NR4A1 suppresses cisplatin-induced ferroptosis in renal proximal tubular epithelial cells by up-regulating the expression of NRF2 / 中华肾脏病杂志

Objective:To explore the role and mechanism of nuclear receptor subfamily 4 group A member 1 ( NR4A1) in suppressing cisplatin nephrotoxicity. Methods:The expression of NR4A1 gene in renal cell subpopulations was analyzed using the "Tabula-muris" single cell transcriptome sequencing database. NR4A1 gene was over-expressed by lentivirus infection in HK-2 cell line and primary renal proximal tubular epithelial cells. Cell counting kit-8 was used to detect the cytotoxicity of cisplatin. The cell death ratio was analyzed using propidium iodide (PI) staining by flow cytometry. The expression of NR4A1 and nuclear factor erythroid 2-related factor 2 ( NRF2) was detected by real-time fluorescent quantitative PCR and Western blotting. Ferroptosis was analyzed by detecting the contents of malondialdehyde (MDA), oxidized glutathione (GSSG) and lipid reactive oxygen species (ROS). Results:The single cell transcriptome sequencing database showed that NR4A1 gene was the lowest expression in renal proximal tubular epithelial cell subsets. Cisplatin (50 μmol/L or 100 μmol/L) could significantly induce MDA, GSSG and lipid ROS production in renal proximal tubular epithelial cells (all P<0.01), and higher cisplatin concentration accompanied with a more increase of MDA, GSSG and lipid ROS. Compared with the control HK-2 cells, the lipid ROS content and iron ion content of HK-2 cells over-expressing NR4A1 were significantly lower (all P<0.01), and the over-expression of NR4A1 inhibited cisplatin-induced cytotoxicity and ferroptosis in renal proximal tubular epithelial cells. Mechanistically, NR4A1 up-regulated the expression of anti-ferroptosis gene NRF2 in proximal renal tubular epithelial cells ( P<0.01). Furthermore, single cell data analysis showed that, similar to the expression of NR4A1 in renal tissue subsets, NRF2 was also the lowest in renal proximal tubular epithelial cells. Conclusions:Cisplatin can induce ferroptosis of renal proximal tubular epithelial cells in a dose-dependent manner. NR4A1 can inhibit cisplatin-induced ferroptosis by up-regulating NRF2 in renal proximal tubular epithelial cells, thereby alleviating the cytotoxicity of cisplatin.

One case of developmental epileptic encephalopathy caused by NR4A2 gene variation and literature review / 中华神经科杂志

Objective:To analyze the clinical characteristics of a child with developmental epileptic encephalopathy caused by NR4A2 gene mutation, and to summarize the clinical phenotypes and genotypes to improve the clinician′s understanding of this disease. Methods:The clinical data of a child with developmental epileptic encephalopathy admitted to Linyi People′s Hospital in August 2022 were collected, video electroencephalogram, craniocerebral magnetic resonance imaging and family whole exon sequencing were improved, and the suspected mutation sites were verified by Sanger sequencing. Relevant literature was consulted to summarize the clinical phenotypes and genetic characteristics of nervous system diseases caused by NR4A2 gene. Results:It was found that there was a heterozygous missense mutation at the locus c.866G>A (p.A289H) of NR4A2 gene in the child, which was a de novo mutation, and both parents were wild type. According to the American Society of Medical Genetics and Genomics variation classification, it was assessed as a suspected pathogenic variation. Through literature review, there were 16 related cases reported internationally, with clinical phenotypes including mental retardation/mental retardation, language disorders, seizures, muscle tone changes and different psychological and behavioral problems. Conclusions:The NR4A2 gene is not only associated with dopa responsive disorders, but also with neurological development, intellectual impairment, language development delay, and epilepsy. The mutation of NR42A gene c.866G>A (p.A289H) is the genetic cause of the patient, and the detection of this locus expands the NR4A2 gene spectrum. NR4A2 gene is one of the pathogenic genes of developmental epileptic encephalopathy.

Role of orphan nuclear receptor Nur77 in tunicamycin-induced injury to hippocampal neurons of mice: relationship with endoplasmic reticulum stress / 中华麻醉学杂志

