Effect of interleukin-6 receptor (IL-6R) antibody on polymethyl methacrylate (PMMA) bone cement-mediated expression of osteoprotegerin (OPG) and receptor activator of nuclear factor-kappaB ligand (RANKL) in synovial fibroblasts was investigated. Synovial tissue obtained from total knee arthroplasty was digested and cultured. Inverted microscope was employed to observe the synovial cells and immunocytochemistry (SABC method) staining was used to identify synovial fibroblasts. This experiment was divided into three groups according to different culture media: PMMA group (75 μg/mL PMMA bone cement particles), IL-6R antibody group (10 ng/mL IL-6R antibody+75 μg/mL PMMA bone cement particles), and control group (no IL-6R antibody or PMMA bone cement particles). Influence of IL-6R antibody and PMMA on proliferation of synovial fibroblasts was measured by cell counting kit-8 (CCK-8). ELISA method was used to measure OPG and RANKL levels in culture solution. Fluorescence quantitative real-time PCR (FQ-PCR) was used to detect the expression of OPG and RANKL mRNA. After three consecutive passages, more than 95% of the primary synovial cells became long spindle fibroblast-like cells. SABC staining results showed that the fibroblast-like cells were negative for anti-CD68 antibody and positive for anti-vimentin antibody, with brown madder stained. CCK-8 test demonstrated that the absorbance (A) value at 450 nm was significantly lower in IL-6R antibody group than in PMMA group and control group (P<0.01), but there was no statistically significant difference in A value at 450 nm between the control group and PMMA group (P>0.05). Results of ELISA indicated that the expression of OPG was significantly higher in IL-6R antibody group than in PMMA group and control group (P<0.01). The expression of RANKL was inhibited (P<0.05), and the ratio of OPG/RANKL was significantly increased in IL-6R antibody group as compared with PMMA group and control group. There was no significant difference in the expression of OPG between control group and PMMA group (P>0.05), but the expression of RANKL was higher in PMMA group than in control group (P<0.05), and there was a significant difference in the ratio of OPG/RANKL between them (P<0.05). Results of FQ-PCR revealed the expression of RANKL mRNA was significantly inhibited (P<0.01) and the expression of OPG mRNA was significantly increased (P<0.01) in IL-6R antibody group as compared with PMMA group and control group. The expression of RANKL mRNA was higher in PMMA group than in control group (P<0.05), but the expression of OPG mRNA had no significant difference between them (P>0.05). IL-6R antibody could significantly increase the expression of OPG, but inhibit the expression of RANKL, which might provide a theoretical basis of molecular biology for the prevention and treatment of aseptic loosening of prosthesis.

Effect of interleukin-6 receptor (IL-6R) antibody on polymethyl methacrylate (PMMA) bone cement-mediated expression of osteoprotegerin (OPG) and receptor activator of nuclear factor-kappaB ligand (RANKL) in synovial fibroblasts was investigated. Synovial tissue obtained from total knee arthroplasty was digested and cultured. Inverted microscope was employed to observe the synovial cells and immunocytochemistry (SABC method) staining was used to identify synovial fibroblasts. This experiment was divided into three groups according to different culture media: PMMA group (75 μg/mL PMMA bone cement particles), IL-6R antibody group (10 ng/mL IL-6R antibody+75 μg/mL PMMA bone cement particles), and control group (no IL-6R antibody or PMMA bone cement particles). Influence of IL-6R antibody and PMMA on proliferation of synovial fibroblasts was measured by cell counting kit-8 (CCK-8). ELISA method was used to measure OPG and RANKL levels in culture solution. Fluorescence quantitative real-time PCR (FQ-PCR) was used to detect the expression of OPG and RANKL mRNA. After three consecutive passages, more than 95% of the primary synovial cells became long spindle fibroblast-like cells. SABC staining results showed that the fibroblast-like cells were negative for anti-CD68 antibody and positive for anti-vimentin antibody, with brown madder stained. CCK-8 test demonstrated that the absorbance (A) value at 450 nm was significantly lower in IL-6R antibody group than in PMMA group and control group (P<0.01), but there was no statistically significant difference in A value at 450 nm between the control group and PMMA group (P>0.05). Results of ELISA indicated that the expression of OPG was significantly higher in IL-6R antibody group than in PMMA group and control group (P<0.01). The expression of RANKL was inhibited (P<0.05), and the ratio of OPG/RANKL was significantly increased in IL-6R antibody group as compared with PMMA group and control group. There was no significant difference in the expression of OPG between control group and PMMA group (P>0.05), but the expression of RANKL was higher in PMMA group than in control group (P<0.05), and there was a significant difference in the ratio of OPG/RANKL between them (P<0.05). Results of FQ-PCR revealed the expression of RANKL mRNA was significantly inhibited (P<0.01) and the expression of OPG mRNA was significantly increased (P<0.01) in IL-6R antibody group as compared with PMMA group and control group. The expression of RANKL mRNA was higher in PMMA group than in control group (P<0.05), but the expression of OPG mRNA had no significant difference between them (P>0.05). IL-6R antibody could significantly increase the expression of OPG, but inhibit the expression of RANKL, which might provide a theoretical basis of molecular biology for the prevention and treatment of aseptic loosening of prosthesis.
Antibodies ; administration & dosage ; immunology ; Bone Cements ; Fibroblasts ; immunology ; Gene Expression ; drug effects ; Humans ; Osteoprotegerin ; biosynthesis ; genetics ; Polymethyl Methacrylate ; administration & dosage ; Prostheses and Implants ; RANK Ligand ; biosynthesis ; genetics ; metabolism ; Receptors, Interleukin-6 ; immunology ; metabolism ; Synovial Fluid ; immunology ; metabolism

