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Objective: To investigate the effect of berberine on programmed necrosis of hepatocytes induced by metabolic-associated fatty liver disease (MAFLD) in mice and its related molecular mechanism. Methods: Twenty male C57BL/6N mice were randomly divided into four groups (n=5 in each group): control group (S), fatty liver group (H), berberine group(B), nuclear factor erythroid 2-related factor 2 inhibitor group (Nrf2), and all-trans-retinoic acid (ATRA) group (A). Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), triglycerides (TG), total cholesterol (TC), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) concentrations were detected at the end of week 12 to calculate fatty liver index (liver mass/body mass ratio). Liver tissue was stained with HE, Masson and Oil Red O, and SAF score was used to evaluate the degree of liver injury. The expression levels of hepatic programmed necrosis-related proteins, namely receptor-interacting protein kinase 3 (RIPK3), phosphorylated mixed series protease-like domain (p-MLKL) and Nrf2 were detected by Western blot method. One-way ANOVA was used for intragroup comparisons and LSD-t tests were used for intergroup comparisons. Results: Compared with S group, H group serum ALT, AST, LDH, TG, TC, TNF-α, IL-1β levels and fatty liver index were significantly increased. The liver tissue was filled with vacuolar-like changes and inflammatory cell infiltration. Numerous red lipid droplets were observed with oil red O staining. Collagen fiber hyperplasia was evident with Masson staining. SAF scores (6.60 ± 0.55 and 0.80 ± 0.45) were significantly increased. The expressions of RIPK3 and p-MLKL were up-regulated. Nrf2 level was relatively increased, and the differences were statistically significant (P < 0.05). Compared with H group, berberine intervention group liver biochemical indexes, lipid levels, pro-inflammatory mediator expression, fatty liver index, and SAF score were significantly reduced, and the expression of RIPK3 and p-MLKL were down-regulated, while Nrf2 levels were further increased, and the differences were statistically significant (P<0.05). Compared with B group, treatment with Nrf2 inhibitor had antagonized the protective effect of berberine on fatty liver. Serum ALT, AST, LDH, TG, TC and TNF-α, IL-1β levels, fatty liver index, and SAF scores were significantly increased and the expressions of RIPK3 and p-MLKL were relatively increased, and the differences were statistically significant (P < 0.05). Conclusion: Berberine can significantly improve the metabolic-associated fatty liver disease injury in mice, and its mechanism is related to activation of Nrf2 and inhibition of programmed necrosis of hepatocytes.
Subject(s)
Animals , Male , Mice , Berberine/therapeutic use , Fatty Liver , Mice, Inbred C57BL , NF-E2-Related Factor 2/metabolism , NecrosisABSTRACT
BACKGROUND: Recurrence of acute leukemia after hematopoietic stem cell transplantation is one of the major problems affecting the long-term survival of patients. Early intervention to prevent ALL recurrence after transplantation can improve disease-free survival, overall survival and reduce post-transplant mortality. Monitoring of minimal residual disease (MRD) by flow cytometry and PCR-based molecular biology techniques is a widely reliable and practicable method. OBJECTIVE: To dynamically monitor the MRD level of acute lymphoblastic leukemia after peripheral blood haploidentical hematopoietic stem cell transplantation and to explore its implications for predicting early relapse. METHODS: A retrospective study was conducted in 53 patients with acute lymphoblastic leukemia who had underwgone peripheral blood haploidentical hematopoietic stem cell transplantation at the First Affiliated Hospital of Zhengzhou University from June 2011 to June 2017. The patients were followed up for postoperative 1, 3, 6, 12 months to observe the relation between MRD levels and relapse after transplantation. RESULTS AND CONCLUSION: (1) The disease-free survival rate of MRD positive group and MRD negative group were 20.0% and 65.8%, respectively; and the overall survival rates were 50.8% and 68.9% in the two groups, respectively. There were significant differences between two groups. (2) Among 16 MRD positive patients accepting clinical intervention after transplantation, 4 patients presented with MRD negative and had no recurrence. (3) Eleven hematologic recurrence patients were given tyrosine kinase inhibitor-targeted therapy, chemotherapy, donor lymphocytes Infusion and secondary transplantation, but they eventually died. The median time from the discovery of MRD positive to hematologic recurrence was 100 (7-190) days, and during this period. Clinical intervention was confirmed to extend the recurrence time. In this study, one case refused clinical intervention, and eventually died of recurrence. Our findings indicate that dynamic monitoring of the MRD level in acute lymphoblastic leukemia patients after peripheral blood haploidentical transplantation can predict recurrence, by which the patients can be given early intervention to reduce the risk of recurrence and improve disease-free survival and overall survival.
