Diallyl disulfide augments sensitivity of DJ-1 overexpressed human gastric cells to 5-FU / 中国药理学通报

Aim To investigate whether diallyl disul-fide (DADS) augments the sensitivity of DJ-1 (protein/ nucleic acid deglycase) overexpressed human gastric SGC7901 cells to 5-FU (5-fluorouracil). Methods The experimental groups include control group, DADS group, VCR (vincristine) group, VCR + DADS group, DJ-1 group, DJ-1 + DADS group. MTT was used to analyze the effect of DADS on 5 -FU (5 -fluorou- racil) induced proliferation inhibition. Flow cytometry was performed to examine the effect of DADS on cell apoptosis. RT-PCR, Western blot, and immunofluo-rescence were used for determine the effect of DADS on the drug resistance associated gene expression. Results DADS enhanced the proliferation inhibitory effect of 5-FU on DJ-1 overexpressed cells and VCR resistant cells. DADS could induce apoptosis in VCR-resistant cells. DADS downregulated the expression of DJ-1 while inducing apoptosis in DJ-1 overexpressed cells. DJ-1 overexpression upregulated the expression of P-gp (P-glycoprotein), Bcl-2, and XIAP (X-linked inhibitor of apoptosis protein), downregulated the expression of caspase-3. DADS decreased the expression of P-gp, Bcl-2, and XIAP, while increased the expression of caspase-3 in DJ-1 overexpressed cells and VCR-resistant cells. Conclusions DADS can augment the sensitivity of DJ-1 overexpressed cells to 5-FU, which is related to its antagonism against DJ-1 mediated upregula- tion of P-gp, Bcl-2, XIAP, and downregulation of caspase-3.

DJ-1 over-expression promotes proliferation, migration, invasion and epithelial-mesenchymal transformation of human gastric cancer MGC803 cells through PTEN/Akt pathway / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨DJ-1基因过表达对人胃癌MGC803细胞增殖、迁移、侵袭与上皮间质转化（EMT）的影响及其机制。方法：利用基因转染技术构建DJ-1基因过表达MGC803细胞，实验分为MGC803、空载体和DJ-1过表达组。采用MTT、平板克隆形成、细胞划痕和Transwell实验分别检测DJ-1过表达对MGC803细胞增殖、克隆形成、迁移与侵袭的影响；qPCR和WB法检测DJ-1过表达对各组细胞DJ-1、PTEN、Akt、p-Akt、Snail、vimentin、E-cadherin、MMP-9与TIMP-3表达的影响，相差显微镜下观察MGC803细胞形态学的变化。裸鼠荷瘤实验检测DJ-1过表达对MGC803细胞移植瘤体内生长的影响。结果：成功构建DJ-1基因稳定过表达的MGC803细胞。与MGC803组和空载体组比较，DJ-1过表达组细胞的增殖能力与克隆形成数均显著增加（均P<0.05），细胞迁移距离明显增加、划痕距离明显缩短（均P<0.05），迁移与侵袭细胞数显著增多（均P<0.05），DJ-1 mRNA与蛋白表达明显上调、PTEN mRNA与蛋白表达下调（均P<0.05），Akt总蛋白各组比较无明显差异（均P>0.05），p-Akt蛋白表达明显上调（P<0.05），Snail、vimentin与MMP-9表达上调、E-cadherin与TIMP-3表达下调（均P<0.05）。相差显微镜下见长梭形细胞数目增多，圆形与椭圆形细胞减少，异型性更为明显。荷瘤裸鼠体内实验结果表明，与MGC803组相比较，DJ-1过表达组MGC803细胞移植瘤生长速度明显加快、移植瘤质量显著增加（均P<0.05）。结论：DJ-1过表达可通过PTEN/Akt通路在体内外抑制MGC803细胞的增殖、迁移、侵袭与EMT。

DJ-1 mediates resveratrol to attenuate myocardial ischemia/reperfusion injury in rats by protecting activity of mitochondrial complex I / 中国病理生理杂志

