Effect of selective cerebral mild hypothermia on SUMO2/3 modification of Drp1 in a rat model of cerebral ischemia-reperfusion / 中华麻醉学杂志

Objective:To evaluate the effect of selective cerebral mild hypothermia on small ubiquitin-like modifier 2/3 (SUMO2/3) modification of dynamin-related protein 1 (Drp1) in a rat model of cerebral ischemia-reperfusion (I/R).Methods:Sixty clean-grade healthy male Sprague-Dawley rats, aged 6-8 weeks, weighing 240-260 g, were divided into 4 groups ( n=15 each) using a random number table method: sham operation group (S group), cerebral I/R group (I/R group), selective cerebral mild hypothermia group (HT group) and normal temperature group (NT group). The operation was performed under the monitoring of cerebral temperature and rectal temperature.Only the cervical blood vessels were exposed in S group, while focal cerebral I/R was induced by 2 h middle cerebral artery occlusion (MCAO) followed by 24 h reperfusion in anesthetized animals in the other three groups.In HT group and NT group, 4 and 37 ℃ normal saline was perfused through the left internal carotid artery at a rate of 80 ml·kg -1·h -1 for 15 min, respectively. Modified neurological severity score (mNSS) was assessed at 24 h of reperfusion. Then the rats were sacrificed under deep anesthesia, brains were removed, brain tissues were obtained for determination of the percentage of cerebral infarct size (by TTC staining), and the ischemic penumbra tissues in the cerebral cortex were removed for examination of the ultra-structural changes of mitochondria (with a transmission electron microscope) and for determination of the SUMO2/3 modification of Drp1 (by CO-IP), expression of total Drp1 (T-Drp1) and total cytochrome c (T-Cytc) (by Western blot), and expression of mitochondrial outer membrane Drp1 (M-Drp1) and cytoplasmic Cytc (C-Cytc) (by Western blot) after isolation of mitochondria and cytoplasm. Results:Compared with S group, the mNSS and percentage of cerebral infarct size were significantly increased, the expression of M-Drp1, T-Drp1, C-Cytc and T-Cytc was up-regulated, and SUMO2/3 modification of Drp1 in ischemic penumbra area was increased ( P<0.05), the fragmentation of mitochondria was aggravated, and cristae rupture and vacuolation were obvious in the other three groups. Compared with I/R group, the mNSS and percentage of cerebral infarct size were significantly decreased, the expression of M-Drp1, T-Drp1, C-Cytc and T-Cytc was down-regulated, SUMO2/3 modification of Drp1 was increased ( P<0.05), the fragmentation of mitochondria was significantly attenuated, and cristae rupture and vacuolation were weakened in HT group. There were no significant differences in these detection parameters between NT group and I/R group ( P>0.05). Conclusions:The mechanism by which selective cerebral mild hypothermia alleviates the cerebral I/R injury is related to increased SUMO2/3 modification of Drp1, decreased binding of Drp1 to mitochondrial outer membrane, and reduced mitochondrial excessive fission in rats.

Role of PIAS-regulated SUMOylation of PPARγ in endogenous protective mechanism against endotoxin-induced acute lung injury in mice / 中华麻醉学杂志