Objective:To evaluate the role of orphan nuclear receptor Nur77 in tunicamycin(TM)-induced injury to hippocampal neurons and the relationship with endoplasmic reticulum stress in mice.Methods:Mouse HT22 cells were divided into 4 groups ( n=10 each) using a random number table method: control group (C group), Nur77 specific agonist Csn-B group (Csn-B group), endoplasmic reticulum stress inducer TM group (TM group), and TM+ Csn-B group. Cells in C group were cultured for 24 h under normal condition. In Csn-B group, Csn-B at a final concentration of 10 μg/ml was added to the culture medium, and the cells were incubated for 24 h. In TM group, TM at a final concentration of 200 ng/ml was added to the culture medium and the cells were incubated for 24 h to induce cell endoplasmic reticulum stress injury. Cells in TM+ Csn-B group were pretreated with Csn-B at a final concentration of 10 μg/ml for 15 min, then TM at a final concentration of 200 ng/ml was added, and the cells were co-incubated for 24 h. The cell viability was examined by CCK-8 assay kit after treatment in each group. The expression of endoplasmic reticulum stress-related protein CCAAT/enhancer-binding protein homologous protein (CHOP), glucose regulated protein 78 (GRP78)and apoptosis-associated protein Bcl-2, Bax, caspase-3 and cleaved-caspase-3 was detected by Western blot. Results:Compared with C group, the cell viability was significantly decreased, and the expression of CHOP, GRP78, Bax and cleaved-caspase-3 was up-regulated, and the expression of Bcl-2 and caspase-3 was down-regulated in TM group ( P<0.05 or 0.01). Compared with TM group, the cell viability was significantly increased, the expression of CHOP, GRP78, Bax and cleaved-caspase-3 was down-regulated, and the expression of Bcl-2 and caspase-3 was up-regulated in TM+ Csn-B group ( P<0.05 or 0.01). Conclusions:Orphan nuclear receptor Nur77 is involved in TM-induced injury to hippocampal neurons, which is related to activation of the endoplasmic reticulum stress in mice.

The F-box-only protein 44 regulates pregnane X receptor protein level by ubiquitination and degradation

Pregnane X receptor (PXR) is a ligand-activated nuclear receptor that transcriptionally upregulates drug-metabolizing enzymes [e.g., cytochrome P450 3A4 (CYP3A4)] and transporters. Although the regulation of PXR target genes is well-characterized, less is known about the regulation of PXR protein level. By screening an RNAi library, we identified the F-box-only protein 44 (FBXO44) as a novel E3 ligase for PXR. PXR abundance increases upon knockdown of FBXO44, and, inversely, decreases upon overexpression of FBXO44. Further analysis revealed that FBXO44 interacts with PXR, leading to its ubiquitination and proteasomal degradation, and we determined that the F-box associated domain of FBXO44 and the ligand binding domain of PXR are required for the functional interaction. In summary, FBXO44 regulates PXR protein abundance, which has downstream consequences for CYP3A4 levels and drug-drug interactions. The results of this study provide new insight into the molecular mechanisms that regulate PXR protein level and activity and suggest the importance of considering how modulating E3 ubiquitin ligase activities will affect PXR-mediated drug metabolism.

SIRT1 activation synergizes with FXR agonism in hepatoprotection via governing nucleocytoplasmic shuttling and degradation of FXR

Farnesoid X receptor (FXR) is widely accepted as a promising target for various liver diseases; however, panels of ligands in drug development show limited clinical benefits, without a clear mechanism. Here, we reveal that acetylation initiates and orchestrates FXR nucleocytoplasmic shuttling and then enhances degradation by the cytosolic E3 ligase CHIP under conditions of liver injury, which represents the major culprit that limits the clinical benefits of FXR agonists against liver diseases. Upon inflammatory and apoptotic stimulation, enhanced FXR acetylation at K217, closed to the nuclear location signal, blocks its recognition by importin KPNA3, thereby preventing its nuclear import. Concomitantly, reduced phosphorylation at T442 within the nuclear export signals promotes its recognition by exportin CRM1, and thereby facilitating FXR export to the cytosol. Acetylation governs nucleocytoplasmic shuttling of FXR, resulting in enhanced cytosolic retention of FXR that is amenable to degradation by CHIP. SIRT1 activators reduce FXR acetylation and prevent its cytosolic degradation. More importantly, SIRT1 activators synergize with FXR agonists in combating acute and chronic liver injuries. In conclusion, these findings innovate a promising strategy to develop therapeutics against liver diseases by combining SIRT1 activators and FXR agonists.

Development and optimization of a cell screening system for farnesoid X receptor agonist / 生物工程学报

This study aims to develop an improved cell screening system for farnesoid X receptor (FXR) agonists based on a dual luciferase reporter gene system. FXR response element (FXRE) fragments from FXR target genes were cloned and inserted into upstream of firefly luciferase (Luc) gene in the plasmid pGL4-luc2P-Hygro. In combination with the internal reference plasmid containing renilla luciferase, a dual luciferase reporter gene system was developed and used for high throughput screening of FXR agonists. After studying the effects of over-expression of RXR, mouse or human FXR, various FXRE fragments, and different ratio of FXR plasmid amount to reporter gene plasmid, induction efficiency of the screening system was optimized by the known FXR agonist GW4064, and Z factor for the system reached 0.83 under optimized conditions. In summary, an improved cell screening system based on double luciferase reporter gene detection system was developed to facilitate the discovery of FXR agonists, where a new enhanced FXRE element was formed by a superposition of multiple FXRE fragments from FXR target genes, instead of a superposition of traditional IR-1 (inverted repeats-1) fragments.

