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Purpose This study aims to observe the hemodynamic state of the carotid artery in healthy volunteers and diabetes patients via ultrasound vector flow imaging(V-Flow),and to quantitatively analyze the characterization of wall shear stress(WSS)in different degrees of atherosclerosis carotid artery.Materials and Methods This was a cross-sectional study.Forty four diabetic patients diagnosed in Sichuan Provincial People's Hospital from April 2019 to June 2019 were retrospectively analyzed.Routine carotid ultrasound examination and V-Flow imaging were performed on 44 diabetic patients(88 vessels)and 15 controls(30 vessels).According to the results of carotid ultrasound examination,diabetic patients were divided into normal intima-media thickness(IMT)group,IMT thickening group and plaque group.The differences of WSS in different parts of carotid artery between diabetic patients and controls were observed,and the changes of WSS in different groups of diabetic patients were further compared and analyzed.Results In this study,the WSS(including WSSmax and WSSmean)of the bifurcation and middle segment of the common carotid artery in diabetic patients were significantly lower than those in the normal control group(P=0.001,P=0.003).WSS at the bifurcation of common carotid artery was significantly smaller than that at the middle segment of common carotid artery in both normal control group and diabetic group(P=0.001).WSS at the bifurcation of common carotid artery in diabetic patients was changed with the severity of atherosclerosis(P=0.018 and P=0.043),and was highest in the plaque group[WSSmax(2.58±0.69)Pa;WSSmean(0.86±0.25)Pa],followed by IMT thickening group[(2.37±0.69)Pa;(0.77±0.35)Pa].The WSS was lowest in the normal IMT group[(2.13±0.52)Pa;(0.69±0.20)Pa].There was no significant difference in WSS of the middle segment of common carotid artery among the three groups(P>0.05).Conclusion The WSS in the bifurcation and middle section of common carotid artery in diabetic patients is lower than that in controls,and it changed with the increase of atherosclerosis degree.Therefore,the V-Flow technique of ultrasonic vector flow imaging has a certain value in evaluating the progression of carotid atherosclerosis in diabetic patients.
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Objective:pH low insertion peptide (pHLIP)-variant 7 (var7)-fluorescein isothiocyanate (FITC) was used to explore an accurate imaging tool that targeted burn wounds to better perform burn debridement.Methods:Twelve rat models of burn wound were established and pHLIP-var7-FITC with different concentrations (0.5, 1.5 and 2.0 mg/ml) were injected from the rat tail vein for in vivo fluorescence imaging. By determining the concentration of fluorescent conjugates to the burn wound, the scope of wound injury necrosis was judged by combining pathological sections, and its residue and toxicity in important organs such as heart, liver, kidneys, and brain were detected. The Kruskal-Wallis rank sum test, Bonferroni correction method and one-way analysis of variance were used for data analysis. Results:Within 24 h, the fluorescence photons per unit area of the burn wound in the group of 0.5 mg/ml, 1.5 mg/ml and 2.0 mg/ml were 1.49(1.31, 1.65), 2.46(1.88, 2.68), 2.77 (1.94, 3.10)×10 7 p·s -1·cm -2·Sr -1, with significant differences in the overall distribution of fluorescence photons ( H=73.55, P<0.001). The fluorescence intensity was stronger in the group with higher concentration, but with no significant difference in the number of fluorescence photons between the group of 1.5 mg/ml and 2.0 mg/ml ( P=0.263, Bonferroni correction method). At 14 time points (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, 8.0, 12, 24 h), there was no significant difference in the overall mean of fluorescence photons ( F=1.04, P=0.419), and the tissue with burn necrosis seen in tissue sections was highly consistent with the fluorescence imaging region. There was no obvious fluorescence residue in the heart, liver, kidney and brain sections. Conclusion:In superficial second-degree burn tissue, pHLIP-var7-FITC can accurately target and gather on the burn wound within 24 h, showing a clear boundary between burn tissue and normal tissue, which can assist clinical surgical debridement to determine the extent of injury.