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BACKGROUND: In recent years, genetic haploidentical peripheral blood stem cell transplantation has been gradually improved, and haploid allogeneic hematopoietic stem cell transplantation has become an important treatment choice for malignant hematopoietic disease. OBJECTIVE: To observe the clinical efficacy of genetic haploidentical peripheral blood stem cell transplantation for myelodysplastic syndrome. METHODS: The clinical data of 21 myelodysplastic syndrome cases undergoing genetic haploidentical peripheral blood stem cell transplantation were retrospectively analyzed. Modified BU/CY+ATG administration was performed as a pretreatment strategy for haploidentical peripheral blood stem cell transplantation, and the combined use of cyclosporine A+mycophenolate mofetil+short-range methotrexate±basiliximab was adopted to prevent graft-versus-host disease (GVHD). RESULTS AND CONCLUSION: (1) The 21 cases were followed for an median of 333 days (22-1 222 days), with 76% (16/21) infection of granulocyte lack period, 100% (21/21) neutrophil reconstruction, the median implantation time of 12 days (7-17 days), 81% (17/21) platelet engraftment, and the median implantation time of 14 days (7-68 days). (2) The accumulative incidence of GVHD was 52.4% (11/21), including 29% (6/21) of acute GVHD and 24% (5/21) of chronic GVHD. The incidence of hemorrhagic cystitis was 38.1% (8/21). The recurrence rate after transplantation was 4.8% (1/21). (3) The 2-year non-relapse mortality was 48% (10/21), and the 2-year disease-free survival rate was 46.8%. These results show that in the absence of HLA-identical related donors and unrelated donor, genetic haploidentical peripheral blood stem cell transplantation is a safe, effective, feasible and alternative treatment option for myelodysplastic syndrome.
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[Objective] To explore the value of serum procalcitonin (PCT) and percentage of neutrophils (NE％) in early diagnosis of bacterial infection in decompensated liver cirrhosis.[Methods] The clinical data of 244 in the patients with decompensated liver cirrhosis in the Department of Infectious Diseases,The third Affiliated Hospital of Sun Yat-sen University from January 2016 to June 2017 were retrospectively analyzed.According to whether complicated with bacterial infection,patients were divided into infection group (n=120) and non-infection group (n=124).The infection group consisted of 60 cases with spontaneous bacterial peritonitis (SBP),25 cases with pulmonary infection,16 cases with bloodstream infection,19 cases with other kinds of infections.The levels of PCT,peripheral white blood cells (WBC),peripheral neutrophils (NEUT),percentage of neutrophils (NE％) and ratio of WBC to platelets (PLT)(WBC/PLT) in each group were compared.The receiver characteristic curve (ROC) was applied and area under curve (AUROC),sensitivity and specificity were computed.The parallel test was applied to explore the diagnostic value of PCT with NE％.[Results] The levels of PCT,WBC,NEUT,NE％ and WBC/PLT in infection group were higher than those in non-infection group (P＜0.05).The levels of PCT,NE％ and WBC/PLT in bloodstream infection group were higher than in non-blood-stream infection group (P＜0.05).The AUROC of the PCT was 0.947 (95％CI=0.922～0.971).When PCT was≥0.38 ng/mL,the sensitivity,specificity and Youden index(YDI) were 76.7％,97.6％,and 0.742.The AUROC of NE％ was 0.806 (95％CI=0.751～0.861).With a cutoff value of 75.0％ in NE％,the sensitivity and specificity were 83.3％ and 92.7％,respectively.[Conclusion] Serum PCT,with a suggesting cut-off value of 0.38 ng/mL,could be used as an marker for early diagnosis of bacterial infection in decompensated liver cirrhosis.PCT combined with NE％ can improve diagnostic accuracy.
