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Peroxynitrite anion(ONOO?)is one of the most active species of reactive oxygen species(ROS)and reactive nitrogen species(RNS).As an important physiologically active molecule,the abnormal expression of ONOO?will damage the protein and DNA in cells,leading to inflammation and other serious diseases in vivo.In recent years,fluorescence detection technology has been used to realize rapid and sensitive monitoring of bioactive molecules,and imaging tools have been used to conduct high-resolution tracing.Therefore,many organic small molecule fluorescent probes have been developed to detect ONOO?.In this paper,the recent research progresses of ONOO? fluorescence detection methods based on intramolecular charge transfer(ICT),photoinduced electron transfer(PET),fluorescence resonance energy transfer(FRET),excited state intramolecular charge transfer(ESIPT)and other fluorescence response mechanisms were reviewed.
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Objective To investigate the changes of OAT expression in bone cells in chronic renal failure(CRF)and involved mechanism,and to explore the effect of OAT expression on bone metabolism.Methods Ra-ndomly divide the rats into a control group(n=6)and a model group(n=6).The model group established a rat chronic renal failure model using"single nephrectomy+adenine gavage"method,and the red blood cell(RBC)and hemoglobin(HGB)of the rat body were measured using a blood routine analyzer;Measure indicators such as creatinine(Cr),urea nitrogen(BUN),uric acid(UA),blood calcium(Ca2+),and blood phosphorus(P3+)using a fully automated biochemical analyzer;Pathological examination of rat kidneys;X-ray examination of rat tibia;Immunohistochemical examination of bone tissue OAT1 level.Results The bone density of the model group rats is lower than that of the control group;The calcium and phosphorus metabolism of rats in the model group was in metabolic disorder,and the OAT1 value of bone tissue binding was much lower than that of the control group(P= 0.0018),which was statistically significant.(P=0.0018)Conclusion Chronic renal failure affected the binding ability of OAT1 in bone tissue,leading to the metabolic disorder for calcium absorption and phosphorus metabolism,thus aggravating renal osteodystrophy(P<0.05).
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Objective:To investigate the effects and mechanism of exosome (EXO)-loaded kringle V11 (KV11) delivery on corneal neovascularization (CNV).Methods:KV11 was bound to the surface of endothelial cell-derived exosomes by using CP05, an EXO-targeting anchoring peptide, to produce EXO-KV11.The binding efficiency and optimal concentration ratio were determined using the Apogee flow system.A total of 100 8-week-old healthy male SPF grade SD rats were selected, 10 of which were randomly selected as a normal control group without any treatment.The CNV model was established by alkali burn in the other 90 rats, which were randomly divided into three groups, EXO-KV11 group, KV11 group, and normal saline group by the random number table method, with 30 rats in each group.Each group was injected subconjunctivally with 100 μl of EXO-KV11 (25 μg), KV11 (25 μg), or normal saline every other day from the first day after the alkali burn, respectively.The CNV of rats was observed on days 1, 4, 7, and 14 after alkali burn.The CNV area was calculated by ventricular perfusion with fluorescein isothiocyanate-dextran (FITC-dextran) and corneal angiography.The amount of CNV lumen was observed by hematoxylin and eosin staining.The distribution of CD31 in rat corneas was determined by immunohistochemical method.The expression levels of voltage-dependent anion channel 1(VDAC1), endoplasmic reticulum stress, autophagy and apoptosis-associated proteins were detected by Western blot.This study was approved by the Animal Ethics Committee of Peking University People's Hospital (No.20210019). All animal procedures complied with the regulations of the Vision and Ophthalmology Association and the Animal Protection and Use Committee of Peking University.Results:The optimal concentration ratio of KV11 to EXO was 4∶1 and the binding affinity reached up to 87.5% by Apogee flow cytometers.On days 7 and 14 after alkali burn, there were significant differences in CNV area among the four groups ( F=4.613, 15.590; both at P<0.05). On day 7 after alkali burn, the CNV area was smaller in EXO-KV11 group than in KV11 and normal saline groups, with statistically significant differences (both at P<0.05). On day 14 after alkali burn, the CNV area was smaller in EXO-KV11 and KV11 groups than in normal saline group, and smaller in EXO-KV11 group than in KV11 group, showing statistically significant differences (all at P<0.05). The results of quantitative analysis