Objective:To evaluate the role of small ubiquitin-associated modifier (SUMO) E3 ligase (PIAS)-regulated SUMOylation of peroxisome proliferator-activated receptor γ (PPARγ) in the endogenous protective mechanism against endotoxin-induced acute lung injury (ALI) in mice.Methods:Experiment Ⅰ Twenty-four clean-grade wild type male C57BL/6 mice, aged 6-8 weeks, weighing 18-22 g, were divided into 4 groups ( n=6 each) using a random number table method: control group (C group), ALI group, ALI+ PPARγ inducer TZD group (ALI+ T group) and ALI+ TZD+ SUMOylation inhibitor anacardic acid group (ALI+ T+ A group). Lipopolysaccharide (LPS) 15 mg/kg was injected into the tail vein to develop the ALI model. In ALI+ T+ A group, anacardic acid 5 mg/kg was intraperitoneally injected at 1 h before LPS administration. In ALI+ T group and ALI+ T+ A group, TZD 50 mg/kg was intraperitoneally injected at 30 min before LPS administration. The mice were sacrificed at 12 h after LPS administration, and the lung tissues were obtained to examine the pathological changes which were scored and to determine the wet/dry (W/D) weight ratio, and expression of PIAS1, PIAS2, PIAS3 and PIASy protein and mRNA (by Western blot or polymerase chain reaction). Experiment Ⅱ Mouse alveolar macrophages (MH-S cells) were cultured in vitro and divided into 4 groups ( n=5 each) using a random number table method: control group (C group), LPS group, LPS+ PIAS2 siRNA group (L+ P group) and LPS+ Con siRNA group (L+ C group). Cells were routinely cultured in group C. Cells were stimulated with 10 μg/ml LPS to develop the model of endotoxin challenge. PIAS2 siRNA 50 nmol/L and Con siRNA 50 nmol/L were transfected at 48 h before LPS was added in L+ P group and L+ C group, respectively. The cells were collected at 24 h of incubation with LPS to determine the cell viability, levels of M1 and M2 alveolar macrophages (by flow cytometry), expression of PIAS2 and PPARγ (by Western blot), co-expression of PPARγ-SUMO1 (by immunoprecipitation) and expression of tumor necrosis factor-alpha (TNF-α) and interleukin-10 (IL-10) mRNA (by polymerase chain reaction). The ratio of M1/M2 was calculated. Results:Experiment Ⅰ Compared with C group, the lung injury scores and W/D ratio were significantly increased, and the expression of PIAS2 protein and mRNA was up-regulated in the other three groups ( P<0.05). Compared with ALI group, the lung injury scores and W/D ratio were significantly decreased, and the expression of PIAS2 protein and mRNA was up-regulated in ALI+ T group and ALI+ T+ A group ( P<0.05). Compared with ALI+ T group, the lung injury scores and W/D ratio were significantly increased, and the expression of PIAS2 protein and mRNA was down-regulated in ALI+ T+ A group ( P<0.05). There was no significant difference in the expression of PIAS1, PIAS3 and PIASy protein and mRNA in lung tissues among the four groups ( P>0.05). Experiment Ⅱ Compared with C group, the cell viability was significantly decreased, the expression of PPARγ and co-expression of PPARγ-SUMO1 was up-regulated, the levels of M1 and M2 macrophages and M1/M2 ratio were increased, the expression of TNF-α mRNA was up-regulated, and the expression of IL-10 mRNA was down-regulated in the other three groups, and PIAS2 expression was significantly up-regulated in L group and L+ C group ( P<0.05). Compared with L group, the cell viability was significantly decreased, the expression of PIAS2 and PPARγ and PPARγ-SUMO1 co-expression were down-regulated, the M1 macrophage level and M1/M2 ratio were increased, TNF-α mRNA expression was up-regulated, and the expression of IL-10 mRNA was down-regulated in L+ P group ( P<0.05), and no significant change was found in the parameters mentioned above in L+ C group ( P>0.05). Compared with L+ C group, the cell viability was significantly decreased, the expression of PIAS2 and PPARγ and co-expression of PPARγ-SUMO1 were down-regulated, the level of M1 alveolar macrophages and M1/M2 ratio were increased, the expression of TNF-α mRNA was down-regulated, and the expression of IL-10 mRNA was up-regulated in L+ P group ( P<0.05). Conclusions:PIAS2-regulated SUMOylation of PPARγ is the endogenous protective mechanism against endotoxin-induced ALI in mice, which may be related to inhibition of macrophage polarization into M1 type and alleviation of inflammatory responses.

Clinical value of deubiquitination modification in evaluating the prognosis of patients with diffuse large B-cell lymphoma / 中国医师进修杂志

Objective:To observe the expression levels of small ubiquitinated protein specific protease (SENP) 1 and small ubiquitin-related modifier protein (SUMO) 1 in patients with diffuse large B-cell lymphoma (DLBCL), and analyze the clinical value of evaluating prognosis.Methods:The clinical data of 66 patients with DLBCL (DLBCL group) in Inner Mongolia People′s Hospital from February 2017 to October 2020 were retrospectively analyzed, and 60 cases of healthy people in the same period were selected as the healthy control group. The expression levels of SENP1 and SUMO1 were detected by enzyme linked immunosorbent assay (ELISA). The correlation between the expression levels of SENP1, SUMO1 and clinical characteristics was analyzed. The independent risk factors affecting the prognosis were analyzed by Cox multivariate analysis.Results:The SENP1 in DLBCL group was significantly higher than that in healthy control group (50.39 ± 6.86 vs. 7.47 ± 1.32), the SUMO1 in DLBCL group was significantly lower than that in healthy control group (8.84 ± 2.13 vs. 31.49 ± 5.89), and there were statistical differences ( t = 47.640 and 29.210, P<0.01). There were statistical differences in SENP1 and SUMO1 among patients with different clinical stages ( P<0.01). The expression levels of SENP1 and SUMO1 were correlated with clinical stage and international prognostic index (IPI) ( P<0.05), and were not correlated with age, gender, disease site and clinical symptoms ( P>0.05). The 3-year survival rate in patients with high SENP1 expression (30 cases) was significantly lower than that in patients with low SENP1 expression (36 cases), the 3-year survival rate in patients with high SUMO1 expression (38 cases) was significantly higher than that in patients with low SUMO1 expression (28 cases), and there were statistical differences (26.67% vs. 75.00% and 73.68% vs. 39.29%, P<0.05). Cox multivariate regression analysis result showed that clinical stage, IPI, SENP1 and SUMO1 were independent risk factors affecting the prognosis in patients with DLBCL ( HR = 1.352, 1.487, 2.048 and 3.295; 95% CI 1.180 to 1.691, 1.187 to 1.602, 2.536 to 4.023 and 2.752 to 5.325; P<0.05 or <0.01). Conclusions:In patients with DLBCL, SENP1 is highly expressed and SUMO1 is lowly expressed. The expression levels of SENP1 and SUMO1 are closely related to clinical stage and IPI in patients with DLBCL, and they are independent risk factors of the prognosis.

