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ObjectiveTo observe the effect of Huanglian Jiedutang on the inflammatory injury in the mouse model of acute gouty arthritis （AGA） and to explore the mechanism of Huanglian Jiedutang in treating AGA. MethodForty male C57BL/6J mice were randomized into blank， model， colchicine （0.83 mg·kg-1）， and Huanglian Jiedutang （5 g·kg-1） groups. The mouse model of AGA was established by injecting monosodium urate （MSU） crystals into the ankle joint. The swelling degree of the right ankle joint of each mouse was measured every day for 7 days， and the pathological changes of the ankle joint were detected by hematoxylin-eosin （HE） staining after 7 days. The other 40 C57BL/6J mice were grouped as above. After 18 hours of modeling， the right ankle joint was collected， and real-time polymerase chain reaction was employed to measure the mRNA levels of interleukin （IL）-1β， tumor necrosis factor （TNF）-α， IL-6， NOD-like receptor protein 3 （NLRP3）， apoptosis-associated speck-like protein containing a CARD （ASC）， and Caspase-1. The expression levels of IL-1β， TNF-α， and IL-6 were measured by the enzyme-linked immunosorbent assay. Western blot was employed to determine the protein levels of NLRP3 inflammasome， Toll-like receptor 4 （TLR4）， and nuclear factor-κB （NF-κB）. ResultCompared with the blank group， the model group showed swelling right ankle joint （P<0.01）， obvious foreign body granuloma in the ankle joint with inflammatory cell infiltration. After the treatment with Huanglian Jiedutang， the ankle joint swelling was relieved （P<0.05， P<0.01）， and the size of foreign body granuloma was reduced. Compared with the blank group， the model group showed up-regulated mRNA levels of IL-1β， TNF-α， and IL-6 in the ankle joint tissue （P<0.01）， up-regulated mRNA levels of NLRP3 and Caspase-1 in the NLRP3 inflammasome （P<0.05， P<0.01）， and up-regulated protein levels of NLRP3， Caspase-1， TLR4， and NF-κB （P<0.05， P<0.01）. Huanglian Jiedutang down-regulated the mRNA levels of IL-1β， TNF-α， IL-6， NLRP3， and Caspase-1 （P<0.05， P<0.01） and the protein levels of IL-1β， TNF-α， IL-6， NLRP3， Caspase-1， TLR4， and NF-κB （P<0.05 or P<0.01）. ConclusionInjecting MSU crystal resulted in local inflammatory injury of the joints in the mouse model of AGA. The treatment with Huanglian Jiedutang may alleviate the inflammatory injury by regulating the NLRP3 inflammasome and TLR4/NF-κB signaling pathway.
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Objective:To document the expression of aphasia-related progranulin gene (GRN) in mononuclear cells in the peripheral blood (PBMC) of patients with post-stroke aphasia (PSA).Methods:PC12 cells at the logarithmic-growth stage were cultured and divided into a non-specific interference group (the gene control group) and a specific interference group (the gene silencing group) when the cell density reached 30 to 50%. After the expression of GRN was knocked down in the cells, the occurrence of variable splicing events was analyzed using high-throughput transcriptome sequencing (RNA-seq). Meanwhile, 10 PSA patients were selected into a patient group and 10 healthy counterparts were chosen as a control group. Blood was collected from both groups and real-time fluorescence quantitative polymerase chain reactions (RT-qPCR) were employed to determine any changes in GRN mRNA expression and the occurrence of variable splicing events in the nuclear factor related to kappa-B-binding protein (NFRKB) in their PBMCs. The patient group received conventional speech therapy, and immediately after their first and second blood collections their speech functioning was assessed using the Chinese Aphasia Battery (ABC). Pearson correlation coefficients were then computed relating the GRN expression and ABC scores.Results:After knocking down GRN in the PC12 cells, the expression of GRN in the gene knockdown group was significantly different from that in the control group. There were 237 genes with significant differences in variable splicing between the two samples. The number of genes with variable splicing events at the 5′ end was the largest. There were also significant differences between the groups in the average occurrence of NFRKB variable splicing events. And significant diffe-rences were observed in the mRNA expression of GRN between the two blood collections from the patient group, as well as between the first collection from the patient group and the controls. The average oral expression score of the PSA patients improved significantly, particularly the retelling score. The changes in the GRN expression level were positively correlated with the recovery of oral expression ability.Conclusion:GRN can promote the recovery of speech function in PSA patients by regulating the variable splicing of NFRKB.