AIM:From the perspective of regulating mitochondrial complex I activity by DJ-1 protein,this study aims to explore the mechanism of DJ-1-mediated resveratrol(RES)preconditioning in protecting against oxidative stress injury induced by myocardial ischemia-reperfusion(I/R)in rats.METHODS:After intramyocardial injection of lentivirus carrying DJ-1 shRNA(sh-DJ-1)or negative control(NC)shRNA,the myocardial I/R model was constructed by ligating the left anterior descending branch of the rat coronary artery.Sprague-Dawley(SD)rats were randomly divided in-to 6 groups:sham group,I/R group,RES+I/R group,NC+RES+I/R group,sh-DJ-1+RES+I/R group,and IACS-010759(mitochondrial complex I inhibitor)+RES+I/R group,with 10 rats in each group.The rats in RES treatment groups were given RES(20 mg/kg)via gavage for 7 d prior to the myocardial I/R modeling,once daily.Moreover,the rats in sham and I/R groups received an equivalent volume of normal saline via gavage.Myocardial infarction area and cardiac function were assessed by TTC staining and echocardiography,respectively.The MitoSOX fluorescent probe was used to detect levels of mitochondrial reactive oxygen species(ROS)in the myocardium.The levels of malondialdehyde(MDA),superoxide dis-mutase(SOD)and lactate dehydrogenase(LDH)in the serum were detected using kits.Western blot and co-immunopre-cipitation assays were used to observe the interaction between DJ-1 and the two subunits,ND-1 and NDUFA4,of the mito-chondrial complex I.RESULTS:Compared with I/R group,RES pretreatment significantly reduced the myocardial in-farction area,mitochondrial ROS levels,serum LDH activity,and serum MDA content(P＜0.01).It also elevated left ventricular ejection fraction,left ventricular fractional shortening and serum SOD activity(P＜0.01).Pretreatment with RES increased the expression and mitochondrial translocation of DJ-1(P＜0.01),promoted the interaction between DJ-1 and ND-1/NDUFA4,which in turn protected the activity of mitochondrial complex I(P＜0.01).However,when the ex-pression of DJ-1 was suppressed,the protective effects of RES against myocardial I/R injury were significantly inhibited compared with RES+I/R group(P＜0.05 or P＜0.01).CONCLUSION:Pretreatment with RES increases the expression and mitochondrial translocation of DJ-1,and facilitates the interaction of DJ-1 with ND1 and NDUFA4 subunits of mito-chondrial complex I,thus preserving the activity of mitochondrial complex I and attenuating myocardial I/R-induced oxida-tive stress damage.

Recent advances of DJ-1 regulating mitochondrial function / 中国药理学通报

Dj-l is a protein encoded by PARK7 gene, a member of the peptidase C56 protein family. Defects in PARK7 gene may lead to autosomal recessive early-onset Parkinson ' s disease. Dj-1 is a multifunctional protein that acts as an active androgen receptor-mediated transcriptional regulator, a REDOX sensitive molecular chaperone, an oxidative stress sensor, and it also protects neurons from oxidative stress and cell death. In addition, DJ-1 is also associated with mitochondria, energy metabolism, mitochondrial homeostasis, mitophagy mitochondria-associated endoplasmic reticulum membranes and other life processes. However, the precise function of DJ-1 protein is not well understood. This paper reviews the effect, mechanism and molecular basis of DJ-1 protein in regulating mitochondrial function, and discusses its potential value in combination with clinical diseases. It has good timeliness, necessity, innovation and science, and also helps to provide new targets and ideas for clinical drug development.

DJ1 Ameliorates AD-like Pathology in the Hippocampus of APP/PS1 Mice / 生物医学与环境科学（英文）

OBJECTIVE@#To explore whether the protein Deglycase protein 1 (DJ1) can ameliorate Alzheimer's disease (AD)-like pathology in Amyloid Precursor Protein/Presenilin 1 (APP/PS1) double transgenic mice and its possible mechanism to provide a theoretical basis for exploring the pathogenesis of AD.@*METHODS@#Adeno-associated viral vectors (AAV) of DJ1-overexpression or DJ1-knockdown were injected into the hippocampus of 7-month-old APP/PS1 mice to construct models of overexpression or knockdown. Mice were divided into the AD model control group (MC), AAV vector control group (NC), DJ1-overexpression group (DJ1 +), and DJ1-knockdown group (DJ1 -). After 21 days, the Morris water maze test, immunohistochemistry, immunofluorescence, and western blotting were used to evaluate the effects of DJ1 on mice.@*RESULTS@#DJ1 + overexpression decreased the latency and increased the number of platform traversals in the water maze test. DJ1 - cells were cured and atrophied, and the intercellular structure was relaxed; the number of age spots and the expression of AD-related proteins were significantly increased. DJ1 + increased the protein expression of Nuclear factor erythroid 2-related factor 2 (NRF2), heme oxygenase-1 (HO-1), light chain 3 (LC3), phosphorylated AMPK (p-AMPK), and B cell lymphoma-2 (BCL-2), as well as the antioxidant levels of total superoxide dismutase (T-SOD), total antioxidant capacity (T-AOC), and Glutathione peroxidase (GSH-PX), while decreasing the levels of Kelch-like hydrates-associated protein 1 (Keap1), mammalian target of rapamycin (mTOR), p62/sequestosome1 (p62/SQSTM1), Caspase3, and malondialdehyde (MDA).@*CONCLUSION@#DJ1-overexpression can ameliorate learning, memory, and AD-like pathology in APP/PS1 mice, which may be related to the activation of the NRF2/HO-1 and AMPK/mTOR pathways by DJ1.