Hypoxia increases chemotherapy resistance in nasopharyngeal carcinoma via inducing CDK6 deSUMOylation / 中华耳鼻咽喉头颈外科杂志

Objective@#To understand the mechanism of chemotherapy resistance in nasopharyngeal carcinoma under hypoxic conditions through the perspective of protein SUMOylation modification.@*Methods@#Cobalt chloride (CoCl2) was used to establish the hypoxic model of human nasopharyngeal carcinoma CNE1 cells. Then, the cell cycle was detected by flow cytometry, and the expression level of small ubiquitin-related modifier(SUMO) and cyclin-dependent kinase 6 (CDK6) proteins were detected by western blotting. MTT assay was used to determine the median lethal dose (IC50) of cancer cells against cisplatin, and enzyme-linked immunosorbent assay (ELISA) was used to determine lactate dehydrogenase (LDH) level.@*Results@#The cell cycle of CNE1 induced by hypoxia was arrested in G0/G1 phase.The results of Western blot showed that the protein expression level of CDK6 in CNE1 cells was lower than that in the control group (0.83±0.25 vs. 0.43±0.21, t=14.67, P=0.003). The protein level of conjugated SUMO1 was significantly lower than that in the control group (2.69±0.48 vs. 1.38±0.31, t=17.22, P=0.001), while the level of free SUMO1 protein was significantly higher than that in the control group (2.01±0.43 vs. 2.60±0.59, t=15.45, P=0.002).The LC50 of CNE1 cells in the control group was significantly lower than that in the hypoxic group (29.44 μg/ml vs. 97.72 μg/ml, t=12.79, P=0.001). After CNE1 cells received 50 μg/ml cisplatin for 48 h, the LDH content in the supernatant of the control group was significantly higher than that in the hypoxic group ((541.49±64.59) ng/ml vs. (234.67±41.03) ng/ml, t=11.94, P=0.007)). The apoptosis rate of CNE1 cells in the control group was significantly higher than that in the hypoxic group ((76.64±5.37)% vs. (32.84±4.77) ng/ml, t=8.49, P=0.003)).@*Conclusion@#Hypoxia can dissociate the covalent modification of CDK6 and SUMO1, inhibit cell cycle and increase the chemotherapy resistance of nasopharyngeal carcinoma.

IL-21 modulates biological characteristics of NK92 cells by upregulating SENP1 expression / 军事医学

Objective To investigate the roles of SENP1 in regulation of biological characteristics of NK cells.Methods Lentivirus-mediated-Senp1-small-hairpinRNA (shRNA) transduction was applied to NK92 cells.The expression of SENP1 in NK92 cells was determined by real-time PCR and Western blot.The proliferation of NK92 cells was detected by CCK-8 assay.The apoptosis of NK92 cells was determined by Annexin Ⅴ and PI labeling.The cytotoxicity of NK92 cells against K562 cells was evaluated by luciferase reporter assay.Results Treatment of NK92 cells with IL-21 resulted in SENP1 upregulation.Lentivirus mediated SENP1 knockdown reduced proliferation and increased apoptosis in NK-92 cells,but SENP1 inhibition had slight impact on the cytotoxic ability of NK92 cells to kill K562 cells.Conclusion SENP1 mediates the regulatory effect of IL-21 on the proliferation and survival of NK92 cells.

