RESUMEN
Objective To investigate the effect of long non-coding RNA (lncRNA) alpha-2-macroglobulin antisense RNA 1 (A2M-AS1) targeting microRNA (miR) -106b-5p on oxidized low-density lipoprotein (ox-LDL) -induced injury of human brain microvascular endothelial cells. Methods Human brain microvascular endothelial cells (ox-LDL group) were induced by ox-LDL, normal cultured cells were control group (Ctrl); A2M-AS1 overexpression (pcDNAA2M-AS1 group), empty vector (pcDNA group), miR-106b-5p inhibitor (anti-miR-106b-5p group), negative control (anti-miR-NC group), pcDNA-A2M-AS1 with control mimic NC (miR-NC group), pcDNA-A2M-AS1 with miR-106b-5p mimic (miR-106b-5p mimics group) were transfected into cells and treated with ox-LDL, n = 9. Real-time PCR was used to detect the expression levels of A2M-AS1 and miR-106b-5p; Kits were used to detect malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT)); Flow cytometry and TUNEL detected apoptosis; Dual luciferase reporter gene assay detected A2M-AS1 and miR-106b-5p targeting; Western blotting detected Bcl-2 and Bax protein expression. Results Compared with the Ctrl group, the expression level of A2M-AS1 in the ox-LDL group decreased, and the activity of SOD and CAT and the protein level of Bcl-2 decreased (P<0.05), while the expression level of miR-106b-5p and the level of MDA increased (P<0.05), and the rate of apoptosis and the protein level of Bax increased (P<0.05). Overexpressing A2M-AS1 or interfering with miR-106b-5p decreased the MDA level, apoptosis rate and Bax protein level after ox-LDL-induced cells, and increased SOD, CAT activity and Bcl-2 protein level (P<0.05). A2M-AS1 targeted miR-106b-5p; upregulation of miR-106b-5p reversed the effect of overexpressed lncRNA A2M-AS1 on ox-LDL-induced injury of human brain microvascular endothelial cells (P < 0.05). Conclusion A2M-AS1 attenuates ox-LDL-induced injury of human brain microvascular endothelial cells by targeting miR-106b-5p.
RESUMEN
Background & objectives: Type 2 diabetes mellitus (T2DM) is known to induce inflammation and activation of neutrophils causing the release of neutrophil elastase (NE), a pro-inflammatory proteinase. The activity of NE is regulated by endogenous inhibitors alpha1-antitrypsin (?1-AT) and alpha2- macroglobulin (?2-MG). Disrupted proteolytic homeostasis in T2DM patients is one of the causes for vascular complications. This study was carried out for evaluating the levels of plasma NE, ?1-AT, ?2-MG and NE-?1-AT complex to understand their roles in the pathophysiology of diabetic nephropathy (DN) and diabetic retinopathy (DR). Methods: A total of 240 participants (Control, n=60; T2DM, n=60; DN, n=60; and DR, n=60) were recruited after recording history, clinical examination and laboratory investigations. Retinopathy was confirmed by fundoscopy and nephropathy by urinary albumin excretion and serum creatinine levels. NE was measured using STANA. ?1-AT, ?2-MG and NE-?1-AT complex were estimated by ELISA. Results: Baseline clinical and laboratory findings were confirmatory to the study groups. The mean elastase activity was higher (P<0.0005) in diabetes groups (T2DM=0.73±0.31, DN=0.87±0.35, DR=0.76±0.41) than controls (0.35±0.20). The levels of ?1-AT were lower in DR (8.77±2.85) than DN (26.26±6.16) and T2DM (41.13±14.06) when juxtaposed with controls (122.95±25.71). The approximate fold decrease of ?1-AT levels was 15 for DR and four for DN compared to controls. The levels of ?2-MG were lowered in T2DM (167.29±30.45), DN (144.66±13.72), and DR (104.67±11.47) than controls (208.87±31.16). The NE-?1-AT complex levels were: controls (215.83±13.61), T2DM (98.85±23.85), DN (129.26±20.40) and DR (153.25±17.11). Interpretation & conclusions: Homeostasis of NE, ?1-AT and ?2-MG is disrupted in T2DM, DN and DR. Strikingly reduced levels of ?1-AT observed in DR are indicative of its possible role in the pathophysiology of retinopathy and emphasizes ?1-AT as a plausible therapeutic target.