Study on the value of color Doppler ultrasound combined with serum miR-128-3p and DJ-1 in diagnosis and clinical staging of bladder cancer / 中国医学装备

Objective:To investigate the value of color Doppler ultrasound combined with serum miR-128-3p and DJ-1 in the diagnosis and clinical staging of bladder cancer.Methods:A total of 136 patients with suspected bladder cancer were selected and they were divided into bladder cancer group(56 cases)and benign lesion group(80 cases)according to the results of pathological examination.All patients underwent color Doppler ultrasound examination,and the levels of serum miR-128-3p and DJ-1 of them were measured.The receiver operating characteristic(ROC)curve was applied to analyze the diagnostic efficacy of color Doppler ultrasound examination combined with serum miR-128-3p and DJ-1 for bladder cancer and the clinical staging of that.Results:Compared with pathological results as the"gold standard",the sensitivities of color Doppler ultrasound in diagnosing bladder cancer and the clinical staging of that were respectively 75.00%and 80.49%,and the specificities of that for them were respectively 86.25%and 80.00%,and the accuracies of that for them were respectively 81.62%and 80.36%.Compared with benign lesion group,the serum miR-128-3p level decreased and DJ-1 level increased in bladder cancer group,the differences were statistically significant(t=8.842,t=8.844,P<0.05),respectively.In the bladder cancer group,the serum miR-128-3p level of infiltration type patients was lower than that of superficial type patients,and the serum DJ-1 level of them was higher than that of superficial type patients,and the differences were statistical significant(t=5.722,t=6.327,P<0.05),respectively.ROC curve results showed that the area under curve(AUC),sensitivity and specificity of color Doppler ultrasound combined with serum miR-128-3p and DJ-1 were respectively higher than those of single examination indicator in diagnosing bladder cancer and the clinical staging of that,and the differences were statistical significant(Z=4.028,Z=2.535,Z=3.386,Z=2.131,Z=2.137,Z=2.063,P<0.05).Conclusion:Color Doppler ultrasound combined with serum miR-128-3p and DJ-1 can improve the diagnostic efficacy for bladder cancer and the clinical staging of that,which has important clinical significance in diagnosing bladder cancer and judging the clinical staging of that.

Co-editing PINK1 and DJ-1 Genes Via Adeno-Associated Virus-Delivered CRISPR/Cas9 System in Adult Monkey Brain Elicits Classical Parkinsonian Phenotype / 神经科学通报·英文版

Whether direct manipulation of Parkinson's disease (PD) risk genes in the adult monkey brain can elicit a Parkinsonian phenotype remains an unsolved issue. Here, we used an adeno-associated virus serotype 9 (AAV9)-delivered CRISPR/Cas9 system to directly co-edit PINK1 and DJ-1 genes in the substantia nigras (SNs) of two monkey groups: an old group and a middle-aged group. After the operation, the old group exhibited all the classic PD symptoms, including bradykinesia, tremor, and postural instability, accompanied by key pathological hallmarks of PD, such as severe nigral dopaminergic neuron loss (>64%) and evident α-synuclein pathology in the gene-edited SN. In contrast, the phenotype of their middle-aged counterparts, which also showed clear PD symptoms and pathological hallmarks, were less severe. In addition to the higher final total PD scores and more severe pathological changes, the old group were also more susceptible to gene editing by showing a faster process of PD progression. These results suggested that both genetic and aging factors played important roles in the development of PD in the monkeys. Taken together, this system can effectively develop a large number of genetically-edited PD monkeys in a short time (6-10 months), and thus provides a practical transgenic monkey model for future PD studies.