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Objective To investigate the effects of microsurgical varicocelectomy on testicular function and sexual function in patients with varicocele.Methods The clinical data of 90 patients with varicocele admitted to our hospital were retrospectively analyzed,and the patients were divided into the laparoscopic group(received laparoscopic varicocelectomy)and the microscopic group(received microsurgical varicocelectomy)according to different surgical methods,with 45 cases in each group.The testicular function and sexual function related indexes including sperm density,normal sperm ratio,rate of sperm motility(grades a+b),forward motility sperm rate,international index of erectile function-5(IIEF-5)score,and the levels of testosterone,follicle-stimulating hormone,luteinizing hormone,and androgen levels before and 6 months after surgery in the two groups were compared.The incidence of complications and recurrence 6 months after surgery in the two groups were counted.Results Compared with those before surgery,the sperm density,forward motility sperm rate,rate of sperm motility(grades a+b),normal sperm ratio,IIEF-5 score,testosterone level,and androgen level 6 months after surgery of patients in the two groups were significantly increased(P<0.05),and the levels of luteinizing hormone and follicle-stimulating hormone were decreased(P<0.05).Compared with the laparoscopic group,the levels of follicle-stimulating hormone and luteinizing hormone,and incidence of complications 6 months after surgery of patients in the microscopic group were decreased(P<0.05),and the levels of testosterone and androgens,and IIEF-5 score 6 months after surgery were increased(P<0.05).There was no significant difference in the recurrence rate between the two groups(P>0.05).Conclusion Microsurgical varicocelectomy can improve the testicular function and sexual function of patients with varicocele,with a low incidence of complications.
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Objective To explore the safety and effectiveness of bridging therapy in elderly patients with acute stroke due to posterior circulation large vessel occlusion.Methods A total of 160 eld-erly patients with acute stroke caused by posterior circulation large vessel occlusion admitted to our department were prospectively recruited and randomly divided into bridging group(n=80)and control group(n=80).The bridging group received thrombolysis treatment and then mechan-ical thrombectomy.The control group received mechanical thrombectomy directly.Prognosis and adverse reactions were compared between the two groups.Results The NIHSS score and BATMAN score after treatment were significantly decreased in both groups(P＜0.01),and the two scores were obviously lower in the bridging group than the control group(6.54±1.23 vs 7.12± 0.98,2.12±0.34 vs 2.87±0.44,P＜0.01).There was no statistical difference in the conversion rate of bleeding after cerebral infarction between the two groups(5.00％vs 3.75％,P＞0.05).The number of intraoperative thrombus removal was significantly lower in the bridging group than the control group(2.43±0.33 vs 2.98±0.41,P＜0.01).Remarkable difference was observed in the mRS score between the two groups after treatment(P＜0.05),with the proportion of mRS score ranging from 0 to 1 larger in the bridging group than the control group(52.50％vs 27.50％,P＜0.05).Conclusion Bridging thrombolysis can significantly improve the neurological function in elderly patients with acute stroke due to posterior circulation occlusion.
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ObjectiveTo explore the effects of modified Gualou Zhishitang combined with piperacillin sodium and tazobactam sodium on the immune function and serum levels of inflammatory cytokines in the patients with stroke-associated pneumonia （SAP， syndrome of phlegm-heat accumulation in lung）. MethodEighty SAP patients with the syndrome of phlegm-heat accumulation in lung were randomized into a control group （40 cases） and a study group （40 cases）. The SAP patients in the control group were treated with piperacillin sodium and tazobtam sodium， while those in the study group were treated with modified Gualou Zhishitang on the basis of the treatment in the control group for 2 consecutive weeks. The clinical therapeutic effects， immune function indexes， inflammation indexes， and lung function of SAP patients in the two groups before and after treatment were determined and compared. ResultAfter treatment， the scores of lesion， pulmonary rales， cough， fever， phlegm color， and constipation in both groups decreased （P<0.05）. After treatment， the ratio of forced expiratory volume in the first second to forced expiratory volume （FEV1/FVC） and forced expiratory volume in the first second as percentage of predicted value（FEV1%） in both groups improved （P<0.05）， and the study group outperformed the control group （P<0.05）. The treatment decreased the neutrophil to lymphocyte ratio （NLR） in the two groups （P<0.05）， and the study group had lower NLR than the control group after treatment （P<0.05）. The serum levels of procalcitonin （PCT） and hypersensitive C-reactive protein （hs-CRP） in both groups declined after treatment （P<0.05）， and the declines were more significant in the study group than in the control group （P<0.05）. After treatment， the study group was better than the control group （P<0.05）. The treatment in both groups elevated the levels of CD3+， CD4+， and CD4+/CD8+ in the peripheral blood and lowered the level of CD8+ （P<0.05）， and the changes were more significant in the study group than in the control group （P<0.05）. The total response rate of the study group was 95.00% （38/40）， which was higher than that （80.00%， 32/40） of the control group （χ2=4.114，P<0.05）. There was no significant difference in the incidence of adverse reactions between the two groups. ConclusionModified Gualhou Zhishitang combined with piperacillin sodium and tazobactam sodium demonstrates a significant therapeutic effect on the SAP patients with the syndrome of phlegm-heat accumulation in lung. This therapy can mitigate the clinical symptoms， improve the lung function， lower the serum levels of inflammatory cytokines， and improve the immune capacity， with high safety.