Three cases of congenital adrenal hypoplasia with hypogonadotropic hypogonadism due to novel NR0B1 mutation / 中华内分泌代谢杂志

Objective:To advance the understanding of X-linked adrenal hypoplasia congenita(XL-AHC)through genetic analysis.Methods:Genomic DNA was extracted from peripheral blood of three patients with XL-AHC and their family members as well. Pathogenic genes were screened with whole exome sequencing followed by Sanger sequencing and pedigree verification.Results:All three probands were diagnosed as primary adrenal insufficiency at early age and developed hypogonadotropic hypogonadism in adolescence. The proband 1 was hemizygous for c. 420delG(p.R141Gfs*123)mutation in exon 1 of NR0B1 gene. His mother was a heterozygous mutation carrier while his brother did not carry the mutation, which was consistent with the X-linked recessive inheritance. A hemizygous mutation c. 212_213delAA(p.K71Rfs*41)of NR0B1 gene was detected in both proband 2 and proband 3. These two novel mutations were not reported in HGMD database.Conclusions:In this study, two novel NR0B1 mutations, c. 420delG and c. 212_213delAA were identified in 3 patients with XL-AHC. For men with early onset of adrenocortical hypofunction, XL-AHC should be considered. Early genetic screening of NR0B1 gene is helpful for early diagnosis.

Role of hippocampal REV-ERBα in postoperative cognitive dysfunction in rats / 中华麻醉学杂志

Objective:To evaluate the role of hippocampal REV-ERBα in postoperative cognitive dysfunction in rats.Methods:Thirty-two SPF healthy male Sprague-Dawley rats, aged 12-14 weeks, weighing 360-380 g, were divided into 4 groups ( n=8 each) using a random number table method: control group (group C), surgery group (group S), surgery + dimethyl sulfoxide (DMSO) group (group SD), and surgery + SR9009 group (group SS). Exploratory laparotomy was performed under sevoflurane anesthesia in S, SD and SS groups.Normal saline containing 0.1% DMSO was injected into hippocampal CA1 area at 1 h before laparotomy, with 2 μl on each side in group SD, and REV-ERBα agonist SR9009 (in normal saline containing 0.1% DMSO) was injected into hippocampal CA1 area at 1 h before laparotomy, with 2 μl on each side in group S+ SR9009.Morris water maze test was performed at 1 and 3 days after operation.Rats were sacrificed at 1 h after the end of Morris water maze test on day 3 after surgery, and the hippocampal tissues were obtained for determination of the expression of REV-ERBα, Brain and Muscle ARNT-Like 1 (BMAL1) protein, synaptophysin (SYN), postsynaptic density (PSD)-95 protein and N-methyl-D-aspartate receptor 2B subunit (GRIN2B) (by Western blot) and microscopic examination of the morphology of hippocampal neurons and Nissl bodies (by Nissl staining), and the viable neurons were counted. Results:Compared with group C, the percentage of time of staying at the target quadrant was significantly decreased, and the number of crossing platform was reduced on days 1 and 3 after exploratory laparotomy, the expression of REV-ERBα, BMAL1, PSD95, SYN and GRIN2B was down-regulated, and the number of viable neurons was decreased in group S and group SD ( P<0.05). Compared with group S and group SD, the percentage of time of staying at the target quadrant and the number of crossing platform were significantly increased on days 1 and 3 after exploratory laparotomy, the expression of REV-ERBα and PSD95 was up-regulated, the number of viable neurons was increased ( P<0.05), and no significant change was found in the expression of BMAL1, SYN and GRIN2B in group SS ( P>0.05). There was no significant difference in the indexes mentioned above between group S and group SD ( P>0.05). Conclusions:Activation of REV-ERBα can improve postoperative cognitive dysfunction, and the mechanism may be related to up-regulation of PSD95 expression in hippocampus and reduction of neuronal damage in rats.