of corneal fluorescence mounts showed that the relative CNV fluorescence area of the normal saline group, KV11 group and EXO-KV11 group were (8.3±1.7)%, (5.2±1.6)%and (3.4±0.7)%, respectively, showing a statistically significant overall comparison difference ( F=11.735, P<0.01). The relative CNV fluorescence area was larger in KV11 and normal saline groups than in EXO-KV11 group, and larger in normal saline group than in KV11 group, showing statistically significant differences (all at P<0.05). On day 14 after alkali burn, massive neovascular lumens were observed in the matrix of the normal saline group.The number of neovascular lumens in KV11 group was smaller than that in normal saline group.The corneal structure appeared normal in EXO-KV11 group, and neovascular lumens were rare.Numerous CD31-positive cells were observed in the corneal stroma of the normal saline group, which formed into lumen structures.The number of lumens surrounded by CD31-positive cells in the corneal stroma was smaller in KV11 group than in normal saline group, and smaller in EXO-KV11 group than in KV11 group.There were significant differences in the relative expression levels of VDAC1, protein kinase R-like endoplasmic reticulum kinase (PERK), p62, cleaved caspase 3 among the four groups ( F=35.960, 8.947, 17.791, 101.168; all at P<0.01). The relative expression levels of VDAC1, PERK, p62, cleaved caspase 3 were higher in EXO-KV11 group than in KV11 and normal saline groups, showing statistically significant differences (all at P<0.001). There was no significant difference in the relative expression of microtubule-associated proteins 1A/1B light chain 3B (LC3B)Ⅱ/LC3BⅠ protein among all four groups ( F=0.445, P=0.727). Conclusions:EXO-KV11 can inhibit CNV more remarkably than KV11.EXO-KV11 inhibits CNV by promoting the expression of VDAC1 and PERK and suppressing the autophagic flux.
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@#Objective To develop and verify an anion-exchange high-performance liquid chromatography(AEX-HPLC)method for the determination of empty capsid ratio of recombinant adeno-associated virus type 9(rAAV9).Methods AEXHPLC based on the differences in surface charge was used to establish a method for detecting the ratio of empty and full capsid rAAV9 by optimizing the elution gradient of mobile phase,pH,column temperature,flow rate,sample concentration,injection volume and detection wavelength of fluorescence detector. The specificity,linearity,limit of detection(LOD),limit of quantitation(LOQ),precision and accuracy of the method were verified to confirm the feasibility.Results Using a CIMac AAV full/empty-0. 1 mL column,20 mmol/L BIS-Tris propane(BTP)as mobile phase A and 20 mmol/L BTP+1 mol/L NaCl as mobile phase B,gradient elution was performed with pH of 9.0,column temperature of 20 ℃,flow rate of1 mL/min,sample concentration of 4×10~(12)vg/mL,injection volume of 10 μL,excitation wavelength of 280 nm and emission wavelength of 330 nm,which realised the baseline isolation and quantitative detection of empty and full capsid rAAV9. The verification results of the method showed that the preparation buffer had no interference with good specificity;rAAV9 showed a good linear relationship in the range of(1.6-8)×10~(12)vg/mL,r = 0. 993;the LOD was 5×10~(10)vg/mL,and the LOQ was 1×10~(11)vg/mL;the RSD of repeatability and intermediate precision were 2. 95% and 2. 10%,respectively;the accuracy rates were not less than 80%.Conclusion A highly sensitive and rapid AEX-HPLC method for determination of the ratio of empty capsid to full capsid rAAV9 was developed,which could be used for the analysis of empty capsid rate and quality control in gene therapy products.
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Ferroptosis is an iron-dependent cell death caused by phospholipid peroxidation damage of polyunsaturated fatty acids on cell membranes and involves several pathways, including the iron homeostasis regulatory pathway, the cystine glutamate reverse transporter (system Xc) pathway and the voltage-dependent anion channel (VDAC) pathway. Ferroptosis is involved in the development of several diseases (e. g. myocardial infarction, stroke, cancer and degenerative diseases). The ubiquitination is an important post-translational modification of various protein molecules in the organism. Studies have shown that regulating the ubiquitination of ferroptosis pathway-related molecules can control cellular ferroptosis. Targeting the ubiquitination of ferroptosis pathway-related molecules can effectively promote or inhibit ferroptosis, which is expected to be a new strategy for the treatment of cancer or cardiovascular diseases. In this paper we review the progress of the ferroptosis pathways and the ubiquitination modification of ferroptosis-related molecules.