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Objective @#To explore whether the NLRP3 inflammasome is activated after Newcastle disease virus (NDV) exposure to esophageal cancer ECA109 cells , whether its activation is related to K + efflux , and the effect of ATP/P2X7 axis on the activation of NLRP3 inflammasome.@*Methods@#The expression of NLRP3 and IL⁃1β was detected by Western blot; the content of IL⁃1β in the supernatant was detected by ELISA ; the formation of ASC spots was detected by fluorescence immunoassay; the change of intracellular K + concentration was detected by fluorescent probe technology; Interventions with ATPase , ATP and P2X7 receptor inhibitors were used to investigate their role in NLRP3 inflammasome activation.@*Results@#Compared with the control group , the expression of NLRP3 , IL⁃1β and ASC protein in cells was up⁃regulated after NDV F3 infection ; the intracellular potassium concen tration decreased with the prolongation of infection time (P < 0. 05) . After the intervention of P2X7 receptor inhibitor, the efflux of intracellular K + was blocked. With the increase of inhibitor concentration , the efflux of K + was maximally inhibited at 10 μmol/L (P < 0. 05) . The results of ATPase and ATP intervention showed that ATPase inhibited K + efflux , while ATP promoted K + efflux. Western blot results showed that compared with the control group , P2X7 receptor was inhibited , and the expressions of NLRP3 and IL⁃1β were down⁃regulated ; after ATPase intervened cells , the expressions of NLRP3 and IL⁃1β decreased ; After ATP intervention in cells , the protein expressions of NLRP3 and IL⁃1β were up⁃regulated (P < 0. 05) . @*Conclusion@#NDV F3 infection of ECA109 cells can activate the NLRP3 inflammasome , the mechanism may be related to the ATP/P2X7 axis.
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Exosomes (EXOs) are formed by intracellular multivesicular bodies and carry a variety of biomacromolecules such as lipids, proteins, encoding and non-coding RNAs, and mitochondrial DNA. EXOs can be released in vivo by different cell types, including hepatocytes, hepatic stellate cells, and immune cells and play the role of intercellular communication. More and more studies have shown that EXOs are involved in the development, progression, and prognosis of chronic hepatitis B (CHB) and hepatocellular carcinoma (HCC) caused by hepatitis B virus (HBV) infection and are expected to become potential biomarkers for the early diagnosis and prognostic evaluation of HBV-related HCC. This article reviews the role of EXOs in the host infection process of HBV and the importance of EXOs in the development, progression, and prognosis of CHB and HCC, in order to provide new ideas for the basic and clinical research in this field.