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OBJECTIVE@#To observe the characteristics of the interstitial fluid (ISF) drainage in the Alzheimer's disease (AD) rats through magnetic resonance imaging (MRI) tracer gadolinium-diethylene triamine pentacetic acid (Gd-DTPA)spread in the brain extracellular space (ECS) and to discuss the role of aquaporin-4 (Aqp4) in the AD.@*METHODS@#Wild type SD rats (300-350 g) and Aqp4 gene knock out (Aqp4-/-) SD rats (300-350g) were divided into Sham group, AD group, Aqp4-/--Sham group and Aqp4-/--AD group. Sham group and Aqp4-/--Sham group were injected with saline by intraperitoneal each day for 6 weeks, and the AD group and Aqp4-/--AD group were injected with D-galactose by intraperitoneal each day for 6 weeks. MRI tracer Gd-DTPA (10 mmol/L, 2 μL) was injected into the hippocampus of the rats. MRI scan was performed at the end of 0.5 h, 1.5 h, 1 h, 2 h, and 3 h to observe the dynamic distribution of the Gd-DTPA in the hippocampus and the diffusion rate D*, clearance rate k' and half-life t1/2 measured.@*RESULTS@#The diffusion rate D* in Sham group was (2.66±0.36)×10-6 mm2/s, the diffusion rate D* in AD group was (2.72±0.62)×10-6 mm2/s, the diffusion rate D* in Aqp4-/--Sham group was (2.75±0.47)×10-6 mm2/s, the diffusion rate D* in Aqp4-/--AD group was (2.802±0.55)×10-6 mm2/s, and there was no statistically significant difference in the four groups (One-Way ANOVA, P>0.05).The clearance rate k' in Sham group was (4.57±0.14)×10-4/s, the clearance rate k' in AD group was (3.68±0.22)×10-4/s, the clearance rate k' in Aqp4-/--Sham group was (3.17±0.16)×10-4/s, the clearance rate k' in Aqp4-/--AD group was (2.59±0.19)×10-4/s, and there was significant difference in the four groups (One-Way ANOVA, P<0.05). The half-life t1/2 in Sham group was (0.67±0.12) h, the half-life t1/2 in AD group was (0.88±0.08) h, the half-life t1/2 in Aqp4-/--Sham group was (1.12±0.15) h, the half-life t1/2 in Aqp4-/--AD group was (1.58±0.11) h, and there was significance difference in the four groups(one-way ANOVA,P<0.05).@*CONCLUSION@#The ISF drainage is slow after AD and the loss of Aqp4 in the AD makes the ISF drainage obviously slow down, Aqp4 plays an important role in AD to remove the metabolism of waste out of the brain.
Subject(s)
Animals , Rats , Alzheimer Disease/physiopathology , Aquaporin 4/genetics , Brain/physiopathology , Diffusion , Drainage , Extracellular Fluid , Extracellular Space , Gadolinium DTPA , Magnetic Resonance Imaging , Rats, Sprague-DawleyABSTRACT
Both of Zuogui Wan(ZGW) and Liuwei Dihuang Wan(LWDHW) contain ingredients of Sanbufang(SBF), which have been proven to have antiasthmatic effects. In order to study the antiasthmatic effects of the three tonifying kidney-Yin formulas and their mechanisms, BALB/c mice were randomly divided into 5 groups. Chronic asthma was induced by ovalbumin. Mice in treated groups were respectively given 49.0 g•kg⁻¹ZGW, 35.0 g•kg⁻¹LWDHW and 22.4 g•kg⁻¹SBF by gavage. Those in normal and model group were given normal saline. After treatment, sneeze and nose scratching times of mice were observed. Histological lung sections were prepared to determine the basement membrane thickness(BMT), smooth muscle thickness(SMT), collagen area(CA) and numbers of goblet cells(GCN). Western blotting and RT-PCR were used to determine the expression levels of MMP-9, TGF-β1, Smad2, Smad3 and Smad7. The results showed that sneeze and nose scratching times of ZGW group were significantly lower than those of SBF group. Its inhibition degree on airway remodeling was significantly higher than SBF group. Sneeze and nose scratching times of LWDHW group were significantly lower than SBF group. Its CA and GCN were significantly lower than SBF group. Regarding the four airway remodeling related factors, MMP-9, TGF-β1, Smad2 and Smad3 of ZGW group were significantly lower than those of SBF group, and its Smad7 was significantly higher than SBF. Smad7 of LWDHW group was significantly higher than SBF. There was no significant difference in MMP-9 between model group and SBF group. The results indicate that there are significant differences in the antiasthma effect of these tonifying kidney-Yin formulas. The regulatory effects of ZGW and LWDHW on MMP-9 and Smad7 may be correlated with the differences in the inhibitory effect of airway remodeling of the three formulas.