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AIM:To explore whether down-regulation of peroxisome proliferator-activated receptor γ coactivator (PGC)-1α induces podocyte apoptosis and its mechanism.METHODS:The podocytes were cultured under high glucose (HG) condition and the cell apoptosis was analyzed by flow cytometry.The methods of real-time PCR and Western blot were used to analyze the mRNA and protein expression of related molecules in the control, HG-treated or siRNA-treated podocytes.RESULTS:The expression PGC-1α at mRNA and protein levels was significantly decreased in HG-injured podocytes.Down-regulation of PGC-1α expression in vitro by siRNA resulted in podocyte apoptosis.The nuclear protein expression of nuclear factor of activated T-cells (NFAT) was significantly increased in HG injured podocytes, indicating the NFAT activation.Down-regulation of PGC-1α expression also decreased the nuclear protein expression of NFAT.Moreover, silencing of NFAT expression by siRNA significantly abolished PGC-1α deficiency-induced podocyte apoptosis.CONCLUSION: Down-regulation of PGC-1α induces podocyte apoptosis.NFAT mediates PGC-1α deficiency-induced podocyte apoptosis.
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BACKGROUND/AIMS: The hepatitis B virus (HBV) genome contains binding sites for hepatocyte nuclear factors (HNF) 3 and 4 in the core domain of enhancer 1 (Enh1), and mutations in this domain have a strong impact on virus replication. We aimed to identify frequent base-mutation sites in the core domain of Enh1 and to examine the impact of these mutations on viral replication. METHODS: We studied virological characteristics and genetic sequences in 387 patients with chronic hepatitis B. We evaluated functional differences associated with specific mutations within the core domain of Enh1. RESULTS: Mutations in the core domain were found with significant frequency in C1126 (122/387 [31.5%], the binding site for HNF3) and in C1134 (106/387 [27.4%], the binding site for HNF4). A single mutation at nt 1126 (C1126) was identified in 17/123 (13.8%), and 105/123 (85.4%) had double mutations (C1126/1134). The level of HBV DNA (log10 copies/mL) was lower in single mutants (C1126, 5.81+/-1.25) than in wild (6.80+/-1.65) and double mutants (C1126/1134, 6.81+/-1.54). Similarly, the relative luciferase activity of C1126 and C1126/C1134 was 0.18 and 1.12 times that of the wild-type virus, respectively. CONCLUSIONS: Mutations in the HNF3 binding site inhibit viral replication, whereas mutations at the HNF4 binding site restore viral replication.
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Humans , Binding Sites , DNA , Genome , Hepatitis B virus , Hepatitis B, Chronic , Hepatitis, Chronic , Hepatocyte Nuclear Factors , Luciferases , Virus Replication , VirusesABSTRACT
Objective To observe the effect of intra-articular injection of CM-chitosan on nuclear factor κB (NF-κB) activation and nitric synthase expression in rat osteoarthritis cartilage,and to explore the mechanism of inhibition of joint destruction.Methods Thirty-six SD rats were randomly divided into three groups:A,B,C,12 in each group.Group A was the sham group,group B,C rats had the medial collateral ligaments cut off and part of medial meniscus were removed to establish osteoarthristis model.Group C rats were injected with 3％ carboxymethyl chitosan intra-articularly 0.15 mg/kg 5 weeks later,and then repeated injection every 1 week.Animals were sacrificed 11 weeks after surgery.The gross changes of cartilage and the expression of NF-κB (P65) were compared by immunohistochemistry,the protein expression of I-κB and P65 in nucleus were detected by Western-bloting.Inducible nitric oxide synthase (iNOS) mRNA and protein expres-sion were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and western blot.The general score of cartilage was analyzed by H test,the rest data was analyzed by one-way ANOVA.Results The cartilage degeneration scores pf group C (intra-articular injection of CM-chitosan group) were significantly less than those of group B.The protein expression of NF-κB of the articular cartilage in group C (106±7)was significantly lower than group B (147±8),the I-κB degradation was inhibited significantly in group C,and the expression of iNOS mRNA and iNOS protein were reduced in OA art~icular cartilage of arthritis rat chondrocytes,therefore,it had protective effect on articular cartilage.Conclusion CMC may inhibit NF-κB signaling pathway by inhibiting the degradation of I-κB in cartilage,which reduces iNOS mRNA and protein expression in rat osteoarthritis cartilage,thereby protects rat osteoarthritis cartilage cells.