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Objective To investigate the effects of long noncoding RNA(lncRNA)-NRON on apoptosis following myocardial infarc-tion(MI)in mice.Methods The C57BL/6 mice were randomly divided into four groups:sham operation(Sham)group,MI group,MI combined with lncRNA-NRON interference lentivirus(MI+shNRON)group,and MI combined with the negative control(NC)lentivirus(MI+NC)group.The expression of lncRNA-NRON was detected using real-time PCR.In addition,the pathology of the myocardial tissue injury was analyzed using HE staining,the myocardial infarction size was examined using TTC staining,and the extent of apoptosis was assessed using the TUNEL assay,respectively.The RPISeq database was used to predict the probability of interaction between lncR-NA-NRON and the voltage-dependent anionic channel protein(VDAC).The effect of lncRNA-NRON on the expression of VDAC protein was detected using Western blotting.Results The lncRNA-NRON expression was significantly increased in the MI group,and the tar-geted knockdown of lncRNA-NRON resulted in alleviation of the pathological myocardial tissue injury,reduction in the myocardial infarc-tion area,and inhibition of apoptosis.The probability of interaction between lncRNA-NRON and VDAC reached 0.9,indicating a high probability of their association.Additionally,lncRNA-NRON could regulate the protein expression of VDAC.Conclusion Knockdown of lncRNA-NRON could reduce the occurrence of myocardial injury following myocardial infarction.This effect may be attributable to a spe-cific mechanism wherein lncRNA-NRON affects the process of apoptosis by binding to VDAC,consequently suppressing its expression.
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Pendrin is an electroneutral anion exchanger transporter, residing in the apical region of airway epithelium cells.It is responsible for the reabsorption of chloride(Cl -) and the exchange of bicarbonate(HCO 3－)or thiocyanate(SCN -) to the lumen.It is mainly involved in regulating the pH and thickness of airway surface liquid(ASL), mucin secretion, and airway defense, which is of great significance for maintaining the stability of the airway surface microenvironment.The expression of pendrin is significantly up-regulated in bronchial asthma, which is closely related to the pathological processes of the lung in bronchial asthma, such as airway hyperresponsiveness, neutrophil infiltration, and increased mucin secretion.Inhibiting the function of pendrin may be a new target for the treatment of bronchial asthma.
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Objective:To investigate the role of indoxyl sulfate (IS) in myocardial hypertrophy and fibrosis through organic anion transporter-3 (OAT-3) and oxidative stress in H9C2 cells.Methods:Rat myocardial cells (H9C2) were cultured and divided into four groups: Control group, IS group, siRNA negative control group (siOAT-3), and siOAT-3+ IS group. The control group was cultured routinely without IS stimulation. The IS group was stimulated with 250 μmol IS. The siRNA negative control group was transfected with 20 nmol/L OAT-3 siRNA, and the siOAT-3+ IS group was transfected with OAT-3 siRNA 48 hours later, then stimulated with IS for 24 hours. Real-time polymerase chain reaction (RT-PCR) was used to detect and analyze the mRNA expression levels of OAT-3, NADPH oxidase-4 (Nox-4), antioxidant proteins [nuclear factor E2 related factor 2 (Nrf-2), heme oxygenase 1 (HO-1)], myocardial hypertrophy markers [atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), β-myosin heavy chain gene (β-MHC)], and fibrosis markers [transforming growth factor β1 (TGF-β1), Smad homologous protein 3 (Smad-3), collagen type Ⅰ (Collagen Ⅰ)] in each group. H9C2 cells were further divided into three groups: Control group, IS group, and N-acetylcysteine (NAC) group. The NAC group was pre-treated with the antioxidant NAC before stimulation with 250 μmol IS for 24 hours. RT-PCR was used to detect the mRNA expression levels of the above indicators.Results:The mRNA expression level of OAT-3 in the IS group was significantly higher than that in the control group, while the mRNA expression levels of OAT-3 in the siOAT-3 group and the siOAT-3+ IS group were significantly lower than those in the IS group (all P<0.001). Compared with the Control group, the mRNA relative expression levels of Nox-4, ANP, BNP, β-MHC, TGF-β1, Smad-3, and Collagen Ⅰ in H9C2 cells of the IS group were significantly increased (all P<0.001), while the mRNA expression levels of Nrf-2 and HO-1 were significantly decreased (all P<0.001). Compared with the IS group, the mRNA relative expression levels of Nox-4, ANP, BNP, β-MHC, TGF-β1, Smad-3, and Collagen Ⅰ in H9C2 cells of the siOAT-3 group and the siOAT-3+ IS group were significantly lower (all P<0.001), while the mRNA expression levels of Nrf-2 and HO-1 were significantly increased (all P<0.001). Pre-treatment with NAC significantly inhibited the high expression of Nox-4, ANP, BNP, β-MHC, TGF-β1, Smad-3, and Collagen Ⅰ mRNA induced by IS (all P<0.001), while significantly increasing the mRNA expression levels of Nrf-2 and HO-1 (all P<0.01). Conclusions:IS promotes myocardial hypertrophy and fibrotic factor overexpression in H9C2 cells through OAT-3 and oxidative stress.
