OBJECTIVETo study the activation of platelets in children with Kawasaki disease (KD) at molecular level.

METHODSThe expression of platelet surface glycoproteins CD(41), CD(42a), CD(61), CD(62p) and CD(63) in 20 KD patients was measured by flow cytometry before and at 1, 2, 3 week after treatment with aspirin and high-dose (1 approximately 2 g/kg) intravenous gamma-globulin (IVIG).

RESULTSThe expression of glycoprotein CD(41), CD(42a), CD(61), CD(62p) and CD(63) were higher in KD group than in control group. Aspirin and IVIG could not inhibit these high expression of glycoproteins. Higher expression of CD(62p) was observed in patients with coronary artery injury.

CONCLUSIONPlatelets were highly activated in KD patients which may be one of the most important pathophysiological step in KD. It provided a theoretical basis for treatment of KD with antagonist of glycoprotein of platelets. Obviously increase of CD(62p) can be taken as a criterion for predicting coronary artery injury in KD patients.

Aspirin ; pharmacology ; Blood Platelets ; drug effects ; metabolism ; Child ; Child, Preschool ; Coronary Artery Disease ; complications ; metabolism ; Female ; Humans ; Injections, Intravenous ; Male ; Mucocutaneous Lymph Node Syndrome ; metabolism ; pathology ; P-Selectin ; metabolism ; Platelet Activation ; physiology ; Platelet Membrane Glycoproteins ; metabolism ; gamma-Globulins ; pharmacology

Objective To evaluate the effect of small interfering RNA (siRNA)targeting survivin gene on the expression of survivin and proliferation,apoptosis,migration and invasion of a human cutaneous squamous cell carcinoma cell line A431 in vitro.Methods A survivin-specific siRNA was designed and synthesized.Cultured A431 cells were divided into 3 groups to be transfected with 50.0 nmol/L liposome complexes containing survivin-specific siRNA (survivin siRNA group),50.0 nmol/L liposome complexes containing unrelated siRNA (negative control group) and 50.0 nmol/L prepared vesicles (blank control group).Real-time quantitative reverse transcription-PCR (RT-PCR) and Western blot analysis were performed to determine the mRNA and protein expression of survivin in A431 cells,respectively.Methyl thiazolyl tetrazolium (MTT) assay was conducted to evaluate cellular proliferative activity,flow cytometry using annexin-V/propidium iodide (PI) staining to detect cell apoptosis,Transwell assay to estimate migratory and invasive activities of A431 cells,and flow cytometry to detect cell cycle changes.Results At 48 hours after transfection,the mRNA and protein expression of survivin both significantly differed among the survivin siRNA group,negative control group and blank control group (mRNA:0.56 ± 0.15,0.88 ± 0.37,0.90 ± 0.43,F =276.67,P ＜ 0.001;protein:0.59 ± 0.04,0.86 ± 0.05,0.91 ± 0.07,F =243.61,P ＜ 0.001),the survivin siRNA group showed significantly lower mRNA and protein expression of survivin compared with the negative control group and blank control group (all P ＜ 0.05),and there were no significant differences between the negative control group and blank control group (both P ＞ 0.05).Repeated measures analysis of variance showed that the transfection with survivin siRNA could significantly inhibit the proliferation of A431 cells (F =13.19,P =0.004),the proliferation inhibition rate was significantly higher in the survivin siRNA group than in the negative control group and blank control group (both P ＜ 0.05),and no significant difference was observed between the negative control group and blank control group (P ＞ 0.05).At 24 hours after transfection,the apoptosis rate significantly differed among the 3 groups (F =83.97,P =0.002).The survivin siRNA group showed a significantly higher apoptosis rate compared with the negative control group and blank control group (both P ＜ 0.05),and there was no significant difference between the negative control group and blank control group (P ＞ 0.05).At 48 hours after transfection,the survivin siRNA group showed a significantly higher proportion of cells at G2/M phase,but lower number of migratory cells and invasive cells compared with the negative control group and blank control group (all P ＜ 0.05).Conclusion Survivin-specific siRNA can inhibit the expression of survivin gene and the proliferation of A431 cells,promote cell apoptosis,and suppress cell migration and invasion,indicating that survivin may serve as a genetic target for the treatment of cutaneous squamous cell carcinoma.