Relationship between Rev-erbα and NLRP3 inflammasome during myocardial ischemia-reperfusion in diabetic rats / 中华麻醉学杂志

Objective:To evaluate the relationship between nuclear receptor subfamily 1, group D, member 1 (Rev-erbα) and NOD-like receptor protein 3 (NLRP3) inflammasome during myocardial ischemia-reperfusion (I/R) in diabetic rats.Methods:SPF-grade healthy male Sprague-Dawley rats, weighing 210-240 g, in which 1% streptozotocin 60 mg/kg was intraperitoneally injected to develop the model of type 1 diabetes mellitus.Eighteen non-diabetic rats were divided into 2 groups by the random number table method: non-diabetic sham operation group (NS group, n=6) and non-diabetic myocardial I/R group (NIR group, n=12). Thirty diabetic rats were divided into 3 groups by the random number table method: diabetic sham operation group (DS group, n=6), diabetic myocardial I/R group (DIR group, n=12), and diabetic myocardial I/R + Rev-erbα inhibitor SR8278 group (DIR+ SR group, n=12). Myocardial I/R model was developed by ligation of left anterior descending coronary artery for 30 min followed by reperfusion for 120 min.In DIR+ SR group, SR8278 2 mg/kg was injected via the femoral vein at 1 h before ischemia.At the end of reperfusion, blood samples from the right carotid artery were collected for determination of serum creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH) and cardiac troponin I (cTnI) levels (by enzyme-linked immunosorbent assay). Then the rats were sacrificed, hearts were removed and myocardial tissues were obtained for determination of the percentage of myocardial infarct size (by TTC method) and expression of Rev-erbα, NLRP3 and IL-1β (by Western blot) and for microscopic examination of pathologic changes (by HE staining). Results:Compared with sham-operated rats, the serum concentrations of CK-MB, LDH and cTnI were significantly increased, the expression of Rev-erbα, NLRP3 and IL-1β in myocardial tissues was up-regulated ( P<0.05), and the pathological injury of myocardial tissues was obvious in myocardial I/R rats.Compared with NIR group, the percentage of myocardial infarct size and levels of serum CK-MB, LDH and cTnI were significantly increased, the expression of Rev-erb α, NLRP3 and IL-1β was up-regulated ( P<0.05), and the pathological injury of myocardial tissues was aggravated in DIR group.Compared with DIR group, the percentage of myocardial infarct size and serum CK-MB, LDH and cTnI levels were significantly decreased, the expression of Rev-erbα, NLRP3 and IL-1β was down-regulated ( P<0.05), and the pathological injury of myocardial tissues was reduced in DIR+ SR group. Conclusions:Rev-erbα can promote activation of NLRP3 inflammasome and is involved in the pathophysiological mechanism of myocardial I/R injury in diabetic rats.

Expression and clinical significance of lncRNA NR2F2-AS1 and miR-129-5p in glioma / 中国医师杂志

Objective:To detect the expression level of long non-coding RNA (lncRNA) nuclear receptor subfamily 2 group F member 2-antisense RNA 1 (NR2F2-AS1) and microRNA-129-5p (miR-129-5p) in glioma tissues and to explore its clinical significance.Methods:The glioma tissues of 103 patients with glioma who underwent surgical treatment in the neurosurgery department of Shanxi Provincial People′s Hospital from January 2015 to September 2016 and 50 normal brain tissues removed due to craniocerebral surgery in the same period were selected as the research objects. Real time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression levels of lncRNA NR2F2-AS1 and miR-129-5p in tissue samples. The correlation between the two indexes and the relationship between the expression changes of the two and clinicopathological parameters of glioma patients were analyzed. Kaplan Meier curve was used to analyze the 5-year cumulative survival rate of glioma patients with different expression levels of lncRNA NR2F2-AS1 and miR-129-5p. Cox regression analysis was used to analyze the factors affecting the poor prognosis of glioma patients.Results:Compared with normal brain tissue, the relative expression of lncRNA NR2F2-AS1 in glioma was higher and the relative expression of miR-129-5p was lower (all P<0.05). There was significant negative relationship between the relative expression of lncRNA NR2F2-AS1 and miR-129-5p in glioma ( r=-0.756, P<0.05). The expression of lncRNA NR2F2-AS1 and miR-129-5p in glioma was related to World Health Organization (WHO) grade and tumor length (all P<0.05). The 5-year cumulative survival rate of patients in lncRNA NR2F2-AS1<2.89 group was higher than that in lncRNA NR2F2-AS1≥2.89 group, and the 5-year cumulative survival rate of patients in miR-129-5p<0.55 group was lower than that in miR-129-5p≥0.55 group (all P<0.05). Multivariate analysis showed that high expression of lncRNA NR2F2-AS1, low expression of miR-129-5p, WHO grade Ⅲ-Ⅳ and tumor length were the risk factors affecting the prognosis of patients with glioma (all P<0.05). Conclusions:The increased expression of lncRNA NR2F2-AS1 and the decreased expression of miR-129-5p in glioma tissues are involved in the clinical biological progress of glioma and are closely related to the prognosis of patients.