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Objective To evaluate the effect of the activity of Ca2+/CaN-NFATc on the activation and prolfferation of lymphocyte in asthmatic rats.Methods The rats of the asthma group and the CsA group were sensitized and challenged by ovalbumin.So did the control group with saline instead.Lymphocyte was separated from spleen and cultured for 24 hours,and PHA-p (5 μg/ml) was added to the culture medium in every group,CsA ( 1.0 μg/ml) was added to the CsA group,respectively.The concentration of [ Ca2+ ] i,the activity of CaN,the protein expression of dephosphorylated NFATc and Cyclin E in T lymphocyte were assayed.The level of IL-4 and IL-2 in culture supernatants was measured,and the cell cycle distribution of lymphocyte was analyzed.ResultsWhen compared to the control group,the activity of Ca2+/CaN-NFATc [ (81.21 4-14.39) vs (63.66 ±9.02) ] was increased and the protein expression of CyclinE [ (0.9327 ±0.0370) vs (0.8374 ±0.0637) ] was higher in Lymphocyte of the asthma group ( P＜0.05,P ＜0.01,respectively).The percentage of lymphocyte in the S phase [ (7.8600±2.8241) vs (4.0270 ± 1.8650) ] and S + G2/M phase [ ( 10.6700±3.3850) vs (5.8740 ± 1.4389) ] was higher;however,the percentage of G0/G1 phase [ (89.3300 ± 3.3850) vs (94.1260± 1.4389 )] was lower in asthma group ( all P ＜ 0.01 ).The level of IL-4 [ ( 1.55 ± 0.19) pg/ml vs (0.99 ± 0.12 ) pg/ml ] and the IL-4/ IL-2 ratio [ (0.81 ±0.12) vs (0.49 ±0.49) ] in culture supernatants of the asthma group were higher than those of the control group ( all P ＜0.01 ).While the activity of Ca2+/CaN-NFATc (47.19 ±7.16)was decreased and the protein expression of Cyclin E (0.6840 ± 0.0485 ) was reduced in lymphocyte in CsA group(all P ＜0.01 ),the percentage of lymphocyte in the S phase (4.8600 ± 1.9595) and S + G2/M phase (7.9900 ± 1.9405) was decreased and the percentage of G0/G1 phase (92.2100 ± 1.9267) was increased ( all P ＜ 0.05 ),and the level of IL-4 (0.47 ± 0.09 ) pg/ml and the ratio of IL-4/ IL-2 (0.78 ±0.20) was lower in culture supernatants in the CsA group than that of the asthma group( all P ＜ 0.01 ).There was a positive correlation between the protein expression of dephosphorylated NFATc and the protein expression of CyclinE in Lymphocyte,so did between the protein expression of NFATc and the level of IL-4 in culture supernatants( r =0.711,P ＜0.01 and r =0.749,P ＜0.01.respectively).Conclusions The activity of Ca2+/CaN-NFATc was increased in lymphocyte of the asthmatic rats.Its increasing might result in the imbalance of Th1/Th2 by promoting the expression of IL-4 and might lead to the proliferation of lymphocyte by promoting the Cyclin E expression.