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Abstract The cold storage of milt implies potentials alterations in its quality because the storage generates as main process, free radicals that produce spermatozoa membrane lipids damage with the consequent motility and fertilising capacity disruptions. To decrease the damage generated by free radicals the cells have antioxidant defences (proteins, enzymes, and low molecular weight substances). The objective of the present study evaluated the time storage effect and different antioxidants prepared in spermatic diluents on sperm viability of O. mykiss milt stored at 4°C. The two-way ANOVA denoted that the time storage and antioxidant influence have significant effects separated or combined on viability parameters (sperm motility and viability, proteins concentrations and superoxide dismutase enzymatic activity in seminal plasma). In contrast, only the storage time affected the fertilising capacity and catalase enzymatic activity in seminal plasma. The resulting analysis can conclude that the antioxidant presence improves the viability of cold stored milt, especially the transport conditions and the antioxidants allow the fecundity despite motility decrease.
Resumo O armazenamento a frio de leite implica potenciais alterações em sua qualidade, pois gera como processo principal radicais livres que provocam danos aos lipídios da membrana dos espermatozoides, com as consequentes alterações na motilidade e na capacidade de fertilização. Para diminuir os danos causados pelos radicais livres, as células têm defesas antioxidantes (proteínas, enzimas e substâncias de baixo peso molecular). O presente estudo avaliou o efeito do tempo de armazenamento e diferentes antioxidantes preparados em diluentes espermáticos no armazenamento de viabilidade de O. mykiss milt a 4°C. A ANOVA de duas vias denotou que o armazenamento no tempo e a influência antioxidante têm efeitos significativos separados ou combinados nos parâmetros de viabilidade (motilidade espermática, viabilidade espermática, concentrações de proteínas e atividade enzimática da superóxido dismutase no plasma seminal), enquanto apenas o tempo de armazenamento afetou a capacidade de fertilização e atividade enzimática da catalase no plasma seminal. A análise resultante pode concluir que a presença de antioxidante melhora a viabilidade do leite frio, especialmente as condições de transporte, e os antioxidantes permitem a fecundidade apesar da diminuição da motilidade.
Subject(s)
Animals , Male , Semen Preservation/veterinary , Oncorhynchus mykiss , Sperm Motility , Spermatozoa , Cryopreservation , AntioxidantsABSTRACT
The cold storage of milt implies potentials alterations in its quality because the storage generates as main process, free radicals that produce spermatozoa membrane lipids damage with the consequent motility and fertilising capacity disruptions. To decrease the damage generated by free radicals the cells have antioxidant defences (proteins, enzymes, and low molecular weight substances). The objective of the present study evaluated the time storage effect and different antioxidants prepared in spermatic diluents on sperm viability of O. mykiss milt stored at 4°C. The two-way ANOVA denoted that the time storage and antioxidant influence have significant effects separated or combined on viability parameters (sperm motility and viability, proteins concentrations and superoxide dismutase enzymatic activity in seminal plasma). In contrast, only the storage time affected the fertilising capacity and catalase enzymatic activity in seminal plasma. The resulting analysis can conclude that the antioxidant presence improves the viability of cold stored milt, especially the transport conditions and the antioxidants allow the fecundity despite motility decrease.
O armazenamento a frio de leite implica potenciais alterações em sua qualidade, pois gera como processo principal radicais livres que provocam danos aos lipídios da membrana dos espermatozoides, com as consequentes alterações na motilidade e na capacidade de fertilização. Para diminuir os danos causados pelos radicais livres, as células têm defesas antioxidantes (proteínas, enzimas e substâncias de baixo peso molecular). O presente estudo avaliou o efeito do tempo de armazenamento e diferentes antioxidantes preparados em diluentes espermáticos no armazenamento de viabilidade de O. mykiss milt a 4°C. A ANOVA de duas vias denotou que o armazenamento no tempo e a influência antioxidante têm efeitos significativos separados ou combinados nos parâmetros de viabilidade (motilidade espermática, viabilidade espermática, concentrações de proteínas e atividade enzimática da superóxido dismutase no plasma seminal), enquanto apenas o tempo de armazenamento afetou a capacidade de fertilização e atividade enzimática da catalase no plasma seminal. A análise resultante pode concluir que a presença de antioxidante melhora a viabilidade do leite frio, especialmente as condições de transporte, e os antioxidantes permitem a fecundidade apesar da diminuição da motilidade.