The Research on Ferritinophagy, Ferroptosis and Related Diseases / 中国生物化学与分子生物学报

Iron, an important cofactor for heme, mitochondrial respiratory chain complexes, and various biologically important enzymes, participates in biological processes including oxygen transport, redox reactions, and metabolite synthesis. Ferritin is an iron storage protein that maintains iron homeostasis in the body by sequestering and releasing iron. Ferritinophagy is a selective type of autophagy that mediates ferritin degradation, releasing free iron when increased intracellular iron level is needed. Moderate rates of iron autophagy maintain intracellular iron content homeostasis. Excessive ferritinophagy will release a large amount of free iron, causing lipid peroxidation and cell damage via reactive oxygen species (ROS) produced by the Fenton reaction. Therefore, ferritinophagy plays a vital role in maintaining cellular iron homeostasis. Nuclear receptor co-activator 4 (NCOA4) acts as a key regulator of ferritinophagy by targeting ferritin binding and delivery to lysosomes for degradation, leading to release of free iron. Thus, NCOA4-mediated ferritinophagy is an important contributor to iron metabolism. Recent research reveals that NCOA4 is regulated by factors including iron content, autophagy, lysosomes, and hypoxia. NCOA4-mediated ferritin degradation is related to ferroptosis (an autophagic cell death process) . Ferritinophagy acts as an upstream mechanism driving ferroptosis by regulating cellular iron homeostasis and ROS production, which are closely correlated with the occurrence and development of anemia, neurodegenerative diseases, cancer, ischemia / reperfusion injury, and other diseases. In this study, the functional characteristics of NCOA4-mediated ferritinophagy in ferroptosis and the role of NCOA4 in these diseases were reviewed, which may provide new avenues for the treatment of related diseases.

Effects of Gelsemium elegans Combined with Mussaenda pubescens on Transporter BCRP and Drug-metabolizing Enzyme CYP3A11 / 中国实验方剂学杂志

ObjectiveTo explore the effect of Gelsemium elegans combined with Mussaenda pubescens on efflux transporter breast cancer resistance protein （BCRP） and cytochrome P450 3A11 （CYP3A11） and their attenuation mechanism， and to investigate whether the nuclear receptors were involved in such regulation by intervening it with nuclear receptor activators. MethodC57BL/6 mice were divided into the blank group， G. elegans （GE， 0.25 g·kg-1）group， GE + M. pubescens （MP） （0.25 g·kg-1+10 g·kg-1） group， GE + pregnane X receptor （PXR） activator （rifampicin）（GE + Rif，0.25 g·kg-1+50 mg·kg-1） group， GE + MP + Rif （0.25 g·kg-1+10 g·kg-1+50 mg·kg-1） group， GE + constitutive androstane receptor （CAR） activator （1，4-Bis ［2-（3，5-Dichloropyridyloxy）］ benzene， TCPOBOP）（GE + TCP， 0.25 g·kg-1+0.5 mg·kg-1） group， and GE + MP + TCP （0.25 g·kg-1+10 g·kg-1+0.5 mg·kg-1） group. The medication lasted for 14 successive days. One hour after the last administration， the mice were sacrificed by cervical dislocation and the liver tissue was harvested. The left liver tissue was stained with hematoxylin- eosin （HE） for observing the pathological changes. The right liver tissue was used for BCRP and CYP3A11 mRNA and protein expression detection by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot. ResultThe survival rates of mice in the GE + Rif group， GE group， and GE + MP group were 25% （the lowest）， 40%， and 80%， respectively， and no death was observed in the other groups. Compared with the obvious lesions in the liver cells of the GE group， the pathological changes in liver cells of the GE + MP group were alleviated， while those in the GE + Rif group were worsened. Compared with the GE + Rif group， the GE + MP + Rif group exhibited relieved pathological changes in liver cells. Both the GE + TCP group and the GE + MP + TCP group showed mild liver lesions. The comparison with the GE + MP group revealed that the pathological changes in the GE + MP + TCP group were slightly relieved. Compared with the blank group， the expression of BCRP protein and mRNA in GE group were significantly decreased （P<0.05，P<0.01）.The expression of CYP3A11 protein in GE group were significantly decreased （P<0.01）. Compared with the GE group， the GE + MP group displayed remarkably up-regulated BCRP protein and mRNA expression （P<0.05，P<0.01） and CYP3A11 protein expression （P<0.05）， but slightly up-regulated CYP3A11 mRNA expression. Compared with the GE group， the GE + Rif group exhibited down-regulated BCRP protein expression （P < 0.05）. The protein and mRNA expression levels of BCRP were lower in the GE + MP + Rif group than in the GE + MP group （P<0.05，P<0.01）. The PXR activator rifampicin regulated BCRP before and after the combination of G. elegans with M. pubescens. The CYP3A11 protein and mRNA expression levels in the GE + TCP group were higher than those in the GE group （P<0.05，P<0.01）. Compared with the GE + MP group， the GE + MP + TCP group showed up-regulated CYP3A11 protein and mRNA expression （P<0.05，P<0.01）. CAR activator TCPOBOP also had a regulatory effect on CYP3A11 before and after the compatibility of G. elegans with M. pubescens. ConclusionThe attenuated toxin after the combination of G. elegans with M. pubescens is closely related to the efflux transporter BCRP and the drug-metabolizing enzyme CYP3A11.