Non-fusion soluble expression of broad-spectrum antivirus protein in Escherichia coli by translational-coupling with SUMO / 军事医学

Objective To design and construct a new non-fusion soluble expression vector pTIG-mSUMO(small ubiq-uitin-related modifier) using the widely used solubility promoting protein SUMO and based on the translational coupling phenomenon in order to enable the non-fusion soluble expression of the broad-spectrum antiviral protein RA in Escherichia coli by pTIG-mSUMO.Methods The smt3 gene coding for SUMO protein was cloned from yeast genome DNA by PCR. After directed-site silent mutation to eliminate the EcoRⅠsite, the mutant mSUMO was inserted into pET-22b to obtain the translational coupling expression vector pTIG-mSUMO.The RA was subject to PCR amplification and cloned into the pTIG-mSUMO to obtain the expression plasmid pTIG-mSUMO/RA which was supposed to direct the soluble expression of RA by the translational coupling with mSUMO.Results A translational coupling expression vector pTIG-mSUMO which could di-rect/drive the SUMO and heterogonous protein non-fusion expression simultaneously was designed and constructed.The Western blotting result indicated that pTIG-mSUMO could direct the high-level expression of RA, around 40%of which was soluble.Conclusion A translational coupling expression vector pTIG-mSUMO is obtained.After coupling with SUMO, RA is highly expressed in E.coli and both the expression level and solubility are greatly improved.pTIG-mSUMO might contrib-ute to soluble expression of other proteins.

Effects of angiotensin Ⅱand losartan on expression of SUMO in rat glomerular mesangial cells / 重庆医学

Objective To investigate the effects of angiotensin Ⅱ (Ang Ⅱ ) and losartan on the expression of small ubiquitin-related modifier(SUMO) protein (SUMO1 ,SUMO2/3) in cultured rat glomerular mesangial cells(GMCs) .Methods In vitro cul-tured HBZY-1 rat GMCs were divided into 5 groups:normal control group(NC group) ,different concentrations of Ang Ⅱinterven-tion groups(A1 ,A2 ,A3 groups) and losartan treatment group(MT group) .The expression of SUMO1 and SUMO2/3 protein and mRNA of each group was measured by Western blot and RT-PCR .Results Compared with the NC group ,the expression of SU-MO1 and SUMO2/3 protein and mRNA in the Ang Ⅱintervention groups and the losartan treatment group was increased signifi-cantly (P<0 .01);Compared with the Ang Ⅱintervention groups ,the expression of SUMO1 and SUMO2/3 protein and mRNA in the losartan treatment group had no statistically significant difference .Conclusion Ang Ⅱ up-regulates the expression of SUMO protein in rGMCs by a dose-dependent manner in certain range ,this effect is not blocked by losartan ,Ang Ⅱ may be involved in the pathogenesis of diabetic nephropathy .

Study on expression of SENP1 and c-myb gene in patients with acute lymphoblastic leukemia / 重庆医学

Objective To investigate the SENP1 and c-myb gene expression and their correlations in bone marrow specimens in the patients with acute lymphoblastic leukemia (ALL ) to provide the basis for expounding the role ,mechanism and prognosis of SENP1 and c-myb in ALL .Methods 31 patients diagnosed with ALL (22 cases of B-ALL ,1 case of T-ALL and 8 cases of uncate-gorized ALL ;6 cases in the low/medium risk group ,25 cases in the high risk group) and 31 patients with proliferative bone marrow and hyperplastic anemia diagnosed by the morphology were taken as the control group .The real-time PCR and immunocytochemical staining(SP method) were adopted to detect the mRNA and protein expressions of SENP1 and c-myb in the bone marrow specimens of the ALL patients and the control group .Results The expression of SENP1 and c-myb were both increased in the bone marrow specimens and smears of ALL patients ,which showed the statistical difference compared with the control group (P< 0 .05) ,the Pearson correlation analysis found that the high expression of SENP1 and c-myb had correlation .The expression of SENP1and c-myb in the low/medium risk group were lower than that in the high risk group ,but the difference had no statistical significance . Conclusion The high expression of SENP1 and c-myb exists in the bone marrow specimens of the ALL patients ,SENP1 and c-myb could possibly have the correlation with the occurrence and development of ALL ;but now the differences of SENP1 and c-myb ex-pression among different risk groups of ALL patients are yet to be proven .

Small ubiquitin-like modifier 4 M55V polymorphism is not associated with diabetic nephropathy in Iranian type 2 diabetes patients.