Co-editing PINK1 and DJ-1 Genes Via Adeno-Associated Virus-Delivered CRISPR/Cas9 System in Adult Monkey Brain Elicits Classical Parkinsonian Phenotype / 神经科学通报·英文版

Whether direct manipulation of Parkinson’s disease (PD) risk genes in the adult monkey brain can elicit a Parkinsonian phenotype remains an unsolved issue. Here, we used an adeno-associated virus serotype 9 (AAV9)-delivered CRISPR/Cas9 system to directly co-edit PINK1 and DJ-1 genes in the substantia nigras (SNs) of two monkey groups: an old group and a middle-aged group. After the operation, the old group exhibited all the classic PD symptoms, including bradykinesia, tremor, and postural instability, accompanied by key pathological hallmarks of PD, such as severe nigral dopaminergic neuron loss (>64%) and evident α-synuclein pathology in the gene-edited SN. In contrast, the phenotype of their middle-aged counterparts, which also showed clear PD symptoms and pathological hallmarks, were less severe. In addition to the higher final total PD scores and more severe pathological changes, the old group were also more susceptible to gene editing by showing a faster process of PD progression. These results suggested that both genetic and aging factors played important roles in the development of PD in the monkeys. Taken together, this system can effectively develop a large number of genetically-edited PD monkeys in a short time (6–10 months), and thus provides a practical transgenic monkey model for future PD studies.

DJ-1 interacting proteins analyzed by tandem affinity purification mass spectrometry / 中国医师杂志

Objective:RNA interference technology (siRNA) was used to inhibit the expression of DJ-1 gene in lung squamous cell carcinoma HTB-182 cells, then, tandem affinity purification mass spectrometry (TAP-MS) was performed to screen the interacting proteins of DJ-1 in lung cancer cell line of HTB-182.Methods:The siRNA lentivirus vector targeting DJ-1 gene was constructed to infect HTB-182 cells (DJ-1 siRNA group), and the lentivirus vector control group (control siRNA group) and blank control group were established. The expression level of DJ-1 protein was detected by Western blot, and the endogenous DJ-1 protein silenced si-DJ-1-HTB-182 cells were established. The specific primers of DJ-1 were designed, and the DJ-1 expression plasmid pNTAP-DJ-1 with streptomycin binding peptide label (SBP) and calmodulin binding peptide label (CBP) was constructed. The cell line DJ-1 siRNA HTB-182 was stably transfected with liposome, and the positive clones were screened by G418. The positive clones were verified by Western blot, and the interacting proteins of DJ-1 were found by TAP-MS.Results:The protein expression of DJ-1 in DJ-1 siRNA interference group was significantly lower than that in empty plasmid group and blank control group ( P<0.05); HTB182 cell line stably expressing pNTAP-DJ-1 plasmid was successfully constructed; Three proteins interacting with DJ-1 were screened by TAP-MS: cytokeratin 1 (keratin 1), cytokeratin 10 (keratin 10) and NADPH oxidase activating protein P47 (P47 Px). Conclusions:Keratin 1, Keratin l0 and P47 Px protein may be DJ-1 interactions protein.

The effects mutant DJ-1 and sumoylation of DJ-1 protein on DJ-1 mitochondrial localization / 中风与神经疾病杂志