Subject(s)
Animals , Catalase/analysis , Cryopreservation/methods , Oncorhynchus mykiss , Semen/drug effects , Analysis of VarianceABSTRACT
Abstract The cold storage of milt implies potentials alterations in its quality because the storage generates as main process, free radicals that produce spermatozoa membrane lipids damage with the consequent motility and fertilising capacity disruptions. To decrease the damage generated by free radicals the cells have antioxidant defences (proteins, enzymes, and low molecular weight substances). The objective of the present study evaluated the time storage effect and different antioxidants prepared in spermatic diluents on sperm viability of O. mykiss milt stored at 4°C. The two-way ANOVA denoted that the time storage and antioxidant influence have significant effects separated or combined on viability parameters (sperm motility and viability, proteins concentrations and superoxide dismutase enzymatic activity in seminal plasma). In contrast, only the storage time affected the fertilising capacity and catalase enzymatic activity in seminal plasma. The resulting analysis can conclude that the antioxidant presence improves the viability of cold stored milt, especially the transport conditions and the antioxidants allow the fecundity despite motility decrease.
Resumo O armazenamento a frio de leite implica potenciais alterações em sua qualidade, pois gera como processo principal radicais livres que provocam danos aos lipídios da membrana dos espermatozoides, com as consequentes alterações na motilidade e na capacidade de fertilização. Para diminuir os danos causados pelos radicais livres, as células têm defesas antioxidantes (proteínas, enzimas e substâncias de baixo peso molecular). O presente estudo avaliou o efeito do tempo de armazenamento e diferentes antioxidantes preparados em diluentes espermáticos no armazenamento de viabilidade de O. mykiss milt a 4°C. A ANOVA de duas vias denotou que o armazenamento no tempo e a influência antioxidante têm efeitos significativos separados ou combinados nos parâmetros de viabilidade (motilidade espermática, viabilidade espermática, concentrações de proteínas e atividade enzimática da superóxido dismutase no plasma seminal), enquanto apenas o tempo de armazenamento afetou a capacidade de fertilização e atividade enzimática da catalase no plasma seminal. A análise resultante pode concluir que a presença de antioxidante melhora a viabilidade do leite frio, especialmente as condições de transporte, e os antioxidantes permitem a fecundidade apesar da diminuição da motilidade.
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Resumen: La interpretación gasométrica de forma rápida es muy útil en urgencias, ya que establecer de manera oportuna un diagnóstico es de suma importancia. Existen herramientas que nos permiten igualar la sensibilidad de los métodos complejos de Stewart. Evaluar la gasometría de manera tradicional, dependiendo de la escuela preferida: utilizando sólo el bicarbonato o el exceso de base; tiene baja sensibilidad diagnóstica cuando nos enfrentamos a escenarios donde existe más de un trastorno metabólico asociado. Proponemos los siguientes cinco pasos: evaluar el pH, déficit de base, anión gap, exceso de base y el índice cloro/sodio, que son importantes para identificar de manera rápida y sensible una gasometría en pacientes críticos.
Abstract: The interpretation of a gasometry of rapid form is very useful in an emergency department since to establish in an opportune way a diagnosis performs supreme importance. There exists hardware that allows us to equal the sensibility of the complex m ethods of Stewart. To evaluate the gasometry of a traditional way, depending on the favorite school: using only the bicarbonate or the base excess, it has low diagnostic sensibility when we face stages where more than one metabolic associate disorder exists. We propose the following five steps: to evaluate the pH, deficit of base, anion gap, excess of base, and the index chlorine/sodium, which are important to identify rapidly and sensitively a gasometry in critical patients.
Resumo: A interpretação gasométrica rápida é muito útil em emergências, pois é de suma importância estabelecer um diagnóstico em tempo hábil. Existem ferramentas que nos permitem igualar a sensibilidade dos métodos complexos de Stewart. Avaliar a gasometria da forma tradicional, dependendo da escola preferida: usando apenas bicarbonato ou excesso de base, tem baixa sensibilidade diagnóstica diante de cenários onde há mais de um distúrbio metabólico associado. Propomos as 5 etapas a seguir: avaliação do pH, déficit de base, lacuna aniônica, excesso de base e relação cloreto/sódio, importantes para identificar de forma rápida e sensível os gases sanguíneos em pacientes críticos.