RESUMEN
Introduction. Fetal obstructive uropathy can be treated early in pregnancy by intrauterine vesicoamniotic shunting, immediately after diagnosis by ultrasound, in specific cases with a favorable prognosis in renal function using fetal urine beta2 microglobulin. Objective: To determine the changes of beta-2 microglobulin in consecutive samples of fetal urine in 48 - 72 hour-intervals in fetuses with obstructive uropathy at 16 weeks of gestation. Methods: We designed a descriptive and longitudinal study, including 15 pregnant women whose fetuses were diagnosed with obstructive uropathy without chromosomal abnormalities, performing vesicocentesis for urinary biometry. Results: Beta-2 microglobulin values were higher than 4 mg/dL in the first vesicocentesis of all 15 cases and decreased to less than 4 mg/dL in 7 cases (46.6%) after the second vesicocentesis. In all cases when beta-2 microglobulin was less than 8 mg/dL in the first vesicocentesis, there was a decrease to nearly 4 mg/dL (maximum 4.3 mg/dL) in the second vesicocentesis. Conclusions: Vesicoamniotic shunting should be performed in all cases of fetal obstructive uropathy when the values of beta-2 microglobulin are less than 8 mg/dL in the first vesicocentesis.
Antecedentes y objetivos. La uropatía obstructiva fetal puede ser tratada precozmente mediante la colocación de derivación vesicoamniótica intrauterina. Los criterios de tratamiento incluyen la ausencia de malformaciones mayores asociadas, cariotipo normal y función renal conservada demostrada por microglobulina beta-2 4 mg/dL en los 15 casos con la primera vesicocentesis, y disminuyeron a < 4 mg/dL en 7 casos (46,6%) en la segunda vesicocentesis. En todos los casos cuando la microglobulina beta-2 fue < 8 mg/dL en la primera vesicopunción, siempre disminuyó a valores muy próximos a 4 mg/dL (máximo de 4,3 mg/dL) en la segunda vesicopunción. Conclusiones. Se plantea la colocación in útero de derivaciones vesicoamnióticas en los casos de uropatía obstructiva fetal cuando la microglobulina beta-2 sea < 8 mg/dL en la primera vesicopunción, omitiendo realizar una segunda vesicopunción.
RESUMEN
[Objective]To explore the effect of alpha2-macroglobulin(α2M)on superoxide anion(.O2-)content, superoxide dismutase(SOD)activity and the process of cell-to-myofibroblast transformation in human skin fibroblasts (HSF)after X-ray irradiation.[Methods]HSF cells were irradiated with 0,5,10,15 and 20 Gy X-ray.The change of. O2- content and SOD activity in the supernatant of cell culture medium were measured on the first day after irradiation. The protein expression of alpha-smooth muscle actin(α-SMA)was detected by Western blot on the fifth day after irradia-tion.The most sensitive radiation dose is selected.HSF cells were irradiated with the above sensitive dose.Respectively, 1h before irradiation,1 h after irradiation,the experimental group cell culture medium was added to a final concentration of 0.25 mg/mL,0.5 mg/mL of α2M.The change of.O2-content,SOD activity and the protein expression of α-SMA were detected.[Results]HSF cells were irradiated with 5~20 Gy doses of X-ray..O2- content increased,SOD activity de-creased and α-SMA protein expression increased gradually(P<0.05).The addition of α2M at 1 h after 10Gy X-ray irradi-ation reduced the.O2- content,increased the SOD activity and downregulated the protein expression of α-SMA in HSF cells(P<0.05). There was no significant change in the administration at 1 h before irradiation.[Conclusion]HSF cells increased.O2-content significantly,while SOD activity decreased,and the tendency to transform myofibroblasts after X-ray irradiation.α2M can reduce the.O2-content,increase the SOD activity in HSF cells and inhibit the transformation of fibroblasts into myofibroblasts after irradiation.Indicating that α2M can play a role in radiation protection by anti-oxida-tion and anti-fibrosis.
RESUMEN
AIM:To evaluate the effect of α2-macroglobulin(α2M) against X-ray induced obstacle on osteo-genic differentiation of human bone marrow mesenchymal stem cells(hBMMSCs). METHODS:hBMMSCs were cultured in vitro. The 4th generation of hBMMSCs was irradiated with 8 Gy X-ray,then induced osteogenic differentiation and trea-ted with different concentrations of α2M(0.5 and 1.0 g/L). The alkaline phosphatase(ALP) activity and the mRNA ex-pression of runt-related transcription factor-2 (RUNX2) were detected on day 7 after osteogenic induction. The protein ex-pression of osteoglycin (OGN) was evaluated by Western blot on day 14 after osteogenic induction. The formation of calci-um nodules was detected by alizarin red staining on day 21 after osteogenic induction. The activity of superoxide dismutase (SOD) and the protein expression of MnSOD of irradiated hBMMSCs with 8 Gy X-ray were determined at 24 h after α2M treatment. RESULTS:Compared with 8 Gy X-ray group,the activity of ALP,the mRNA expression of RUNX2,the pro-tein expression of OGN and MnSOD,as well as SOD activity were higher than those in the hBMMSCs treated with α2M at 0.5 and 1.0 g/L after 8 Gy X-ray irradiation,and the calcium nodules were also increased. CONCLUSION:α2M signifi-cantly improves the osteogenic differentiation ability,the SOD activity and MnSOD protein expression of hBMMSCs after ra-diation injury.