@#Objective To investigate the effects of DJ-1 A39S mutation in Parkinson’s disease（PD）and SUMO modification of DJ-1 on DJ-1 mitochondrial localization. Methods Five COS-7 cell lines that can stably express wild type，A39S mutant，K130R mutant，A39S K130R double mutant DJ-1 protein and SUMO-1 protein were constructed. The transfection results were detected by Western blot，double enzyme digestion and PCR sequencing. The expression and subcellular localization of DJ-1 protein were observed by Western blotting，confocal microscope and immunofluorescence.Results Immunofluorescence showed that DJ-1 protein was mainly distributed in the cytoplasm and also in the nucleus.Some wild-type and K130R DJ-1 proteins were distributed in mitochondria，most of A39S and A39S-K130R mutant DJ-1 proteins were transferred to mitochondria，and SUMO-1 was mainly distributed in the nucleus and around the nucleus.Immunofluorescence co localization showed that four DJ-1 proteins were colocalize with SUMO-1 protein. Conclusion The pathogenic mutation A39S of DJ-1 transferred DJ-1 protein from cytoplasm to mitochondria；DJ-1 and SUMO-1 have colocalization，and they may interact.mitochondrial localization.Methods Five COS-7 cell lines that can stably express wild type，A39S mutant，K130R mutant，A39S K130R double mutant DJ-1 protein and SUMO-1 protein were constructed. The transfection results were detected by Western blot，double enzyme digestion and PCR sequencing.The expression and subcellular localization of DJ-1 protein were observed by Western blotting，confocal microscope and immunofluorescence.Results Immunofluorescence showed that DJ-1 protein was mainly distributed in the cytoplasm and also in the nucleus.Some wild type and K130R DJ-1 proteins were distributed in mitochondria，most of A39S and A39S-K130R mutant DJ-1 proteins were transferred to mitochondria，and SUMO-1 was mainly distributed in the nucleus and around the nucleus.Immunofluorescence colocalization showed that four DJ-1 proteins were colocalize with SUMO-1 protein.Conclusion The pathogenic mutation A39S of DJ-1 transferred DJ-1 protein from cytoplasm to mitochondria；DJ-1 and SUMO-1 have colocalization，and they may interact.

A comparative study of rapid eye movement sleep behavior disorders on Parkinson’s disease and multiple system atrophy Parkinsonism type / 中风与神经疾病杂志

@#Objective To compare the differences in incidence of rapid eye movement sleep behavior disorder（RBD）on Parkinson’s disease（PD） and multiple system atrophy parkinsonism type（MSAP），and to investigate the correlation factors of the RBD screening questionnaire （RBDSQ） scores.Methods Thirty-four PD patients and 30 MSAP patients without dementia within 5 years of onset were included. Baseline data were collected in detail，and UPDRS Ⅲ score，HY staging score and RBDSQ score were performed between the two groups to compare the difference in the incidence of RBD between the two groups，as well as the correlation factors of RBDSQ score.Results The incidence of RBD in prodrome MSA （40.0%） was significantly higher than that in prodrome PD （2.9%，χ2=11.330，P=0.001）.The incidence of RBD in the clinical stage of MSAP（63.3%）was significantly higher than that in the clinical stage of PD（29.4%，χ2=7.401，P=0.007）.The RBDSQ score of MSAP patients was significantly higher than that of PD patients （6.6±3.9 vs.4.7±3.4，Z=-2.084，P=0.037）.There was no clear correlation between RBDSQ scores in PD group or MSAP group and disease course，UPDRS Ⅲ score，MMSE score and disease progression rate. Conclusion Compared with PD group，the incidence of RBD in prodrome and clinical MSAP group was significantly higher than that in PD group. In MSAP patients and PD patients with normal cognitive function，there was no clear correlation between RBDSQ score and UPDRS Ⅲ score and MMSE score.

DJ-1 promotes proliferation and epithelial-mesenchymal transition of colorectal cancer cells by regulating p53 signaling pathway / 肿瘤

Objective: To investigate the effects of up-regulating or silencing DJ-1 gene expression on the apoptosis, migration and invasion of colorectal cancer (CRC), and to explore the possible molecular mechanism. Methods: The expression level of DJ-1 in CRC tissues and cells was detected by immunohistochemistry, Western blotting and real-time fluorescent quantitative PCR, respectively. The SW480 and HCT116 cells were transfected with the recombinant lentiviral vector carrying human DJ-1 gene to obtain DJ-1 overexpressed SW480/OE-DJ-1 and HCT116/OE-DJ-1 cells, while the cells transfected with the empty vector was as the negative control (OE-NC). The SW480 and HCT116 cells were transfected with the recombinant lentiviral vector carrying the specific shRNA targeting DJ-1 gene to generate the SW480/shDJ-1 and HCT116/sh-DJ-1 cells with stable knockdown of DJ-1, while the cells transfected with the empty vector was as the negative control (sh-NC). Subsequently, the expressions of DJ-1 and p53 protein and mRNA were detected by immunohistochemistry and real-time fluorescent quantitative PCR, respectively; and their relationship was analyzed. The expressions of p53 and its downstream apoptosis-related proteins Bax and Bcl-2 in SW480 and HCT116 cells with DJ-1 over-expression or knockdown were detected by Western blotting. The effects of overexpressing and silencing DJ-1 gene expression on the invasion and migration abilities of SW480 and HCT116 cells were detected by Transwell chamber assay. The epithelial-mesenchymal transition (EMT) of CRC cells was induced by transforming growth factor-β1 (TGF-β1), then the expression levels of DJ-1 and EMT-related markers (N-cadherin, β-catenin, vimentin, E-cadherin) were analyzed by Western blotting. Results: DJ-1 was highly expressed in 34 CRC tissues (24/34, 70.59%) (P < 0.001). The overall survival time of the patients with DJ-1 high expression was significantly shorter than that of the patients with DJ-1 low expression (P < 0.001). The high expression of DJ-1 was correlated with TNM stage, tumor location, lymph node metastasis, and degree of differentiation (all P < 0.05). There was a negative correlation between DJ-1 and p53 expressions (r =-0.428, P = 0.015). Silencing DJ-1 increased the expression level of p53 and its downstream apoptotic protein Bax, decreased the expression of anti-apoptotic protein Bcl-2 (all P < 0.05), and decreased the invasion and migration capacities of SW480 and HCT116 cells (both P < 0.01); Conversely, overexpressing DJ-1 decreased the expression level of p53 and Bax, increased the expression of Bcl-2 (all P < 0.05), and increased the invasion and migration capacities of SW480 and HCT116 cells (both P < 0.01). Overexpression of DJ-1 induced by TGF-β1 increased the expressions of N-cadherin, β-catenin and vimentin, and decreased the expression of E-cadherin in the process of EMT (P < 0.05). Conclusion: DJ-1 promotes the apoptosis and invasion of CRC cells by negatively regulating the p53 signaling pathway.