Objective To evaluate effects of antisense oligonucleotides against microRNA-155 (miRNA-155) on the proliferation,apoptosis,migration and invasion of a human cutaneous squamous cell carcinoma cell line A431.Methods A431 cells were divided into 3 groups:nonsense oligonucleotide group transfected with nonsense control oligonucleotides using liposomes,antisense oligonucleotide group transfected with antisense oligonucleotides against microRNA-155 using liposomes,and blank control group treated with Dulbecco's minimum essential medium (DMEM) containing Lipofectamine 2000.Real-time quantitative polymerase chain reaction (qRT-PCR)was performed to determine the expression of miRNA-155 in A431 cells:Methyl thiazolyl tetrazolium (MTT) assay was conducted to estimate cellular proliferative activity at 24,36,72,96 and 120 hours after transfection,flow cytometry to detect apoptosis and cell cycle changes,and Transwell assay to evaluate the migration and invasion of A431 cells.Statistical analysis was carried out by one-way analysis of variance (ANOVA) for intergroup comparisons and by least significant difference (LSD)-t test for multiple comparisons.Results After transfection,there were significant differences in the expression of miRNA-155 among the nonsense oligonucleotide group,antisense oligonucleotide group and blank control group (0.98 ± 0.02,0.28 ± 0.18,1.00 ± 0.01 respectively,F =634.57,P ＜ 0.001),and the expression of miRNA-155 was significantly lower in the antisense oligonucleotide group than in the blank control group and nonsense oligonucleotide group (both P ＜ 0.05).At 72,96 and 120 hours,there were significant differences in the survival rate of A431 cells among the 3 groups (all P ＜ 0.05),and the antisense oligonucleotide group showed a significantly lower survival rate of A431 cells compared with the blank control group and nonsense oligonucleotide group (all P ＜ 0.05).Additionally,the proportions of cells at G0/G1 phase and at S phase,and the cellular proliferative index all significantly differed among the 3 groups (F =23.46,36.81,19.35,respectively,P ＜ 0.01).The antisense oligonucleotide group showed significantly higher proportion of cells at G0/G1 phase (74.63％ ± 2.13％),but lower proportion of cells at S phase (9.88％ ± 1.83％) and cellular proliferative index (25.36 ± 2.13) compared with the blank control group(62.92％ ± 2.56％,18.86％ ± 2.78％,37.08 ± 2.56,respectively,all P ＜ 0.05) and nonsense oligonucleotide group (63.75％ ± 3.06％,18.33％ ± 3.72％,36.25 ± 3.06,respectively,all P ＜ 0.05).Additionally,the antisense oligonucleotide group showed significantly lower numbers of migratory cells and invasive cells compared with the blank control group and nonsense oligonucleotide group (all P ＜ 0.05).Conclusion Transfection of A431 squamous cell carcinoma cells with antisense miRNA-155 oligonucleotides can decrease the expression of miRNA-155,effectively inhibit the proliferation,migration and invasion of A431 cells,and promote cell apoptosis.