INTRODUCTION: We studied the impact of small ubiquitin-like modifier 4 (SUMO4) M55V polymorphism on susceptibility to diabetic nephropathy in Iranian type 2 diabetes patients. MATERIALS AND METHODS: The patient group consisted of 50 Iranian type 2 diabetes patients with nephropathy, and the control group consisted of 50 Iranian type 2 diabetes patients without nephropathy. Genotyping was performed using polymerase chain reaction-restriction fragment length polymorphism method for the M55V. RESULTS: The frequency of SUMO4 AA, AG, and GG genotypes were 23%, 18%, and 9% in the patient group and 10%, 22%, and 18% in the control group. There was no significant difference in frequency of SUMO4 genotypes in patients compared to controls. CONCLUSION: These findings indicate that SUMO4 M55V polymorphism is not associated with diabetic nephropathy in Iranian type 2 diabetes patients.

Expressions of SUMO4, NF-κB and IκB in kidney of rats with type 2 diabetes mellitus and its significance / 心血管康复医学杂志

Objective: To study expressions of small ubiquitin-related modifier protein（SUMO）4 (SUMO4), nuclear factor (NF)- κB and inhibitory factor of NF-κB (IκB) in kidneys of rats with type 2 diabetes mellitus (T2DM). Methods: A total of ten 40-week-old male Goto-Kakizaki (GK) rats （with spontaneous diabetes mellitus）of specific-pathogen free (SPF) grade, and ten 40-week-old male Wistar rats of SPF grade were selected. The lesion of renal tissue was observed by hematoxylin eosin (HE) staining. Expressions of SUMO4, NF-κB and IκB in renal tissue were observed by immunohistochemistry methods. Results: In the GK rats, glomerular capillary ball hypertrophy, basilar membrane slightly thickening; glomerular mesangial cells hyperplasia, hypertrophy and renal tubular epithelial cells hypertrophy were observed. Compared with normal Wistar rats, expression levels of NF-κB [(0.232±0.034) vs. (0.634±0.058)], IκB [(0.242±0.027) vs. (0.712±0.078)] and SUMO4 [(0.160±0.031) vs. (0.545±0.045)] significantly increased in renal tissue of GK rats (P<0.01 all). Conclusion: Compared with Wistar rats, expressions of NF-κB, IκB and SUMO4 significantly increase in renal tissue of GK rats, suggesting that SUMO inhibiting transcriptional activity of NF-κB may exist in kidneys of T2DM rats. Therefore, sumoylation may be a new therapeutic target for inhibit renal microvascular lesion of diabetic disease.

Expressions of SUMO1, NF-κB and TNF-α in myocardium of rats with type 2 diabetes mellitus / 心血管康复医学杂志

Objective: To study effect of small ubiquitin related modifier protein 1 (SUMO1) in inflammatory reactions mediated by tumor necrosis factor (TNF)-α and nuclear factor (NF)-κB in myocardial damage of rats with type 2 diabetes mellitus (T2DM). Methods: A total of 20 Goto-Kakizaki (GK) rats with spontaneous diabetes mellitus (DM) were randomly divided into group DM1 (pure DM group, n=10) and group DM2 (DM+high-fat diet group, n=10), and another 10 normal Wistar rats were regard as healthy control group. Expressions of SUMO1, TNF-α and NF-κB were measured by immunohistochemical method. Results: 1. Levels of blood glucose and TG in group DM1 and group DM2 were significantly higher than those of healthy control group, and those of DM2 group were higher than of DM1 group ,P<0.05 all; 2. Myocardial cells lined up in order and there was no hypertrophy in group DM1; but those in group DM2 showed cells loosely lined up and hypertrophy under light microscope; 3 Immunohistochemical assay indicated that expression of SUMO1 in group DM2 and DM1 group were significantly higher than those of healthy control group [(44.5±1.1) vs. (27.2±2.2) vs. (21.7±3.0)], and of group DM2 was significantly higher than that of DM1 group (P<0.01 all); expression of TNF-α in group DM2 and group DM1 were significantly higher than that of healthy control group [(27.5±1.5) vs. (20.2±2.7) vs. (13.1±1.6)], and of DM2 group was significantly higher than that of group DM1 (P<0.01 all)；expression of NF-κB in group DM2 and group DM1 were significantly higher than that of healthy control group [（30.1±1.7）vs.40.7±1.5）vs.（16.0±2.6）], but of group DM1 was significantly higher than that of group DM2 (P<0.01 all). Conclusion: There are obvious metabolic disorders of glucose and lipid in T2DM rats, and complicated morphological changes of myocardial tissues similar to myocardial lesions in DM humans; the expressions of SUMO1, NF-κB and TNF-α significantly increase, suggest SUMO1 takes part in inflammatory reaction mediated by NF-κB, TNF-α in myocardial lesion of rat with T2DM，and may inhibit NF-κB, possesses effect of protect myocardium.