Salivary α-synuclein and DJ-1 for the value of the diagnosis of Parkinson disease / 中国神经精神疾病杂志

Objective To investigate the diagnostic value of quantitative detection of α-synuclein and DJ-1 protein in saliva for Parkinson Disease. Methods Twenty seven patients diagnosed with primary Parkinson's disease and 27 healthy controls were studied.The clinical data of all subjects were collected.Each participant received a disease evaluation including Hohn-Yahr stage, unified Parkinson Disease Rating Scale (UPDRS)-Ⅱ / Ⅲ, 12 Item odor identification test from Sniffin'Sticks (SS-12), Montreal cognitive assessment (MoCA), Mini Mental State Examination (MMSE).α-syn and DJ-1 protein in saliva were examined by using enzyme linked immunosorbent assay (ELISA). The statistical analysis was used to test the difference in these two protein levels between patient and control groups and the correlation with age, gender and course of disease. Results There were significant changes in mean concentration of salivary α-syn (1269.02±16.09 pg/mL、1350.51±25.79 pg/mL,P=0.010) and DJ-1 protein (6.07±3.23 ng/mL、8.43±4.33 ng/mL,P=0.027) between patient and control groups. The sensitivity of α-syn and DJ-1 protein levels in PD diagnosis was 55.56% and 77.8%,and the specificity was 89.19% and 55.6%.The area under the ROC curve in finding the PD of α-syn and DJ-1 was 0.671 and 0.649, respectively. In Parkinson disease group, age, gender, UPDRS-Ⅱ / Ⅲ, Hohn-Yahr stage, SS-12, MMSE and MoCA of Parkinson's disease group were not related to the concentration of α-syn and DJ-1 protein in saliva (P>0.05). Conclusion The detection of α-syn and DJ-1 protein levels in saliva may be an auxiliary tool for diagnosis of PD.

Progress of DJ-1 protein as target for liver fibrosis treatment / 中国药理学通报

Liver fibrosis occurs prior to cirrhosis and the patho-logical feature is the transitional deposition of extracellular matrix and activation of hepatic stellate cells. Varieties of cytokines and related signaling pathways have been shown to participate in the process of hepatic fibrosis or activation of hepatic stellate cells. In recent years,studies have emerged to reveal the role of tran-scriptional factor DJ-1 in hepatic fibrosis. DJ-1 can relieve the process of hepatic fibrosis by regulating the level of oxidative free radicals in hepatocytes,Kupffer cells and inflammatory cell infil-suggesting DJ-1 as a potential molecular target for hepatic fibrosis treatment. This article provides a comprehensive review of DJ-1 in hepatic fibrosis treatment, including liver fibrosis and oxidative stress, DJ-1 structure and function, mechanism of DJ-1 in the treatment of hepatic fibrosis, and therapeutic prospect of targeting DJ-1.