RESUMEN
Objective @# To investigate the changes of α2-macroglobulin in different stages of radiotherapy in patients with nasopharyngeal carcinoma, and to explore its feasibility as a marker of serum markers reflecting radiotherapy injury.@*Methods @#We collected the blood samples of 23 cases of newly diagnosed patients with nasopharyngeal carcinoma before the simple radiotherapy, the 10, 20, 30 and 33 times after simple radiotherapy, detected the α2- macroglobulin levels. The difference among the five stages was analysed by paired t-test using SPSS17.0 software package.@*Results @#The serum level of α2- macroglobulin elevated with the increase of number of radiotherapy. After 10 times’ radiotherapy, the serum α2-MG concentration in patients with nasopharyngeal carcinoma was significantly higher than that before radiotherapy (12.04 ± 5.72 vs. 10.81 ± 5.38 U/L), the difference was statistically significant (t=4.818, P < 0.05). After 20 times’ radiotherapy, the serum α2-MG concentration in patients with nasopharyngeal carcinoma was significantly higher than that before radiotherapy (12.26 ± 5.77 vs. 10.81 ± 5.38 U/L), and the difference was statistically significant (t=5.237, P < 0.001). After 30 times’ radiotherapy, the serum α2-MG concentration in patients with nasopharyngeal carcinoma was significantly higher than that before radiotherapy (12.91 ± 5.55 vs. 10.81 ± 5.38 U/L), the difference was statistically significant (t=6.076, P < 0.05). At the end of radiotherapy, the serum α2-MG concentration in nasopharyngeal carcinoma patients was significantly (13.43 ± 6.05 vs. 10.81 ± 5.38 U/L) higher than that before radiotherapy (t=5.189, P < 0.05).@*Conclusion@#The serum level of α2- macroglobulin changes with the radiotherapy, so it can be a serum marker reflecting the damage of maxilla induced by ionizing radiation.
RESUMEN
Objective To establish an assay for detecting α2-macroglobulin activity in Cohn fraction Ⅳ.Methodsα2-M reacted with trypsin to form α2-M-trypsin complex.After the chromogenic substrate Na-benzoyl-DL-arginine 4-nitroanilide hydrochloride ( BAPNA) was added, absorption at 410 nm was detected with the microplate reader .α2-M activity in Cohn fractionⅣwas quantitatively detected according to the established standard curve of plasma α2-M activity. Result Several critical parameters in this assay were optimized .A standard curve of plasma α2-M activity was established . According to this standard curve ,α2-M activity in Cohn fraction Ⅳsample was detected to be 1.578 PU/ml.Conclusion Using normal human plasma as the reference material , theα2-M activity in Cohn fractionⅣcan be detected through chro-mogenic substrate assay.This study provides a simple method to detect α2-M activity during the purification process of α2-M from Cohn fraction Ⅳ.
RESUMEN
A través del presente estudio se analizaron plasmas sanguíneos de seis especies, incluyendo el humano tanto en estado gestante como no gestante, identificándose por primera vez en plasma, la glicoproteína α2-Macroglobulina (α2-M) de ovino de pelo (Ovis aries) y de búfalo (Bubalus bubalis). La presencia de esta proteína en el plasma sanguíneo de todas las especies en estudio se demostró mediante electroforesis en gel de poliacrilamida usando sodio dodecilsulfato como agente denaturante (SDS PAGE) al 7.5% identificándose como bandas de 180 kDa y en forma no denaturante PAGE 5% como bandas de 720 kDa. Estas últimas bandas fueron claramente intercambiables de la forma tetramérica a la forma monomérica en los ensayos electroforéticos. Como controles se usaron la α2-M (tetramérica) y la proteína de la zona de gestación (PZP) (dimérica) purificadas a un 98%; así como, las bandas de estas dos proteínas en el plasma humano. El análisis de la secuencia del dominio N-terminal de la (α2-M) de ovino de pelo, fue muy similar al de la proteína humana purificada. Tanto la α2-M humana como la bovina llegaron a ser activadas a la forma rápida por medio de la reacción con metilamina. Lo anterior demuestra diferencias en la reactividad de las α2-M animales con la amina primaria cuando se comparan los resultados con la forma rápida de la α2-M humana. Será necesario unificar los métodos de purificación de esta proteína en todas las especies, de tal manera que los dominios sensibles de las α-macroglobulinas (tioιster y región señuelo) tengan el mismo tratamiento y el mismo grado de desnaturalización para todas las preparaciones de α2-M.