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Sleep exerts important functions in the regulation of cognition and emotion. Recent studies have found that sleep disorder is one of the important risk factors for Alzheimer's disease (AD), but the effects of chronic sleep deprivation on the cognitive functions of AD model mice and its possible mechanism are still unclear. In the present study, 8-month-old male APP/PS1/tau triple transgenic AD model (3xTg-AD) mice and wild type (WT) mice (n = 8 for each group) were subjected to chronic sleep deprivation by using the modified multiple platform method, with 20 h of sleep deprivation each day for 21 days. Then, open field test, elevated plus maze test, sugar water preference test, object recognition test, Y maze test and conditioned fear memory test were performed to evaluate anxiety- and depression-like behaviors, and multiple cognitive functions. In addition, the immunohistochemistry technique was used to observe pathological characteristics in the hippocampus of mice. The results showed that: (1) Chronic sleep deprivation did not affect anxiety- (P = 0.539) and depression-like behaviors (P = 0.874) in 3xTg-AD mice; (2) Chronic sleep deprivation exacerbated the impairments of object recognition memory (P < 0.001), working memory (P = 0.002) and the conditioned fear memory (P = 0.039) in 3xTg-AD mice; (3) Chronic sleep deprivation increased amyloid β (Aβ) deposition (P < 0.001) and microglial activation (P < 0.001) in the hippocampus of 3xTg-AD mice, without inducing abnormal tau phosphorylation and neurofibrillary tangles. These results indicate that chronic sleep deprivation exacerbates the impairments of recognition memory, working memory and conditioned fear memory in 3xTg-AD mice by aggravating Aβ deposition and the excessive activation of microglia in the hippocampus.
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Animales , Masculino , Ratones , Enfermedad de Alzheimer , Péptidos beta-Amiloides , Precursor de Proteína beta-Amiloide/genética , Cognición , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Ratones Transgénicos , Presenilina-1 , Privación de Sueño , Proteínas tauAsunto(s)
Humanos , Acupuntura , Puntos de Acupuntura , Terapia por Acupuntura , Venodisección , Agujas , Accidente Cerebrovascular/terapiaRESUMEN
OBJECTIVE@#To describe a family with intellectual developmental disorder with autism and speech delay (IDDAS) caused by a splice variant of TBR1 gene.@*METHODS@#A pregnant women with mental retardation, who also had a family history of mental retardation, was admitted to Prenatal Diagnosis Center of WanBei Coal and Electricity Group General Hospital Corporation in April 2019. Molecular genetic tests were performed on the pregnant women and ten other family members to analyze the pathogenic genotype. Functional assays of the pathogenic variant was carried out by minigene technology. With the determination of the genotype, prenatal diagnosis was carried out by amniotic fluid sampling.@*RESULTS@#Through whole exome sequencing, a novel splicing variant (c.1129-1G>C) was identified in the TBR1 gene of the proband, which has co-segregated with the disease phenotype in the family. The results of minigene assay showed abnormal splicing of exon 5. The variant was not detected in the fetal amniotic fluid. Fetal growth and development were normal one year after the birth.@*CONCLUSION@#The c.1129-1G>C variant of the TBR1 probably underlay the disease in of the pedigree. Timely prenatal genetic diagnosis and consultation can help to stop the transmission of the pathogenic variant.
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Femenino , Humanos , Lactante , Embarazo , Trastorno Autístico/genética , China , Discapacidades del Desarrollo , Discapacidad Intelectual/genética , Trastornos del Desarrollo del Lenguaje , Linaje , Proteínas de Dominio T Box/genéticaRESUMEN