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To investigate the effects of Yupingfeng granule (YPF) on immune factors of the rats with allergic rhinitis (AR) induced by ovalbumin(OVA). OVA 0.3 mg, Al(OH)3 30 mg and saline 1 mL were mixed and intraperitoneally injected for the initial immunization, 4% OVA 200 μg (50 μL) was given to the nose on the 15th day for the second immunization to establish the allergic rhinitis model. Sixty male SD rats were randomly divided into allergic rhinitis(AR) model group, Yupingfeng granule three dose (2.7,1.35,0.68 g•kg⁻¹) groups, control drug Biyankang (0.4 g•kg⁻¹) and normal control group. After 14 days, efforts were made to collect blood from abdominal aorta, and take nasopharynx tissues and fasten them into 10% formaldehyde for a pathological examination. The levels of HIS, IgE, IL-4 and TNF-α in serum were examined by radioimmunoassay, and nasal mucosa tissues were examined by HE staining. According to the results, the levels of HIS, IgE, IL-4 and TNF-α in serum of Yupingfeng granule groups were significantly lower than that of AR model group (P<0.05, P<0.01). Nasal mucosa tissues showed slight morphological changes and inflammatory cell infiltration, with unobvious necrosis. Yupingfeng granule can improve the pathological changes of nasal mucosa tissues, and reduce the production and release of immune factors during allergic rhinitis (AR) process in vivo by OVA, which may be the important curative mechanism of allergic rhinitis.
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<p><b>BACKGROUND</b>Lung cancer is the leading cause of cancer-related death in China. Mutation analysis reveals that LKB1 inactivation is present in 30% of non-small-cell lung cancer (NSCLC), indicating its role as a tumor suppressor. However, the molecular mechanism is still not clear. Our study attempted to establish LKB1 stable knockdown NSCLC cell line, detect alterations in gene expression and identify the genes regulated by LKB1.</p><p><b>METHODS</b>LKB1 stable knockdown H1299 cell line was established using a lentiviral short hairpin RNA. To identify the knockdown effect, LKB1 mRNA and protein expression level were evaluated with quantitative real-time PCR and Western blotting. We treated the cell lines with 2-deoxyglucose to determine if LKB1 protein function was impacted. Gene microarray analysis was performed to detect the gene expression alterations in LKB1 stable knockdown H1299 cells.</p><p><b>RESULTS</b>LKB1 mRNA and protein expression were significantly suppressed in LKB1 stable knockdown H1299 cell line. 2-DG treatment had little impact on the phosphorylation of AMPK, which is the downstream target of LKB1, indicating the loss of function of LKB1. The microarray data showed that LKB1 knockdown resulted in expression alterations of 1243 kinds of genes, including those involved in cell migration, cell proliferation and cell apoptosis.</p><p><b>CONCLUSIONS</b>The establishment of LKB1 stable knockdown H1299 cell line provides us with a great tool to investigate various genes regulated by LKB1 through microarray. The discovery of cell proliferation and migration-related genes regulated by LKB1 is critical for unraveling molecular mechanisms of LKB1's role in the development and metastasis of lung cancer.</p>
Subject(s)
Humans , Blotting, Western , Cell Line, Tumor , Gene Expression Profiling , Methods , Lung Neoplasms , Genetics , Metabolism , Protein Serine-Threonine Kinases , Genetics , Metabolism , Real-Time Polymerase Chain ReactionABSTRACT