Preparation of DJ-1 overexpressing cell model by using lentiviral vector / 基础医学与临床

Objective To prepare gene overexpressing cell model of human wild-type DJ-1 and its L166P mutant, and to investigate the role of lentiviral vector in gene overexpressing cell model .Methods Wild type DJ-1 and L166P mutant DJ-1 lentiviral vector plasmids were respectively constructed .After sequencing and comparing cor-rectly, the plasmid was amplified and transfected into HEK 293T cell line.Expression of WT DJ-1 and L166P mu-tant DJ-1 in cell lines was detected by fluorescence and Western blot .After determining the accurate expression of the target protein, a large amount of HEK293T cells was transfected and packaged to produce lentiviral particles. The PC12 cells were infected with the titer of virus supernatant.The fluorescence intensity of GFP and the expres-sion of target protein were observed by fluorescence microscope and Western blot method ,and the infection effi-ciency of the virus was determined .Results Lentiviral vectors carrying wild type DJ-1 and its mutants were suc-cessfully constructed .The virus vector can be transfected into HEK 293T cells and the target protein can be correctly expressed.The viral titers of LV-DJ-1 and LV-DJ-1/L166P were 2×109 TU/mL and 2×108 TU/mL, respectively. Virus supernatant can efficiently infect PC 12 cells, and most cells can express target proteins .The protein expres-sions of exogenous wild-type DJ-1 and L166P mutants were 315% and 285% of endogenous content ,respectively. Conclusions Lentivirus vector can infect cells efficiently , and it is a good way to prepare gene over expressing cell model.A cell model overexpressing DJ-1 or its L166P mutant is successfully prepared .The model can be used for subsequent DJ-1 function research .

Effect of tert-butylhydroquinone on DJ-1∕Nrf2 pathway during renal ischemia-reperfusion in diabet-ic rats / 中华麻醉学杂志

Objective To evaluate the effect of tert-butylhydroquinone ( t-BHQ) on DJ-1∕nuclear factor erythroid 2-related factor 2 (Nrf2) pathway during renal ischemia-reperfusion (I∕R) in diabetic rats. Methods Forty SPF healthy adult male Sprague-Dawley rats, weighing 200-220 g, were divided into 4 groups (n＝10 each) using a random number table method: control group (group C), diabetes mellitus group (group D), diabetes mellitus plus renal I∕R group (I∕R group) and t-BHQ group (group T). Diabe-tes mellitus was induced by intraperitoneal streptozotocin 60 mg∕kg and confirmed by fasting blood glucose level＞16. 7 mmol∕L 72 h later. t-BHQ 50 mg∕kg was intraperitoneally injected in 3 times at an interval of 8 h starting from 24 h before surgery in group T, while the equal volume of normal saline was given instead in D and I∕R groups. Blood samples were collected from the apex of the heart at 24 h of reperfusion for deter-mination of serum creatinine (Cr), cystatin C (Cys C) and β2-microglobulin (β2-MG) concentrations. The rats were then sacrificed, and kidneys were removed for determination of pathological changes of kidneys (with a light microscope) and for detection of the expression of DJ-1, Nrf2 and heme oxygenase-1 (HO-1) in renal tissues (by Western blot). Results Compared with group C, the concentrations of serum Cr, Cys C and β2-MG and pathological scores were significantly increased, and the expression of DJ-1, Nrf2 and HO-1 was up-regulated in D, I∕R and T groups ( P＜0. 05). Compared with group D and group I∕R, the concentrations of serum Cr, Cys C and β2-MG and pathological scores were significantly decreased, and the expression of DJ-1, Nrf2 and HO-1 was up-regulated in group T ( P＜0. 05). Conclusion t-BHQ can at- tenuate renal I∕R injury by activating DJ-1∕Nrf2 pathway in diabetic rats.

MPTP-induced vulnerability of dopamine neurons in A53T α-synuclein overexpressed mice with the potential involvement of DJ-1 downregulation