Objective To investigte the efficacy of docetaxel combined with androgen deprivation therapy for the treatment of metastatic hormone-sensitive prostate cancer based on Chinese population.Methods A total of 497 patients were enrolled from January 2004 to July 2018 in the Changhai Hospital.459 patients received androgen deprivation therapy alone and 38 patients received androgen deprivation therapy combined with docetaxel.The mean age was (72.1 ± 8.7)years.The median PSA level was 100.0 ng/ml,ranging 42.3-999.0 ng/ml.Patients of clinical T2,T3,T4 stage were 213 (42.9％),160 (32.2％),124(24.9％),respectively.Patients of clinical N0,N1,Nx stage were 319 (64.2％),144 (29.0％),34 (6.8％),respectively.Patients of clinical M0,M1a,M1b,M1c,Mx stage were 100(20.1％),51 (10.3％),332 (66.8％),9 (1.8％),5 (1.0％),respectively.Gleason scores of biopsy showed that 146 (29.4％) patients was ≤7,103(20.7％) was 8 and 248(49.9％)was ≥9.Propensity score matching was used to match the baseline between groups.Caliper value was set at 0.02.SPSS 22 software was used to achieve a 1:1 match between the two groups.There were no statistical difference in the age(P =0.102),PSA (P =0.713),T stage (P =0.113),N stage (P =0.226),M stage (P =0.514),Gleason score (P =0.612),tumor loading(P =0.812) between the two groups.The castration resistance-free rate and cancer specific survival rate of the two groups were compared by log-rank and breslow-wilcoxon test.Furthermore,forest plots were used to display the analysis results of different subgroups such as age,PSA,clinical stage,Gleason score,tumor load,whether patients had received palliative resection,and the differences in castration resistance-free rate were compared between the subgroups with high tumor load.Results The median follow-up time was 22.6 months in the androgen deprivation therapy group and 13.7 months in the combined therapy group.The number of patients with castration resistance in the two groups was 23 and 17,respectively.There were 3 and 6 deaths,respectively.There was no statistically significant difference in the overall progression time to castration resistance between the two groups (10.3 m vs.16.5 m,P ＞ 0.05),and no statistically significant difference in the prostate cancer specific survival rate (21.9 m vs.14.8 m,P ＞ 0.05).When subgroup analysis was performed,it was found that patients in the high-metastasis-volume subgroup who received the combination therapy had a significantly longer castration resistance free lifetime (10.6 m vs.7.2 m,P =0.044),but there was no significant difference in the low-metastasis-volume subgroup (10.5 m vs.12.6 m,P ＞ 0.05).Conclusion Docetaxel combined with androgen deprivation therapy can improve the castration resistance free rate in patients with high metastasis volume,but not in low metastasis volume group.
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Tirofiban is a selective non-peptide glycoprotein Ⅱb/Ⅲa receptor inhibitor that reversibly inhibits fibrinogen-dependent platelet aggregation and subsequent thrombosis. Thrombosis caused by platelet activation plays a vital role in the occurrence and development of ischemic cerebrovascular events. Some studies have evaluated the use of tirofiban in intravenous thrombolysis and endovascular therapy. Although the optimal dose, conditions of use, and target patients have not been fully identified, the good tolerability of tirofiban and its efficacy in improving recanalization and long-term functional outcomes have been validated. This article reviews the mechanism of tirofiban and its application in the treatment of acute ischemic stroke reperfusion.
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BACKGROUND:During the percutaneous vertebroplasty, the optimal dose of bone cement that can bring favorable cement dispersion and remodel the biomechanical balance of the fractured vertebrae remains controversial. OBJECTIVE:To investigate the dispersion degree of small dose of bone cement in vertebroplasty. METHODS: In this experiment, 18 sheep selected with the same condition were randomly divided into three groups (group A, group B, group C), 6 in each group. A model of thoracolumbar vertebral compression fracture (T12, L1, L2) was made in each sheep. The injected volume of bone cement in groups A, B, C was 15％, 20％, 25％ of the average volume of adjacent vertebral bodies, respectively. Postoperative CT images were used to evaluate the bone cement dispersion. Dispersion degree of bone cement among the three groups was compared by the Kruskal-Wallis test. RESULTS AND CONCLUSION:There was no statistical difference in the dispersion degree of bone cement among the three groups, and the excellent and good rate of dispersion was over 80％. To conclude, the optimal dose of bone cement injected into the fractured vertebra is 15％ of the average volume of adjacent vertebral bodies, which can achieve good dispersion degree and restore the biomechanical stability of the vertebral body.