Crosstalk between CYP2E1 and PPARα substrates and agonists modulate adipose browning and obesity

Although the functions of metabolic enzymes and nuclear receptors in controlling physiological homeostasis have been established, their crosstalk in modulating metabolic disease has not been explored. Genetic ablation of the xenobiotic-metabolizing cytochrome P450 enzyme CYP2E1 in mice markedly induced adipose browning and increased energy expenditure to improve obesity. CYP2E1 deficiency activated the expression of hepatic peroxisome proliferator-activated receptor alpha (PPARα) target genes, including fibroblast growth factor (FGF) 21, that upon release from the liver, enhanced adipose browning and energy expenditure to decrease obesity. Nineteen metabolites were increased in Cyp2e1-null mice as revealed by global untargeted metabolomics, among which four compounds, lysophosphatidylcholine and three polyunsaturated fatty acids were found to be directly metabolized by CYP2E1 and to serve as PPARα agonists, thus explaining how CYP2E1 deficiency causes hepatic PPARα activation through increasing cellular levels of endogenous PPARα agonists. Translationally, a CYP2E1 inhibitor was found to activate the PPARα-FGF21-beige adipose axis and decrease obesity in wild-type mice, but not in liver-specific Ppara-null mice. The present results establish a metabolic crosstalk between PPARα and CYP2E1 that supports the potential for a novel anti-obesity strategy of activating adipose tissue browning by targeting the CYP2E1 to modulate endogenous metabolites beyond its canonical role in xenobiotic-metabolism.

MiR-20a Promotes Prostate Cancer Cell Proliferation By Targeting NR4A3 / 中国生物化学与分子生物学报

Prostate cancer is a common malignant tumor in male patients. It is of great clinical significance to explore the pathogenesis of prostate cancer and find suitable therapeutic targets. NR4A3 is derived from the nuclear hormone receptor superfamily of steroids, and NR4A3 plays an important role in the malignant progression of a variety of tumors. However, its role in prostate cancer has not yet been elucidated. Therefore, this project intends to investigate the role of NR4A3 in prostate cancer and screen for miRNAs that target NR4A3, which may help find potential target for the diagnosis and treatment of prostate cancer. The GEPIA website predicts that NR4A3 is under-expressed in prostate cancer tissues, and qRT-PCR data confirmed downregulation of NR4A3 in prostate cancer cells (P<0. 01). CCK8 and clone formation experiments show that overexpression of NR4A3 can significantly inhibit the viability, the number and size of colonies of prostate cancer cells (P < 0. 01). The bioinformatics website predicts that NR4A3 may be the target gene of miR-20a, and qRT-PCR showed that miR-20a expression was elevated in prostate cancer cells (P<0. 01). Furthermore, dual luciferase reporter gene experiment confirmed that miR-20a can target two sites of 3′-UTR of NR4A3 (P<0. 05, P<0. 001). Western blot results showed that miR-20a can inhibit the expression of NR4A3. CCK8 experiments further found that miR-20a inhibitor can significantly reduce the viability of prostate cancer cells(P<0. 05), while miR-20a mimic has the opposite effect (P<0. 05, P<0. 01). CCK8 and clone formation experiments showed that when co-transfected with miR-20a mimic and pcDNA3. 1-NR4A3 recombinant plasmids, up-regulation of NR4A3 could partially offset the viability, the number and size of colonies of PC3 cells promoted by miR-20a mimic (P <0. 05). In summary, miR-20a promotes the proliferation of prostate cancer cells by targeting NR4A3.

Effect of Acupuncture-rehabilitation Therapy on Neural Function and Expression of Forkhead Box P3 and Retinoic Acid-related Orphan Receptor γt in Mice with Cerebral Ischemia / 中国康复理论与实践