Familial Parkinson's disease (PD) has been linked to point mutations and duplication of the α-synuclein (α-syn) gene. Mutant α-syn expression increases the vulnerability of neurons to exogenous insults. In this study, we developed a new PD model in the transgenic mice expressing mutant hemizygous (hemi) or homozygous (homo) A53T α-synuclein (α-syn Tg) and their wildtype (WT) littermates by treatment with sub-toxic (10 mg/kg, i.p., daily for 5 days) or toxic (30 mg/kg, i.p., daily for 5 days) dose of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Tyrosine hydroxylase and Bcl-2 levels were reduced in the α-syn Tg but not WT mice by sub-toxic MPTP injection. In the adhesive removal test, time to remove paper was significantly increased only in the homo α-syn Tg mice. In the challenging beam test, the hemi and homo α-syn Tg mice spent significantly longer time to traverse as compared to that of WT group. In order to find out responsible proteins related with vulnerability of mutant α-syn expressed neurons, DJ-1 and ubiquitin enzyme expressions were examined. In the SN, DJ-1 and ubiquitin conjugating enzyme, UBE2N, levels were significantly decreased in the α-syn Tg mice. Moreover, A53T α-syn overexpression decreased DJ-1 expression in SH-SY5Y cells. These findings suggest that the vulnerability to oxidative injury such as MPTP of A53T α-syn mice can be explained by downregulation of DJ-1.

Effects of kaempferol on learning and memory in rats with chronic cerebral ischemia and possible mechanisms / 中国药理学通报

Aim To investigate the effects and possible mechanisms of kaemperol in the rats with chronic cere-bral ischemia.Methods Chronic cerebral hypoperfu-sion model was produced by permanent occlusion of bi-lateral common carotid arteries (2VO)in rats.After KAE treatment,the rats underwent Morris water maze and prehensile traction test.Neuronal morphology was observed using Nissl and HE staining.The activity of SOD and the content of MDA in brain tissue were de-termined.The DJ-1 protein expression was assayed by Western blot.Results Compared with 2VO model group,KAE significantly improved learning and memo-ry and the grasping ability.In addition,KAE signifi-cantly reduced brain tissue pathological injury induced by 2VO. Furthermore, KAE significantly increased SOD activity and enhanced antioxidant protein DJ-1 ex-pression in brain tissue.Conclusions KAE could sig-nificantly attenuate the cognitive impairment,limb bal-ance dysfunction and pathological injury in rats with chronic cerebral ischemia.The mechanism may be re-lated to improving the antioxidant system in vivo.

20C,a new bibenzyl compound, plays a significant role in rotenone-induced oxidative insult / 中国药理学与毒理学杂志

20C, a bibenzyl compound isolated from Gastrodia elata, possesses antioxidative properties in PC12 cells, but its in-depth molecular mechanisms against rotenone-induced neurotoxicity remains unknown. Recent studies indicate that without intact DJ- 1, nuclear factor erythroid 2- related factor (Nrf2) protein becomes unstable, and the activity of Nrf2-mediated downstream antioxidant enzymes are thereby suppressed. Therefore, increasing the nuclear translocation of Nrf2 by DJ-1 may present a helpful means for the prevention and treatment of chronic diseases related to oxidative stress. Our results showed that 20C clearly protected PC12 and SH-SY5Y cells against rotenone-induced oxidative injury in a concentration-dependent manner. Furthermore, 20C markedly up-regulated the levels of DJ-1, which in turn activated phosphoinositide-3-kinase (PI3K)/Akt signaling and inhibited glycogen synthase kinase 3β (GSK3β) activation, eventually promoting Nrf2 nuclear translocation and inducing the expression of Nrf2-mediated downstream antioxidative enzymes such as HO-1. The antioxidative effects of 20C could be partially blocked by ShRNA-mediated knockdown of DJ-1 and inhibition of the PI3K/Akt pathways with Akt1/2 kinase inhibitor in PC12 and SH-SY5Y cells, respectively. Conclusively, our findings confirm that DJ- 1 is necessary for 20C- mediated protection against rotenone- induced oxidative damage, at least in part, by activating PI3K/Akt signaling, and subsequently enhancing the nuclear accumulation of Nrf2. The findings from our investigation suggest that 20C should be developed as a novel candidate for preventing or alleviating the consequences of PD in the future.

DJ-1 regulates the function of mitochondria in Parkinson’s disease / 中国药理学通报

Mitochondrial dysfunction plays an important role in the process of PD, DJ-1 participates in regulating the function of mitochondria,which has an effect on the protection of mitochon-dria. DJ-1 mutations can lead to the decrease of the activity of mitochondrial complex Ⅰ, the decrease of mitochondrial mem-brane potential and then mitochondrial fragmention and mitoph-agy, and then further damage neurons and trigger PD. This re-view presents the role of DJ-1 in regulating the function of the mitochondria in the pathogenesis of Parkinson's disease(PD).