OBJECTIVETo study the expression and localization of nuclear transcription factor Sp1 in macrophages after stimulated by silicon dioxide in vivo and in vitro.

METHODSForty Sprague Dawley rats were randomly divided into the control group and the silica exposure group, 20 in each group. The rat silicosis models were established by direct tracheal instillation of silica into rat lung (0.2 g/kg) only once while the control group was instilled with equal amount of saline. Animals were killed at 1st, 7th, 14th, 21st and 28th day after instillation. Dynamic changes of Sp1 protein expression and its cellular localization were detected by immunohistochemistry in pulmonary macrophages. In vitro, Sp1 mRNA and protein expression and their dynamic changes were monitored by RT-PCR and western blotting after stimulated by silicon dioxide in cultured RAW264.7 macrophages respectively. Cellular localization of Sp1 protein was characterized by immunocytochemistry.

RESULTSCompared to the control group, the Sp1 protein expression was increased in pulmonary macrophages and reached the peak at the 14th day in the silica exposure group. In vitro, the Sp1 mRNA level began to rise at 30 minutes after the administration of silicon dioxide and reached the peak at 240 minutes and then decreased to the minimal level at 960 minutes. The Sp1 total protein and nuclear protein also exhibited the similar trend. The former reached the peak at 240 minutes and the latter at 480 minutes. The significant nuclear translocation of Sp1 protein was observed at 120 minutes after the administration of silicon dioxide and became most significant at 480 minutes.

CONCLUSIONSilicon dioxide can activate nuclear transcription factor Sp1 in macrophages in vivo and in vitro. Sp1 might play an important pathogenic role in the development of silicosis.

Animals ; Cells, Cultured ; Gene Expression Regulation ; drug effects ; Immunohistochemistry ; Macrophages, Alveolar ; drug effects ; metabolism ; Macrophages, Peritoneal ; drug effects ; metabolism ; Male ; RNA, Messenger ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Silicon Dioxide ; pharmacology ; Sp1 Transcription Factor ; biosynthesis ; genetics

OBJECTIVETo study the effects of ulinastatin on coagulation in children who underwent open-heart surgery with cardiopulmonary bypass (CPB).

METHODSFifty children who underwent open-heart surgery for ventricular septal defect were randomly divided into two groups: ulinastatin treatment and control. Before CPB, ulinastatin (1.0×10(4) U/kg) was added to CPB priming fluid only in the ulinastatin treatment group. Activated partial thromboplasin time (APTT), prothrombin time (PT), thrombin time (TT), fibrinogen and international normalized ratio (INR) were measured both before and at 1 hr, 6 hrs and 24 hrs after CPB.