Objective To explore the effect of acupuncture-rehabilitation therapy on the expression of transcription factor forkheadbox P3 (Foxp3) and retinoic acid-related orphan receptor γt (RORγt) protein in cerebral ischemic mice. Methods Forty-five female C57BL/6 mice were randomly assigned to sham operation group, model group, acupuncture group, rehabilitation group, and acupuncture-rehabilitation group, with nine mice in each group. Subsequently, each group was divided into three days, seven days and 14 days subgroups. The permanent middle cerebral artery occlusion models were established by the suture method, except the sham operation group. The sham operation group and the model group received no treatment. The acupuncture group received scalp cluster acupuncture, the rehabilitation group received treadmill training, and the acupuncture-rehabilitation group received scalp cluster acupuncture combined with treadmill training. Three days, seven days and 14 days after modeling, the modified Neurological Severity Score (mNSS) was obtained, and the expression of Foxp3 and RORγt in brain tissue of ischemic side was analyzed by Western blotting. Results The mNSS in the sham operation group was 0, and was higher in the model group than in the sham operation group at each postoperative time point. Three days after operation, the mNSS decreased in the rehabilitation group and the acupuncture-rehabilitation group, compared to the model group (P < 0.05). Fourteen days after operation, the mNSS decreased in the acupuncture-rehabilitation group, compared to the model group and acupuncture group (P < 0.05). The expression of Foxp3 protein was significantly lower in the acupuncture-rehabilitation group than in other groups at all time points after surgery( P < 0.05). Three days after operation, the expression of RORγt was higher in the acupuncture-rehabilitation group than in other groups (P < 0.05). Seven days after operation, the expression of RORγt was higher in the acupuncture-rehabilitation group than in the acupuncture group and sham operation group (P < 0.05). Conclusion Acupuncture-rehabilitation therapy may improve the tissue injury of cerebral ischemia mice, and promote the recovery of neural function, possibly by regulating Foxp3 and RORγT expression to reduce the level of inflammation, and then exert neuroprotective effects.

Effect of Acupuncture-rehabilitation Therapy on Neural Function and Expression of Forkhead Box P3 and Retinoic Acid-related Orphan Receptor γt in Mice with Cerebral Ischemia / 中国康复理论与实践

Objective To explore the effect of acupuncture-rehabilitation therapy on the expression of transcription factor forkheadbox P3 (Foxp3) and retinoic acid-related orphan receptor γt (RORγt) protein in cerebral ischemic mice. Methods Forty-five female C57BL/6 mice were randomly assigned to sham operation group, model group, acupuncture group, rehabilitation group, and acupuncture-rehabilitation group, with nine mice in each group. Subsequently, each group was divided into three days, seven days and 14 days subgroups. The permanent middle cerebral artery occlusion models were established by the suture method, except the sham operation group. The sham operation group and the model group received no treatment. The acupuncture group received scalp cluster acupuncture, the rehabilitation group received treadmill training, and the acupuncture-rehabilitation group received scalp cluster acupuncture combined with treadmill training. Three days, seven days and 14 days after modeling, the modified Neurological Severity Score (mNSS) was obtained, and the expression of Foxp3 and RORγt in brain tissue of ischemic side was analyzed by Western blotting. Results The mNSS in the sham operation group was 0, and was higher in the model group than in the sham operation group at each postoperative time point. Three days after operation, the mNSS decreased in the rehabilitation group and the acupuncture-rehabilitation group, compared to the model group (P < 0.05). Fourteen days after operation, the mNSS decreased in the acupuncture-rehabilitation group, compared to the model group and acupuncture group (P < 0.05). The expression of Foxp3 protein was significantly lower in the acupuncture-rehabilitation group than in other groups at all time points after surgery( P < 0.05). Three days after operation, the expression of RORγt was higher in the acupuncture-rehabilitation group than in other groups (P < 0.05). Seven days after operation, the expression of RORγt was higher in the acupuncture-rehabilitation group than in the acupuncture group and sham operation group (P < 0.05). Conclusion Acupuncture-rehabilitation therapy may improve the tissue injury of cerebral ischemia mice, and promote the recovery of neural function, possibly by regulating Foxp3 and RORγT expression to reduce the level of inflammation, and then exert neuroprotective effects.

Computational study of the potential molecular target for antibreast cancer activity of limonoid derivatives from chisocheton sp

Limonoid is a class of natural compounds that are originated from lemon and other citrus fruits. However, derivativesof limonoids are also produced in other plants, such as Chisocheton sp. Limonoids from Chisocheton sp. showedvarious biological activities, including anticancer. Nevertheless, the molecular target for anticancer activity of thesecompounds is still unclear. Many studies suggested nuclear receptors (NR) as the protein target for limonoids. In thisstudy, we investigated the possible NR as a molecular target for limonoids from Chisocheton sp. using moleculardocking and molecular dynamics (MD) simulation. The docking study was done on AutoDock Vina. Two out of 11NR expressed in breast tissue, i.e., progesterone receptor (PR) and glucocorticoid receptor, was used as the mostpotential target for limonoids. The docking pose was further observed by MD simulation. Both receptors showedstable molecular interactions with limonoids, indicated with a low deviation of binding site residues. Interestingly,simulations of PR showed the alteration of Helix-12, which is one of the key factors to the antagonist action of theligand. It is hoped that the findings could shed insight into the further molecular assay development of anticanceragents based on limonoids.