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Wuzi-Yanzong-Wan (WZYZW) is a classic prescription for male infertility. Our previous investigation has demonstrated that it can inhibit sperm apoptosis via affecting mitochondria, but the underlying mechanisms are unclear. The purpose of the present study was to explore the actions of WZYZW on mitochondrial permeability transition pore (mPTP) in mouse spermatocyte cell line (GC-2 cells) opened by atractyloside (ATR). At first, WZYZW-medicated serum was prepared from rats following oral administration of WZYZW for 7 days. GC-2 cells were divided into control group, model group, positive group, as well as 5%, 10%, 15% WZYZW-medicated serum group. Cyclosporine A (CsA) was used as a positive control. 50 μmol·L-1 ATR was added after drugs incubation. Cell viability was assessed using CCK-8. Apoptosis was detected using flow cytometry and TUNEL method. The opening of mPTP and mitochondrial membrane potential (MMP) were detected by Calcein AM and JC-1 fluorescent probe respectively. The mRNA and protein levels of voltage-dependent anion channel 1 (VDAC1), cyclophilin D (CypD), adenine nucleotide translocator (ANT), cytochrome C (Cyt C), caspase 3, 9 were detected by RT-PCR (real time quantity PCR) and Western blotting respectively. The results demonstrated that mPTP of GC-2 cells was opened after 24 hours of ATR treatment, resulting in decreased MMP and increased apoptosis. Pre-protection with WZYZ-medicated serum and CsA inhibited the opening of mPTP of GC-2 cells induced by ATR associated with increased MMP and decreased apoptosis. Moreover, the results of RT-qPCR and WB suggested that WZYZW-medicated serum could significantly reduce the mRNA and protein levels of VDAC1 and CypD, Caspase-3, 9 and CytC, as well as a increased ratio of Bcl/Bax. However, ANT was not significantly affected. Therefore, these findings indicated that WZYZW inhibited mitochondrial mediated apoptosis by attenuating the opening of mPTP in GC-2 cells. WZYZW-medicated serum inhibited the expressions of VDAC1 and CypD and increased the expression of Bcl-2, which affected the opening of mPTP and exerted protective and anti-apoptotic effects on GC-2 cell induced by ATR.
Subject(s)
Animals , Male , Mice , Rats , 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine , Atractyloside/pharmacology , Peptidyl-Prolyl Isomerase F , Matrix Metalloproteinases , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Permeability Transition Pore , RNA, MessengerABSTRACT
Objective:To analyze the distribution of solute carrier organic anion transporter family member 1b1 ( SLCO1B1) and apolipoprotein E ( ApoE) genes in a population from southern Yunnan. Methods:The data of 104 patients who received treatment in Southern Central Hospital of Yunnan Province (The First People's Hospital of Honghe State) between May 2019 and June 2020 were collected. The distribution of SLCO1B1 and ApoE genes and their relationship with nationality, sex, and age were analyzed and compared between different regions. Results:The percentage of patients carrying *1a/*1a, *1a/*1b, *1b/*1b, *1a/*15, *1b/*15, five phenotypes of SLCO1B1 gene, in the population from southern Yunnan was 4.81%, 32.69%, 42.31%, 12.50% and 7.69% respectively. Phenotypes *1a/*5, *5/*5, *5/*15 and *15/*15 were not detected. Normal metabolic phenotype of SLCO1B1 accounted for 79.81%, and intermediate metabolic phenotype of SLCO1B1 accounted for 20.19%. Weak metabolic phenotype was not detected. The percentage of patients carrying E2/E2, E2/E3, E3/E3, E3/E4, E4/E4, five phenotypes of ApoE gene in the population from southern Yunnan was 0.96%, 16.35%, 70.19%, 11.54% and 0.96% respectively. E2/E4 phenotype was not detected. The percentage of patients with ApoE protective phenotype, ApoE normal phenotype, and ApoE risk phenotype was 17.31%, 70.19% and 12.50% respectively. The observed polymorphism mutation frequency of SLCO1B1 and ApoE genes was consistent with the Hardy-Weinberg equilibrium ( P > 0.05), suggesting constancy and a population representation. The Fisher test showed that SLCO1B1 gene distribution differed significantly between ethnic minorities and Han nationality in southern Yunnan ( P = 0.013). There was no significant difference in SLCO1B1 gene distribution between different sexes and between different ages (all P > 0.05). There was no significant difference in ApoE gene distribution between ethnic minorities and Han nationality, between different sexes, and between different ages in the population from southern Yunnan (all P > 0.05). Conclusion:SLCO1B1 gene distribution is related to nationality in the population from southern Yunnan, but it is unrelated to sex and age. ApoE gene distribution is unrelated to nationality, sex and age.