OBJECTIVE： To study the effects of ligustrazine on miR-20b/VEGF and BMP2/Smad1 pathways in subchondral bone of knee osteoarthritis （KOA） model rats， and to investigate the mechanism of ligustrazine for KOA prevention and treatment. METHODS： Totally 18 healthy male SD rats were randomly divided into normal control group， model group and ligustrazine group， with 6 rats in each group. The rats in the latter two groups were used to establish KOA model by intra-articular injection of 4％ papain solution. From the 2nd day after the last injection， ligustrazine group was given intragastrical administration of Ligustrazine suspension （100 mg/kg） 2 mL； normal control group and model group were given intragastrical administration of isometrical normal saline， once a day， for consecutive 6 weeks. After the last after medication， the situation of bilateral knee articular cartilage of rats were observed after exposure. The knee joints of rats were sectioned and stained with HE. The pathological change of articular cartilage were observed by microscope and scored by modified Mankin’s score. mRNA expression of VEGF， BMP2 and Smad1， and the expression of miR-20b were detected by RT-PCR； the protein expression of VEGF， BMP2 and Smad1 were detected by Western blot assay. RESULTS： Model group and ligustrazine group suffered from cartilage injury of knee joint at varying degrees. Compared with normal control group， Mankin’s scores of knee joint and cartilage tissue were increased significantly in model group （P＜0.01）； mRNA and protein expression of BMP and Smad1， the expression of miR-20b in subchondral bone of model group were decreased significantly， while mRNA and protein expression of VEGF were increased significantly （P＜0.01）. Compared with model group， Mankin’s score of cartilage tissue were decreased significantly in ligustrazine group （P＜0.01）； mRNA and protein expression of BMP and Smad1， the expression of miR-20b in subchondral bone were increased significantly， while mRNA and protein expression of VEGF were decreased significantly （P＜0.05 or P＜0.01）. CONCLUSIONS： Ligustrazine can repair damaged articular cartilage in KOA model rats， the mechanism of which may be associated with inhibiting the protein expression of VEGF and activating BMP-2/Smad1 signaling pathway via up-regulating the expression of miR-20b， and promoting the degradation of VEGF mRNA in subchondral bone.

OBJECTIVE：To ex plore the protective effects of Longbie capsule contained serum （called“LBJN”for short ）on the apoptosis of chondrocytes induced by YAP inhibitor verteporfin and its mechanism. METHODS ：Primary human knee osteoarthritis（OA）chondrocytes were extracted by two-step enzymatic digestion ，and then identif ied by toluidine blue staining and type Ⅱ collagen immunofluorescence staining. The effects of 2，5 μmol/L verteporfin alone or combined with 5％LBJN on cell apoptosis were detected by flow cytometry. Solvent control （0.1％ DMSO）and 5％ LBJN were set. Western blot assay was adopted to detect the expression of apoptosis related proteins （YAP，Bcl-2，cleaved-caspase-3） after treated with 0.1％DMSO（solvent control ），2 μmol/L verteporfin，2 μmol/L verteporfin+5％LBJN 和 0（blank control ），2.5％ LBJN and 5％ LBJN for 48 h. The expression of autophagy related proteins （mTOR，Beclin-1，LC3A/B） after treated with 0 （blank control ），2.5％，5％ LBJN for 48 h were det ected by Western blot assay. RESULTS ：The isolated cells accorded with the characteristics of chondrocytes. Compared with 0.1％DMSO， the apoptosis rates of cells were increased significantly after treated with 2，5 μmol/L verteporfin（P＜0.05），and the effects of the two concentrations were similar （P＞0.05）. Compared with verteporfin alone ，2，5 μmol/L verteporfin combined with 5％LBJN could significantly decrease the apoptotic rate of cells （P＜0.05）. Compared with 0.1％DMSO，the protein expression of YAP and Bcl-2 were decreased significantly after treated with 2 μ mol/L verteporfin （P＜0.05）， while the protein expression of cleaved-caspase-3 were increased significantly （P＜0.05）. Compared with 2 μmol/L verteporfin，protein expression of YAP and Bcl-2 were increased significantly after treated with 2 μmol/L verteporfin+5％LBJN（P＜0.05），while the protein expression of cleaved-caspase-3 were decreased significantly （P＜0.05）. Compared with blank control ，the protein expression of YAP ，Bcl-2 and Beclin-1 were increased significantly after treated with 2.5％，5％LBJN（P＜0.05），while protein expression of cleaved-caspase- 3 and mTOR were decreased significantly （P＜0.05）. CONCLUSIONS ：LBJN can block the apoptosis of chondrocytes induced by YAP inhibitor verteporfin ，and its mechanism may be related to regulating the expression of apoptosis related proteins and enhancing autophagy of chondrocytes.