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Objective Using Cerenkov radiation energy transfer (CRET) effect of rare earth nanoparticles (RENPs) to enhance and convert Cerenkov luminescence into Cerenkov luminescence excited fluorescence,and to compare the accuracy of Cerenkov luminescence tomography (CLT) and Cerenkov luminescence excited fluorescence tomography (CLFT).Methods 68Ga (0.74 MBq) was used to respectively excite Y2O3 ∶Eu3+,Er2O3 and Eu2O3(10 mg/ml).Various radioactivities of 68Ga (3.70,1.85,0.92,0.46,0.23 MBq) were used to respectively excite Y2O3 ∶Eu3+ with a fixed concentration (10 mg/ml).A fixed radioactivity of 68Ga (3.70 MBq) was used to excite Y2O3 ∶Eu3+ with different concentrations (10.0,5.0,2.5,1.2,0.6 mg/ml) in order to find the relationships between the optical intensity and the radioactivity of 68Ga or the concentration of Y2O3 ∶Eu3+.Polyethylene tubes containing 68Ga (0.74 MBq) and 68Ga (0.74 MBq) +Y2O3 ∶Eu3+(1 mg) were respectively implanted into two nude mice,then PET/CT and optical imaging were acquired.Three-dimensional reconstruction was proceeded.One-way analysis of variance,two-sample t test,linear correlation analysis were used for data analysis.Results Y2O3 ∶Eu3+ could significantly and stably enhance the Cerenkov optical signal (F=53.35,q =17.03,P＜0.001).The enhanced optical signal intensity had linear relationships with the radioactivity of 68Ga or the concentration of Y2O3 ∶Eu3+(r values:0.99and 0.93).Three-dimensional reconstruction result showed that CLFT had significantly higher similarity than CLT (0.43±0.14 vs 0.16±0.06,t =5.090,P＜0.05).Conclusion CLFT could reflect the distribution of radiopharmaceuticals more precisely than CLT,and therefore might have potential in biologic optical imaging.
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This study was designed to evaluate the effects of drilling through the growth plate and using adipose-derived stem cells (ADSCs) and bone morphogenetic protein-2 (BMP-2) to treat femoral head epiphyseal ischemic necrosis,which can be done in juvenile rabbits.Passage-four bromodeoxyuridine (BrdU)-labeled ADSCs were cultured,assayed with MTT to determine their viability and stained with alizarin red dye to determine their osteogenic ability.Two-month-old,healthy male rabbits (1.2 to 1.4 kg,n=45) underwent ischemic induction and were randomly divided into five groups (group A:animal model control;group B:drilling;group C:drilling & ADSCs;group D:drilling & BMP-2;and group E:drilling & ADSCs & BMP-2).Magnetic resonance imaging (MRI),X-ray imaging,hematoxylin and eosin staining and BrdU immunofluorescence detection were applied 4,6 and 10 weeks after treatment.Approximately 90％ of the ADSCs were labeled with BrdU and showed good viability and osteogenic ability.Similar results were observed in the rabbits in groups C and E at weeks 6 and 10.The animals of groups C and E demonstrated normal hip structure and improved femoral epiphyseal quotients and trabecular areas compared with those of the groups A and B (P＜0.01).Group D demonstrated improved femoral epiphyseal quotients and trabecular areas compared with those of groups A and B (P＜0.05).In summary,drilling through the growth plate combined with ADSC and BMP-2 treatments induced new bone formation and protected the femoral head epiphysis from collapsing in a juvenile rabbit model of femoral head epiphyseal ischemic necrosis.
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This study was designed to evaluate the effects of drilling through the growth plate and using adipose-derived stem cells (ADSCs) and bone morphogenetic protein-2 (BMP-2) to treat femoral head epiphyseal ischemic necrosis,which can be done in juvenile rabbits.Passage-four bromodeoxyuridine (BrdU)-labeled ADSCs were cultured,assayed with MTT to determine their viability and stained with alizarin red dye to determine their osteogenic ability.Two-month-old,healthy male rabbits (1.2 to 1.4 kg,n=45) underwent ischemic induction and were randomly divided into five groups (group A:animal model control;group B:drilling;group C:drilling & ADSCs;group D:drilling & BMP-2;and group E:drilling & ADSCs & BMP-2).Magnetic resonance imaging (MRI),X-ray imaging,hematoxylin and eosin staining and BrdU immunofluorescence detection were applied 4,6 and 10 weeks after treatment.Approximately 90％ of the ADSCs were labeled with BrdU and showed good viability and osteogenic ability.Similar results were observed in the rabbits in groups C and E at weeks 6 and 10.The animals of groups C and E demonstrated normal hip structure and improved femoral epiphyseal quotients and trabecular areas compared with those of the groups A and B (P＜0.01).Group D demonstrated improved femoral epiphyseal quotients and trabecular areas compared with those of groups A and B (P＜0.05).In summary,drilling through the growth plate combined with ADSC and BMP-2 treatments induced new bone formation and protected the femoral head epiphysis from collapsing in a juvenile rabbit model of femoral head epiphyseal ischemic necrosis.