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Objective:To investigate the protective effect of intermittent fasting on radiation-induced cognitive impairment and the possible underlying mechanism.Methods:A total of 36 male 7-week old c57BL/6J mice were divided into Sham-irradiation and ad libitum (Sham-AL) group, irradiation and ad libitum (IR-AL) group, and irradiation add intermittent fasting (IR-IF) group according to the random number table method, with 12 mice in each group. The cognitive function of mice was assessed by novel object recognition task. The expressions of autophagy gene 5 (ATG5), microtubulesas sociated protein light chain II (LC3II), voltage dependent anion channel protein 1 (VDAC1), interleukin-1β (IL-1β), synaptophysin (SYP), synapsin I (SYN-1), and postsynaptic density 95 (PSD95) were tested by Western blot. The location of VDAC1 in mice hippocampus was detected by immunofluorescence.Results:The discrimination index (-22.45 ± 16.76) of IR-AL group was significantly ( t=3.032, P<0.05) lower than that of Sham-AL group (30.02 ± 9.05). Compared to Sham-AL group, IR-AL group had a decreased expressions of autophagy-related proteins (ATG5 and LC3II), mitochondrial marker (VDAC1), inflammatory factors (IL-1β) as well as synapse-associated proteins SYP, SYN-1 and PSD95 ( t=2.49, 2.19, 2.40, 3.47, 2.87, 2.25, 2.17, 2.31, P<0.05). Compared to IR-AL group, IR-IF group had an increased discrimination index (21.22 ± 5.62) and the increased expressions of ATG5, LC3II, VDAC1, IL-1β, SYP, SYN-1, and PSD95 ( t=2.70, 2.88, 2.71, 3.18, 3.18, 3.11, 3.30, 3.35, 2.53, P<0.05). The immunofluorescence assay revealed that VDAC1 was co-expressed with the markers of astrocytes (GFAP) and microglia (IBA-1), but not with neurons (NEUN). Conclusions:Intermittent fasting could greatly improve the cognitive function of irradiated mice possibly by upregulating VDAC1 expression, induce autophagy, and inhibit the release of inflammatory factors and protecting the synapticplasticity in the hippocampus.
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Ischemic-type biliary lesion (ITBL) refers to biliary tract injury caused by insufficient blood supply of hepatic artery, which is one of the main factors affecting the long-term survival and quality of life of liver transplant recipients. The incidence of ITBL is associated with cold and warm ischemia, acute and chronic rejection, cytomegalovirus infection and the bile effect, etc. The occurrence of ITBL is a complicated process involving with multiple factors and steps. The therapeutic option of ITBL is extremely limited. A large proportion of ITBL patients should undergo repeated liver transplantation. ITBL has become one of the most critical factors preventing further advancement of liver transplantation. Hence, it is of significance to strengthen prevention and explore more effective modalities. Recent studies have found that toxic injury of bile salts plays a central role in ITBL. Active regulation of bile components, regulation of bile acid-related receptor expression and blockage or activation of bile acid-related signaling pathways probably have potentials in the prevention and treatment of ITBL. In this article, the cytotoxicity of bile salts and the mechanism of bicarbonate umbrella in the incidence and progression of ITBL after liver transplantation were reviewed, aiming to provide reference for the diagnosis and treatment of ITBL.
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AIM: To explore the effects of inflammatory conditions on the pharmacokinetics of methotrexate (MTX) and its related mechanisms. METHODS: The model of adjuvant induced arthritis (AIA) was established. The expression of organic anion transporter 3 (OAT3) in kidney was detected by immunohistochemistry, Western blotting and QPCR. The plasma concentration of MTX was detected by LC-MS/MS, and the pharmacokinetics of MTX after different administration time were compared by isolated rat kidney perfusion, kidney slices, in vitro cell uptake and transport experiments. RESULTS: The expression of OAT3 was significantly increased in the kidneys of AIA rats by immunohistochemistry, Western blotting and QPCR. At the same time, the concentration of MTX was detected by the optimized LC-MS/MS. The results showed that the uptake of MTX in the kidney slices of AIA rats was significantly increased, and Pro could reduce the excretion of MTX by inhibiting OAT3. Furthermore, it was demonstrated in vitro that inflammatory pathology can promote renal excretion of MTX by increasing the expression and functional activity of OAT3.CONCLUSION: Under inflammatory pathological conditions, it can increase the expression of OAT3 in the kidney, enhance its functional activity, accelerate the uptake of MTX by the kidney, and promote the excretion of MTX.