Targeting castration-resistant prostate cancer with a novel ROR

Prostate cancer (PCa) patients who progress to metastatic castration-resistant PCa (mCRPC) mostly have poor outcomes due to the lack of effective therapies. Our recent study established the orphan nuclear receptor ROR

Early use of dexamethasone increases Nr4a1 in Kupffer cells ameliorating acute liver failure in mice in a glucocorticoid receptor-dependent manner / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

BACKGROUND AND OBJECTIVE@#Acute liver failure (ALF) is a type of disease with high mortality and rapid progression with no specific treatment methods currently available. Glucocorticoids exert beneficial clinical effects on therapy for ALF. However, the mechanism of this effect remains unclear and when to use glucocorticoids in patients with ALF is difficult to determine. The purpose of this study was to investigate the specific immunological mechanism of dexamethasone (Dex) on treatment of ALF induced by lipopolysaccharide (LPS)/D-galactosamine (D-GaIN) in mice.@*METHODS@#Male C57BL/6 mice were given LPS and D-GaIN by intraperitoneal injection to establish an animal model of ALF. Dex was administrated to these mice and its therapeutic effect was observed. Hematoxylin and eosin (H&E) staining was used to determine liver pathology. Multicolor flow cytometry, cytometric bead array (CBA) method, and next-generation sequencing were performed to detect changes of messenger RNA (mRNA) in immune cells, cytokines, and Kupffer cells, respectively.@*RESULTS@#A mouse model of ALF can be constructed successfully using LPS/D-GaIN, which causes a cytokine storm in early disease progression. Innate immune cells change markedly with progression of liver failure. Earlier use of Dex, at 0 h rather than 1 h, could significantly improve the progression of ALF induced by LPS/D-GaIN in mice. Numbers of innate immune cells, especially Kupffer cells and neutrophils, increased significantly in the Dex-treated group. In vivo experiments indicated that the therapeutic effect of Dex is exerted mainly via the glucocorticoid receptor (Gr). Sequencing of Kupffer cells revealed that Dex could increase mRNA transcription level of nuclear receptor subfamily 4 group A member 1 (Nr4a1), and that this effect disappeared after Gr inhibition.@*CONCLUSIONS@#In LPS/D-GaIN-induced ALF mice, early administration of Dex improved ALF by increasing the numbers of innate immune cells, especially Kupffer cells and neutrophils. Gr-dependent Nr4a1 upregulation in Kupffer cells may be an important ALF effect regulated by Dex in this process.

Early use of dexamethasone increases Nr4a1 in Kupffer cells ameliorating acute liver failure in mice in a glucocorticoid receptor-dependent manner / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Background and objective: Acute liver failure (ALF) is a type of disease with high mortality and rapid progression with no specific treatment methods currently available. Glucocorticoids exert beneficial clinical effects on therapy for ALF. However, the mechanism of this effect remains unclear and when to use glucocorticoids in patients with ALF is difficult to determine. The purpose of this study was to investigate the specific immunological mechanism of dexamethasone (Dex) on treatment of ALF induced by lipopolysaccharide (LPS)/d-galactosamine (d-GaIN) in mice. Methods: Male C57BL/6 mice were given LPS and d-GaIN by intraperitoneal injection to establish an animal model of ALF. Dex was administrated to these mice and its therapeutic effect was observed. Hematoxylin and eosin (H&E) staining was used to determine liver pathology. Multicolor flow cytometry, cytometric bead array (CBA) method, and next-generation sequencing were performed to detect changes of messenger RNA (mRNA) in immune cells, cytokines, and Kupffer cells, respectively. Results: A mouse model of ALF can be constructed successfully using LPS/d-GaIN, which causes a cytokine storm in early disease progression. Innate immune cells change markedly with progression of liver failure. Earlier use of Dex, at 0 h rather than 1 h, could significantly improve the progression of ALF induced by LPS/d-GaIN in mice. Numbers of innate immune cells, especially Kupffer cells and neutrophils, increased significantly in the Dex-treated group. In vivo experiments indicated that the therapeutic effect of Dex is exerted mainly via the glucocorticoid receptor (Gr). Sequencing of Kupffer cells revealed that Dex could increase mRNA transcription level of nuclear receptor subfamily 4 group A member 1 (Nr4a1), and that this effect disappeared after Gr inhibition. Conclusions: In LPS/d-GaIN-induced ALF mice, early administration of Dex improved ALF by increasing the numbers of innate immune cells, especially Kupffer cells and neutrophils. Gr-dependent Nr4a1 upregulation in Kupffer cells may be an important ALF effect regulated by Dex in this process.