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Objective To investigate whether advanced glycation end products (AGEs) can induce the expression of Ros,JC-1 and its apoptosis-related proteins in glomerular mesangial cells under high glucose environment,induce apoptosis and injury of glomerular mesangial cells.Methods Rat glomerular mesangial cell line HBZY-1 was cultured in vitro.The cells were cultured with different concentrations of AGEs for 0,12,24 and 48 hours respectively.MTT assay was used to observe the cell proliferation ability.After the optimal time and concentration of AGEs were selected,the caspase enzyme inhibitor Z-VAD-fmk and reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC) were cultured and the apoptosis rate was detected by cell death detection apoptosis ELISA plus and Annexin V-FITC/PI kit.JC-1 staining was used to detect the changes of mitochondrial membrane potential (MMP).Cell ROX deep red flow cytometry was used to detect the total ROS level.The expression of anti-apoptotic protein Bcl-2,pro-apoptotic protein BAX,caspase-9,caspase-3 and poly ADP-ribose polymerase (PARP)-activated fragments was detected by Western blotting.Results AGEs could decrease the activity of glomerular mesangial cells in a time and concentration-dependent manner,and induce cell death.The percentage of apoptotic cells in glomerular mesangial cells was significantly increased after treatment with 250 mg/L AGEs for 24 h (P ＜ 0.01),and Z-VAD-fmk could significantly alleviate AGEs-induced glomerular mesangial cell apoptosis (P ＜ 0.01).Compared with the control group,AGEs increased the level of intracellular reactive oxygen species and decreased MMP in a time-dependent manner,and the two time points that AGEs significantly caused the change were 1 h and 2 h (all P ＜ 0.01).AGEs also reduced the expression of antiapoptotic protein Bcl-2 and increased the expression of pro-apoptotic protein Bax,cleaved caspase-9,cleaved caspase-3 and cleaved PARP (all P ＜ 0.01).Compared with AGEs group,NAC could significantly stabilize MMP (P ＜ 0.01),increase Bcl-2 expression (P ＜ 0.01),and decrease the expression of BAX,cleaved caspase-9,cleaved caspase-3 and cleaved PARP (all P ＜ 0.01).Conclusion AGEs induce mitochondrial pathway apoptosis in glomerular mesangial cells by increasing intracellular ROS level and destroying MMP.
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Objective To investigate the effects of abated microRNA-21 (miRNA-21) on phosphatase and tensin homologue on chromosome ten protein (PTEN) and PI3K/Akt/mTOR pathway,as well as their further influence on the autophagy in high glucose (HG,25.0 mmol/L) induced rat glomerular mesangial cells.Methods MiRNA-21 inhibitor and negative control were transfected by liposome 2000 into rat glomerular mesangial cells (HBZY-1).The cells were divided into normal glucose (5.5 mmol/L) group,normal glucose + negative control group,normal glucose +miRNA-21 inhibitor group,HG group,HG+negative control group and HG+miRNA-21 inhibitor group.Cell proliferation and hypertrophy were assayed by MTT and the ratio of total protein to cell number respectively.The miRNA-21 expression was detected using real time PCR.The expressions of PTEN/ Akt/mTOR signaling signatures,autophagy-associated protein (p62 and LC3 Ⅱ) and collagen Ⅰ was detected by Western blotting and real time PCR.Autophagosomes were observed using electron microscopy.Results Compared with those in normal glucose group,in HG group cells had hypertrophy and proliferation,up-regulated miRNA-21 expression,and down-regulated PTEN protein and mRNA expressions (all P ＜ 0.01).Also there were and up-regulated p-Akt,p-mTOR,p62 and collagen Ⅰ expression,and lower LC3 Ⅱ expression and autophagosomes (all P ＜ 0.01).Further,compared with those in HG group,cells hypertrophy and proliferation in HG+miRNA-21 inhibitor group were reduced,expressions of p-Akt,p-mTOR,p62 and collagen Ⅰ were down-regulated,while expressions of PTEN and LC3 Ⅱ and autophagosomes were up-regulated (all P ＜ 0.01).Conclusions MiRNA-21 inhibitor up-regulates PTEN expression,which inhibits the activation of Akt/mTOR signaling pathway,ameliorates cell hypertrophy,proliferation and enhances autophagy to reduce extracellular matrix accumulation.