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Objective To investigate the role of RANKL/RANK/OPG system in bone metabolism of ankylosing spondylitis (AS) by detecting bone mineral density, bone metabolism factors such as osteoprotegerin (OPG), soluble receptor activator of nuclear factors-κB ligand (sRANKL) and the expression of membrane-bound (mb) RANKL in the peripheral blood T lymphocytes. Methods Bone mineral density of AS patients were measured by dual-energy X-ray absorptiometry (DEXA) and serum levels of OPG, sRANKL,tartrate resistant acid phosphatase 5b (TRACP-5b) and bone alkaline phosphatase (BALP) were determined by enzyme-linked immunosorbent assay (ELISA). The percentages of CD4+/RANKL+ and CD8+/RANKL+ in the peripheral blood were detected with flow cytometry. T-test, x2-test were used for statisical analysis. Results ① The incidence of osteopenia and osteoporosis in AS was 47% and 37% respectively. ② Serum RANKL,TRACP-5b levels and RANKL/OPG ratio were higher in AS patients than those in normal controls (P<0.05).But there was no significant difference in OPG and BALP between AS patients and normal controls. ③There were positive linear correlation between serum levels of RANKL and OPG, sRANKL and TRACP-5b, OPG and TRACP-5b in AS (P<0.01). ④ The prevalence of CD4+/RANKL+ cells in the peripheral blood of AS patients was significantly higher than that in the normal controls (P<0.05). Conclusion There is a high incidence of bone loss in AS patients. Increased bone resorbtion is the feature of bone metabolism in AS.RANKL/RANK/OPG system may play an important role. The imbalance of RANKL/RANK/OPG system may be one of the bone loss mechanisms of AS. CD4 + T lymphocyte may play an important role in osteoclasts differentiation and bone resorption in AS by up-regulating the expression of RANKL.
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No abstract available.
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Humans , Osteoporosis , Spondylitis, Ankylosing , Tumor Necrosis Factor-alphaABSTRACT
BACKGROUND: To determine the bone mineral density (BMD), serum soluble receptor activator of the nuclear factors kappa B ligand (sRANKL) and the osteoprotegerin (OPG) levels in patients with ankylosing spondylitis (AS), and to determine their relationship with disease activity indexes. METHODS: The disease activity was evaluated by the Bath Ankylosing Spondylitis Disease Activity Score Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI), Bath Ankylosing Spondylitis Metrology Index (BASMI) and Bath Ankylosing Spondylitis Patient Global Score (BAS-G). The BMD was measured by dual energy X-ray absorptiometry. Serum levels of sRANKL and OPG were measured by the sandwich enzyme-linked immunosorbent assay. RESULTS: Osteoporosis and osteopenia of the femoral neck were found in 33% and 41%, respectively. BMD of femoral neck showed a negative correlation with disease activities. The serum levels of sRANKL were higher in patients with AS than in controls, and the ratio of sRANKL to OPG was also elevated in AS, but had no correlation with disease activity. The sRANKL/OPG ratio tended to be higher in patients with lower BMD. CONCLUSIONS: BMD was reduced in 79% of AS patients and reflected disease status. The sRANKL/OPG ratio was upregulated in patients with AS and it appears to be related to BMD and radiological changes. These results suggest that the imbalance between RANKL and OPG might be involved in the pathogenesis and clinical courses of AS.
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Humans , Absorptiometry, Photon , Baths , Bone Density , Bone Diseases, Metabolic , Enzyme-Linked Immunosorbent Assay , Femur Neck , Osteoporosis , Osteoprotegerin , Spondylitis, AnkylosingABSTRACT
Objective To observe the effect of sinomenine(SN) in vitro on nuclear factor-?B(NF-?B) DNA binding activity and nuclear translocation of synoviocytes in collagen-induced arthritis(CIA) rats and explore its antiinflammatory mechanisms.Methods The experimental model of CIA rats was used and synoviocytes were collected.Cells were divided into five groups:normal control,CIA,CIA+10 ?mol/L methotrexate(MTX),CIA+50 ?mol/L SN,CIA+500 ?mol/L SN.Nuclear translocation of NF-?B p65 subunit and NF-?B DNA binding activity of synoviocytes were investigated by fluorescence labelling laser confocal scanning microscopy and electrophoretic mobility shift assay(EMSA) respectively.Results Compared to normal control,significant nuclear translocation of NF-?B p65 subunit was observed and NF-?B DNA binding activity was increased in synoviocytes of CIA rats(P
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The expressions of HNF-4? and HNF-1? gene were assayed by RT-PCR in the livers of insulin-resistant rats and type 2 diabetic rats. mRNA levels of HNF-4? and HNF-1? in insulin-resistant rats were lower than those in control rats, and these levels were further decreased in diabetic rats. Changes in expressions of these 2 genes seem to be associated with type 2 diabetes mellitus.