin secretion,stem cell proliferation and differentiation,pancreatic β-cell function,and programmed cell death with apoptosis and autophagy.m TOR is central element for the protein complexes m TOR Complex 1（m TORC1） and m TOR Complex 2（m TORC2） and is a critical component for a number of signaling pathways that involve phosphoinositide 3-kinase（PI 3-K）,protein kinase B（Akt）,AMP activated protein kinase（AMPK）,silent mating type information regulation 2 homolog 1（Saccharomyces cerevisiae）（SIRT1）,Wnt1 inducible signaling pathway protein 1（WISP1）,and growth factors.As a result,m TOR represents an exciting target to offer new clinical avenues for the treatment of DM and the complications of this disease.Future studies directed to elucidate the delicate balance m TOR holds over cellular metabolism and the impact of its broad signaling pathways should foster the translation of these targets into effective clinical regimens for DM.
文摘BACKGROUND： Acute organophosphate poisoning can cause injuries of multiple visceras; especially,central nervous system injury can increase risk factors of patients with severe acute organophosphate poisoning. An application of modem image may increase diagnostic rate of brain injury in an earlier period and provide evidences for clinical treatment.OBJECTIVE： To reveal imaging manifestations, pathological characteristics and multi-ways injured mechanism of brain injury due to acute organophosphate poisoning.DESIGN： Contrast observational study.SETTING： Department of Medical Image, the Second Hospital of Hebei Medical University.MATERIALS： The experiment was carried out in the Department of Nerve Molecule Imaging Medicine and Laboratory of Neurology, the Second Hospital of Hebei Medical University from August 2003 to February 2004. A total of 30 healthy cats weighing 2.8 - 3.5 g and of both genders were selected from Animal Experimental Center of Hebei Medical University.METHODS： Thirty healthy cats were randomly divided into control group （n =5） and intoxication group （n=25）. Cats in the control group were subcutaneously injected with 0.3 mL/kg saline at four points; while, cats in the intoxication group were subcutaneously injected with 400 g/L 0.3 mL/kg O,O-dimethyl-S-（methoxycarbonylmethyl） thiophosphate at four points. Two minutes after intoxication, cats received muscular injection with 0.5 mg/kg atropine sulfate, and then, brain tissues were collected from parietal lobe, basal ganglia, hippocampus, cerebellum and brain stem were observed at 3, 6, 24 hours, 3 and 7 days after intoxication respectively under optic microscope and electron microscope and expressions of acetylcholinesterase （AChE）, choline acetyltransferase （ChAT）, glial fibrillary acidic protein （GFAP）,glutamic acid （Glu） and γ-amino butyric acid after immunohistochemical staining.MAIN OUTCOME MEASURES： Results of MRI examinations; histological changes under optic microscope and electron microscope; expressions of AChE, ChAT, GFAP, Glu and γ -amino butyric acid after immunohistochemical staining.MAIN OUTCOME MEASURES： Results of MRI examinations; histological changes under optic microscope and electron microscope; expressions of AChE, ChAT, GFAP, Glu and γ -amino butyric acid after immunohistochemical staining.RESULTS： All 30 healthy cats were involved in the final analysis. ① Imaging and pathological observation： Image manifestations of brain injury induced by acute organophosphate poisoning showed as cerebral edema and symmetry signal abnormality of bilateral basal ganglia; while, pathological manifestations also showed as cerebral edema. ② Observation of immunohistochemical staining： As compared with the control group, after organophosphate poisoning, area of AChE immune-positive cells was decreased obviously （P〈0.01）, but area of ChAT immune-positive cells was not changed （P〉0.05）; in addition, positive cells of GFAP were increased remarkably （P〈0.01）, positive cells of γ -amino butyric acid in cerebral cortex were increased obviously （P〈0.05）, but numbers of positive cells of Glu were not changed （P〈0.05）.CONCLUSION： Multi-ways injured mechanism invovled in acute organophosphate poisoning. An application of modern image can increase diagnostic rate of brain injury in an earlier period and provide evidences for clinical treatment.