Blood plasma from six different non pregnant and pregnant species, including human blood plasma, was analyzed for detection of α2-Macroglobulin (α2-M). The tropical hair sheep (Ovis aries) and the buffalo (Bubalus bubalis) were studied for the first time in Colombia. The presence of the α2-M in plasma of all the species was demonstrated by SDS 7.5% PAGE as bands of 180 kDa as well as by non-denaturing 5% PAGE with bands of 720 kDa. The tetrameric form α2-M (tetramérica) and the pregnancy zone protein (PZP) (dimeric) purified at 98%, as well as its corresponding bans from human plasma were used as control. The N-terminal sequence of the band of 180 kDa in Tropical hair sheep plasma was very similar to the purified human α2-M. The results indicated the presence of α2-M in blood plasma of all the species tested, while the PZP was present only in the pregnant human plasma. Both human and bovine α2-M became activated with the fast form by reacting with Methylamine. This Fac. demonstrates the differences in the reactivity of the animal’s α2-M with primary amine as compared with the human α2-M. It could be necessary to unify purification methods into one method for all species, so that the sensitive domain of the α-macroglobulins (thiolester and bait region) receives the same treatment and grade of denaturation for all α2-M preparation.
RESUMEN
BACKGROUND: This study aims to detect any causative genetic alterations and to demonstrate any correlations of these genes in the pathogenesis of mostly late-occurring sporadic type of Alzheimer's disease (AD). METHODS: A total of 67 registered cases of autopsy-confirmed brain tissues were analyzed. Included here was sporadic AD (n=41), vascular dementia (n=17), and non-demented physiologically aging control brains (n=9). ApoE genotyping was done with the enzymatic digestion, and allele specific PCR was done to analyze the -491 A/T polymorphism of ApoE. Detection of polymorphism of alpha 2-macroglobulin (A2M) was done with enzymatic digestion and DNA sequencing. RT-PCR products were electrophoresed to detect mRNA expression of alpha 1-antichymotrypsin (ACT). RESULTS: A prevalence rate of ApoE E4 genotype (E3/E4, E4/E4) showed significantly higher in patients with AD than in patients with vascular dementia (43.8% vs. 11.7%, p=0.019). Only 1 out of 4 cases of sporadic AD was associated with the E4/E4 allele. -491A/ T polymorphism of the ApoE promoter was found only in AD (2/41 cases, 4.9%). The incidence of heterozygous allelic polymorphism with 5 bp deletions in exon 18 of A2M-2 was 4.9% (2 out of 41) in AD. Messenger RNA expression of ACT, which is closely associated with the ApoE E4 allele, was increased in AD in comparison with normal control (p=0.0002). CONCLUSIONS: ApoE4 genotype and ACT are closely related to the pathogenesis of late-onset sporadic AD. Neither -491 polymorphism of ApoE promoter nor A2M-2 showed close association with AD in these brain samples.
Asunto(s)
Humanos , Envejecimiento , Alelos , alfa 1-Antiquimotripsina , alfa-Macroglobulinas , Enfermedad de Alzheimer , Apolipoproteína E4 , Apolipoproteínas E , Apolipoproteínas , Encéfalo , Demencia Vascular , Digestión , Exones , Genotipo , Incidencia , Reacción en Cadena de la Polimerasa , Prevalencia , ARN Mensajero , Análisis de Secuencia de ADNRESUMEN
Normal human blood plasma showed hydrolytic activities on several synthetic substrates for proteases, the most effective being H-D-Ile-Pro-Arg-p-nitroanilide, H-D-Pro- Phe-Arg-p-nitroanilide and H-D-Val-Leu-Arg-p-nitroanilide. When plasma was preincubated for 12 h at 37°C, there was no significant alteration of the hydrolytic activities. On incubation for 12 h with king cobra venom (2 μg for 0·1 ml plasma), there was considerable decrease in the activities and complete abolition of the protease binding capacity of α2-macroglobulin. On chromatography on Sephadex G-200, α2-macroglobulin activity and bulk of the protease activity of normal plasma were eluted in the void volume region. A minor protease peak was eluted with a Ve/Vo value of 2·5. With venom treated plasma, there was no decrease with this peak. The major protease peak and α2-macroglobulin activity were drastically reduced. Chromatography on red Sepharose showed that all the a2- macroglobulin activity and bulk of the protease activity in normal plasma were bound to the column. In venom treated plasma there was marked reduction in the bound fraction. The data suggest that cobra venom proteases directly or through proteases generated in plasma in situ causes limited cleavage of α2-macroglobulin as well as α2– macroglobulin bound proteases, inactivating them.