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Objective To investigate whether the effect of transient high glucose on inflammatory factors expression could be continuous in rat glomerular mesangial cell,and its relation with histone methylation modification.Methods Rat glomerular mesangial cells (HBZY-l) were divided into three groups:the high glucose group (25.0 mmol/L glucose),the hypertonic group (MA,5.5 mmol/L glucose+ 19.5 mmol/L mannitol) and the normal-glucose control group (5.5 mmol/L glucose),which were cultured for 24 h respectively.All 3 groups were then changed with normal-glucose medium to culture for 24 h,48 h and 72 h.Their protein,mRNA and supernatant were harvested.The protein expressions of mono-methylation of H3 lysine 4 (H3K4mel) was measured by Western blotting,and the mRNA expressions of NF-κB subunit p65 and set7/9 were determined by real timequantitative PCR.The expression of monocyte chemoattractant protein 1 (MCP-1) and vascular cell adhesion molecule 1 (VCAM-1) were detected by enzyme-linked immunosorbent assay.Results (1)Compared with those in normal control group,the expressions of H3K4mel protein and set7/9 mRNA were first up-regulated in high glucose group,then gradually down-regulated in the following 48 h normal-glucose medium (as compared with those at 0 h,all P ＜ 0.05).At 72 h there was no statistic difference between high glucose group and normal control group (all P ＞ 0.05).(2) Compared with those in normal control group,the up-regulated p65 mRNA,VCAM-1 and MCP-1 sustained at least for 72 h in high glucose group.Conclusions Transient high glucose can induce persistent inflammatory factors expression in rat glomerular mesangial cells,which may via histone modification.
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Objective@#To investigate the effects of abated microRNA-21 (miRNA-21) on phosphatase and tensin homologue on chromosome ten protein (PTEN) and PI3K/Akt/mTOR pathway, as well as their further influence on the autophagy in high glucose (HG, 25.0 mmol/L) induced rat glomerular mesangial cells.@*Methods@#MiRNA-21 inhibitor and negative control were transfected by liposome 2000 into rat glomerular mesangial cells (HBZY-1). The cells were divided into normal glucose (5.5 mmol/L) group, normal glucose+negative control group, normal glucose+miRNA-21 inhibitor group, HG group, HG+negative control group and HG+miRNA-21 inhibitor group. Cell proliferation and hypertrophy were assayed by MTT and the ratio of total protein to cell number respectively. The miRNA-21 expression was detected using real time PCR. The expressions of PTEN/Akt/mTOR signaling signatures, autophagy-associated protein (p62 and LC3 Ⅱ) and collagen Ⅰ was detected by Western blotting and real time PCR. Autophagosomes were observed using electron microscopy.@*Results@#Compared with those in normal glucose group, in HG group cells had hypertrophy and proliferation, up-regulated miRNA-21 expression, and down-regulated PTEN protein and mRNA expressions (all P<0.01). Also there were and up-regulated p-Akt, p-mTOR, p62 and collagen Ⅰ expression, and lower LC3 Ⅱ expression and autophagosomes (all P<0.01). Further, compared with those in HG group, cells hypertrophy and proliferation in HG+miRNA-21 inhibitor group were reduced, expressions of p-Akt, p-mTOR, p62 and collagen Ⅰ were down-regulated, while expressions of PTEN and LC3 Ⅱ and autophagosomes were up-regulated (all P<0.01).@*Conclusions@#MiRNA-21 inhibitor up-regulates PTEN expression, which inhibits the activation of Akt/mTOR signaling pathway, ameliorates cell hypertrophy, proliferation and enhances autophagy to reduce extracellular matrix accumulation.