RESULTSThe PT in the ulinastatin group was more prolonged than in the control group at 1 hr after CPB (18.7 ± 0.7 s vs 15.5 ± 0.5 s) and 6 hrs after CPB (17.5 ± 0.6 s vs 15.0 ± 0.6 s). The APTT in the ulinatatin group was also significantly more prolonged than in the control group at 6 hrs after CPB (38.7 ± 3.1 s vs 35.3 ± 3.1 s) and 24 hrs after CPB (34.2 ± 3.0 s vs 31.1 ± 2.6 s).

CONCLUSIONSUlinastatin may prolong PT and APTT after CPB, and thus affects coagulation in children.

Blood Coagulation ; drug effects ; Cardiac Surgical Procedures ; Cardiopulmonary Bypass ; Female ; Glycoproteins ; pharmacology ; Humans ; Infant ; Male ; Partial Thromboplastin Time ; Prothrombin Time ; Trypsin Inhibitors ; pharmacology

OBJECTIVE: To investigate the role of AP-1 in the secretion of Type I collagen in TGF-beta1-stimulated human lung fibroblasts. METHODS: Human lung fibroblasts cell line (HLF-02) was cultured, and then stimulated with 10 microg/L TGF-beta1 at different time points. Curcumin was added into the culture medium to inhibit the AP-1 activity before incubating with TGF-beta1. AP-1 DNA binding activity was assayed by electrophoretic mobility shift assay (EMSA), and the expression of Type I collagen was detected by Western blot and RT-PCR. RESULTS: TGF-beta1 could induce the transcription and secretion of Type I collagen in HLF-02 cells(P<0.05). TGF-beta1 could upregulate the AP-1 DNA binding activity ( P<0.05). Curcumin ( 5, 10, 15, and 20 micromol/L) could inhibit the AP-1 DNA binding activity in TGF-beta1-stimulated cells (the inhibition ratio was 17.1%, 17.6%, 24.2%, and 31.3%; P<0.05). Curcumin (5, 10, 15, and 20 micromol/L) could also inhibit the secretion of Type I collagen significantly (the inhibition ratio was 62.1%, 58.8%, 62.1%, and 59.6%; P<0.05). CONCLUSION AP-1 is responsible for the secretion of TGF-beta1-induced Type I collagen in human lung fibroblasts.
Cell Line ; Collagen Type I ; metabolism ; Fibroblasts ; cytology ; drug effects ; metabolism ; Humans ; Lung ; cytology ; Signal Transduction ; Transcription Factor AP-1 ; metabolism ; Transforming Growth Factor beta1 ; pharmacology

Objective To explore the compliance and clinical effect of anti-inflammation and antipruritic spray on perianal eczema in patients with diarrhea .Methods One hundred and sixty patients with perianal eczema induced by diarrhea were chosen and randomly divided into the observation group and the control groups , each with 80 cases.The control group received anti-inflammation and antipruritic lotion in bath , and the observation group received anti-inflammation and antipruritic spray in external use .The compliance , the improvement of clinical symptoms and clinical effect were compared between two groups .Results The compliance was 97 .50%in the observation group , and was 85% in the control group , and the difference was statistically significant (χ2 =7.83, P<0.01).The total efficiency was 97.5% in the observation group, and was 80%in the control group, and the difference was statistically significant (χ2 =4.42, P<0.05).The improvement of clinical symptoms on papule , pruritus, erosion, exudation, incrustation and furfuration in the observation group was better than that of the control group , and the difference was statistically significant (χ2 =4.10, 28.27, 10.62, 6,62;P<0.05).Conclusions Anti-inflammation and antipruritic spray is effective in the treatment of perianal eczema and easy to be used .

BACKGROUNDThe cannabinoid receptor-2 (CB2) is important for bone remodeling. In this study, we investigated the effects of CB2 selective antagonist (AM630) on receptor activator of nuclear factor kappa B (RANK) ligand (RANKL) induced osteoclast differentiation and the underlying signaling pathway using a monocyte-macrophage cell line-RAW264.7.

METHODSRAW264.7 was cultured with RANKL for 6 days and then treated with AM630 for 24 hours. Mature osteoclasts were measured by tartrate-resistant acid phosphatase (TRAP) staining using a commercial kit. Total ribonucleic acid (RNA) was isolated and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was done to examine the expression of RANK, cathepsin K (CPK) and nuclear factor kappa B (NF-κB). The extracellular signal-regulated kinase (ERK), phosphorylation of ERK (P-ERK) and NF-κB production were tested by Western blotting. The effect of AM630 on RAW264.7 viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay.