A 39-year-old female had been subject to headache, and intermittent seizures for 9 years and decreasing memory for one year, without obvious neurological signs. An MRI revealed a 2x2 cm contrast-enhanced Wiaa in the frontal lobe, with a cyst and peritumoral edema, which was not attached to the dura or falx. Preoperatively, it was diagnosed as a glioma. Total surgical removal of the lesion led to a favorable result. Post-operative histo-pathological examination showed characteristic Antoni A and B areas consistent with intraparenchymal schwannoma. Intraparenchymal schwannoma is an extremely uncommon lesion, which is seen mostly in young adults and children. The main clinical symptoms include rising-intracranial-pressure-related manifestations and associated seizure disorders. The possible developmental origins, histological, imaging features, and protocols of treatment for this entity are discussed
Subject(s)
Humans , Female , Adult , Neurilemmoma/pathology , Neurilemmoma/therapy , Magnetic Resonance Imaging , Frontal Lobe/pathologyABSTRACT
OBJECTIVE@#To establish a new method of SNP typing.@*METHODS@#Based on the principle of allele specific PCR and capillary electrophoresis technique, 11 diallelic SNP loci were selected and two forward primers with different length were designed for each SNP, with their 3' ends matched to the two alleles, respectively. An artificially mismatched base was also introduced into the third or fourth base in the 3' end area of the two forward primers in order to enhance the specificity of amplification. A common reverse primer was designed 100-300 bp away from the forward primers, and labeled with fluorescence. The PCR products were separated and analyzed by ABI Prism 310 Genetic Analyzer after all of the 11 SNPs were multiply amplified.@*RESULTS@#A single product peak was observed while the SNP was homozygous, and two product peaks with different height were observed while the SNP was heterozygous. The length of PCR products was different with the different SNPs. According to the length of the products and the number of the product peaks, the genotypes of the 11 SNPs can simultaneously be analyzed, and the results were in accordance with the direct sequencing.@*CONCLUSION@#Fragment length discrepant allele specific fluorescence labeled multi-PCR (FLDASFLM-PCR) is a simple, rapid and efficient new method for SNP typing.
Subject(s)
Humans , Alleles , Electrophoresis, Capillary/methods , Forensic Genetics , Polymerase Chain Reaction/methods , Polymorphism, Single Nucleotide/geneticsABSTRACT
<p><b>OBJECTIVE</b>To investigate effect of tumor necrosis factor-alpha (TNF-alpha) on the Src-suppressed C kinase substrate (SSeCKS) in C6 glioma cells.</p><p><b>METHODS</b>Cultured C6 glioma cells were randomly divided into two groups. In time-dependent group, cells were cultured with TNF-alpha (2 ng/mL) for 0 h, 1 h, 3 h, 6 h, 12 or 24 h, respectively; in dose-dependent group, cells were cultured with TNF-alpha (0 ng/mL, 0.02 ng/mL, 0.2 ng/mL, or 2 ng/mL) for 6 h. The expression of SSeCKS was detected by Realtime PCR and Western blot analysis, and immunocytochemistry was used to investigate SSeCKS's subcellular localization.</p><p><b>RESULTS</b>TNF-alpha induced rapid phosphorylations of protein kinase C (PKC) substrates in C6 glioma cells, and upregulated SSeCKS expression in a time and concentration dependent manner. Immunocytochemistry suggested that SSeCKS was localized in the cyroplasm and the leading end of podosomal extensions in control groups, while TNF-alpha induced translocation of SSeCKS perinuclear. This effect could be partly reversed by PKC inhibitor Ro-31-8220.</p><p><b>CONCLUSION</b>TNF-alpha activates PKC and upregulates SSeCKS expression in C6 glioma cells. These effects are associated with PKC activity, suggesting that SSeCKS plays a role in response to glia activation in PKC mediated pathway.</p>