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Glial cells have been implicated in temporal lobe epilepsy in humans and in its models. Astrocytes are lost in several brain regions after acute seizures induced by pilocarpine and may suffer hyperplasia at subsequent time points. This study investigated the effect of N-methyl-(2S,4R)-trans-4-hydroxy-L-proline (NMP) on astrocytes exposed to cytotoxic concentrations of pilocarpine. Astrocytes were incubated with pilocarpine (half maximal inhibitory concentration (IC50)=31.86 mM) for 24 h. Afterwards, they were treated with NMP at concentrations ranging from 3.12 to 100 μg/mL for 24 h. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cytoplasmic reactive oxygen species (ROS) and mitochondrial transmembrane potential (ΔΨm) were analyzed by flow cytometry using 2',7'-dichlorofluorescein diacetate (DCFH-DA) and rhodamine-123 (Rho123), respectively. Expression of glial fibrillary acidic protein (GFAP) and voltage-dependent anion channel-1 (VDAC-1) were measured by western blot. Pilocarpine significantly decreased cell viability and mitochondrial potential and increased ROS concentration significantly by 6.7 times compared to the control. NMP concentrations ≥25 µg/mL protected astrocytes against pilocarpine-induced injury in a concentration-dependent manner. Concomitantly, NMP reduced cytoplasmic ROS accumulation to 27.3, 24.8, and 12.3% in the groups treated with 25, 50, and 100 µg/mL NMP, respectively. NMP also protected mitochondria from pilocarpine-induced depolarization. These effects were associated with improvement of pilocarpine-induced GFAP and VDAC-1 overexpression, which are important biomarkers of astrocyte dysfunction. In conclusion, the improvement of ROS accumulation, VDAC-1 overexpression, and mitochondrial depolarization are possible mechanisms of the NMP protective action on reactive astrocytes.
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Objective To establish an ion chromatography method for the salt form determination of new psychoactive substances （NPS）. Methods The method of conducting qualitative and quantitative analysis of six types of organic acid ions （acetate ion, tartrate ion, maleate ion, oxalate ion, fumarate ion, citrate ion） and five types of inorganic anions （fluoride ion, chloride ion, nitrate ion, sulfate ion, phosphate ion） in NPS sample by ion chromatography was developed. The salt forms of 222 seized NPS samples （103 samples with synthetic cannabinoids, 81 samples with cathinones, 44 samples with phenylethylamines, 12 samples with tryptamines, 7 samples with phencyclidines, 6 samples with piperazines, 2 samples with aminoindenes, 26 samples with fentanyls and 43 samples with other types of NPS） were analyzed by this method. Results Each anion had good linearity in the corresponding linear range, the correlation coefficients （r） were greater than 0.999, the limits of detection were 0.01-0.05 mg/L, and the limits of quantitative were 0.1-0.5 mg/L. Except that 5F-BEPIRAPIM was hydrochloride, the salt forms of the other 102 synthetic cannabinoids were all base. The salt form of 81 cathinone samples, 44 phenylethylamine samples, 7 phencyclidine samples and 2 aminoindene samples were all hydrochloride. The salt forms of tryptamine samples included base, hydrochloride, fumarate and oxalate. The salt forms of piperazine samples included base and hydrochloride. The salt forms of fentanyl samples and samples of other types included base, hydrochloride and citrate. Conclusion Ion chromatography is a simple, accurate and efficient method for determining the salt form of NPS samples, which makes the qualitative and quantitative conclusions of NPS more scientific and rigorous.
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Chromatography, Liquid , Gas Chromatography-Mass Spectrometry , Ions , Psychotropic Drugs/chemistryABSTRACT
The paper aims to establish the method to determine the monosaccharide composition and monosaccharide ratio in propylene glycol alginate sodium sulphate (PSS). Samples were hydrolyzed with trifluoroacetic acid, neutralized with sodium hydroxide solution after the reaction conditions for sample pretreatment were optimized via orthogonal analysis. A high performance anion exchange chromatograghy (HPAEC) coupled with pulsed amperometric detector (PAD) was performed on a CarboPac®PA20, using 200 mmol·L-1 sodium hydroxide solution and 1 mol·L-1 sodium acetate solution as mobile phase. The established HPAEC-PAD method was validated by testing the linear relationship, precision and accuracy, and showed exclusive, sensitive, rapid and wide use. The monosaccharide composition of PSS from different manufacture can be accurately determined with great significance for the structural identification of PSS.