RESULTSAM630 did not affect the viability of RAW264.7. However, this CB2 selective antagonist markedly inhibited osteoclast formation and the inhibition rate was dose-dependent. The dose of ≥ 100 nmol/L could reduce TRAP positive cells to the levels that were significantly lower than the control. AM630 suppressed the expression of genes associated with osteoclast differentiation and activation, such as RANK and CPK. An analysis of a signaling pathway showed that AM630 inhibited the RANKL-induced activation of ERK, but not NF-κB.

CONCLUSIONAM630 could inhibit the osteoclastogenesis from RAW264.7 induced with RANKL.

Animals ; Blotting, Western ; Cell Differentiation ; drug effects ; Cell Line ; Cell Survival ; drug effects ; Indoles ; pharmacology ; Mice ; Osteoclasts ; cytology ; drug effects ; metabolism ; RANK Ligand ; pharmacology ; Receptor, Cannabinoid, CB2 ; antagonists & inhibitors ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; drug effects

OBJECTIVETo investigate the mechanism of silicosis by observing the effects of silica on the expression of plasminogen activator inhibitor-1 (PAI-1) and activator protein-1 (AP-1) in human alveolar epithelial cells type II (A549).

METHODSA549 cell and SiO(2) (200 microg /ml) were co-cultured for 0, 4, 8, 16 and 24 h respectively. The reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting and SP immunocytochemistry were used for detections of the PAI-1 mRNA and protein expression. The nucleoprotein and total protein expression of AP-1 were investigated by Western blotting.

RESULTSThe expression levels of PAI-1 mRNA and protein were increased in a time-dependent manner(r(mRNA) = 0.911, r(protein) = 0.902, P < 0.05). The expressions of PAI-1 mRNA and protein in experimental groups were higher than that in control group (P < 0.05) and was the highest in 24 h group [(0.73 +/- 0.01) vs (0.36 +/- .03)]. The nucleoprotein expressions of c-jun/c-fos in experimental groups were also higher than in control group (P < 0.05), and the nucleoprotein expression level of c-jun was the highest in 4 h group [(1.54 +/- 0.02) vs (0.56 +/- 0.03)]; the nucleoprotein expression level of c-fos was the highest in 8 h group [(0.36 +/- 0.01) vs (0.15 +/- 0.01)]. Both c-jun and c-fos expression were decreased after 16 h, but the total protein expression of c-jun/c-fos had no difference in all experimental groups. The positive signal of PAI-1 was located in cytoplasm and nucleus.

CONCLUSIONSiO(2) could induce PAI-1 expression of A549 in a time-dependent manner, and AP-1 activation can be observed in early time.

Alveolar Epithelial Cells ; drug effects ; metabolism ; Cell Line ; Humans ; Plasminogen Activator Inhibitor 1 ; metabolism ; Silicon Dioxide ; toxicity ; Transcription Factor AP-1 ; metabolism

OBJECTIVETo present the clinical application of composite graft of acellular allo-dermis matrix (ADM) with thin auto-microskin on burn wound.

METHODS8 inpatients with 18 full thickness skin burn wounds were transplanted with allo-ADM after eschar was excised, then the auto-microskin and allo-human skin were covered on the area of the matrix, the wound where no allo-ADM grafting were covered as control groups only with auto-microskin and allo-human skin. The area of donor to wound is 1:5 - 1:8.

RESULTSSurvived rate of 18 pieces composite skin that allo-ADM with auto-microskin were grafted were 94%. After following up for 3 to 13 months, the skins of complex grifting had well elastic and smooth texture compared to auto-microskin grafted, they appeared less cicatrisation and ulceration. 3 months after operation, it was indicated by histological examination that tightknit the epithelial-dermal conjunction and epidermal papilla structure could be identified in the allo-ADM skin and there were orderly collagenous fibres, but scar skin structure was observed in that auto-microskin grifted area.

CONCLUSIONThe graft effectiveness of allo-ADM and auto-microskin was better than that of auto-microskin, and this method could be used on major deep burn wound healing.

Adult ; Burns ; surgery ; Female ; Humans ; Male ; Middle Aged ; Skin Transplantation ; methods ; Tissue Donors ; Transplantation, Autologous ; Transplantation, Homologous ; Treatment Outcome