Aim To observe the influence of CCK-8 on expression of MMPs/TIMP-1 in TNF-α-induced rat fibroblast-like synovial cell line RSC-364.Methods The secretion levels of MMP-1, MMP-3, MMP-9 and TIMP-1 were determined using ELISA;MMP-3 and MMP-9 mRNA expressions were detected by RT-PCR.Results MMP-3 and MMP-9 could not be examined in RSC-364 incubated with CCK-8 and unstimulated RSC-364, which was able to product a little MMP-1, TIMP-1 and express even less MMP-3,-9 mRNA.CCK-8 inhibited the increase in MMP-1, MMP-3, MMP-9 secretion and MMP-3,-9 mRNA expression in TNF-α-induced RSC-364.TIMP-1 production was also increased in TNF-α-induced RSC-364.CCK-8 had no effect on TIMP-1 production in TNF-α-induced RSC-364, but was able to reduce the ratios of MMP-1, MMP-3, MMP-9 to TIMP-1.Conclusion The inhibitory effect of CCK-8 on MMPs activity may be related to the decrease of MMPs mRNA expression, MMPs secretion and the ratios of MMPs to TIMP-1 in TNF-α-induced RSC-364, which indicates that CCK-8 might be a possible regulator in the pathogenesis of rheumatoid arthritis.

Objective To explore the inhibitory effect of harmine on melanoma A375 cells and its mechanism thereof.Methods (1) Melanoma A375 cells were treated with harmine at 0,0.5,1,2,5,10,20,50 and 100 mg/L for 48 h in vitro.CCK-8 method was used to detect the cell viability and confirm the experimental concentrations.(2) After the cells were treated with 0,1,2 mg/L harmine,the scratch and transwell assays were used to detect the cell migration and invasion ability.Western blot assay was used to detect the expression levels of epithelial mesenchymal transition (EMT)-related protein E-cadherin,N-cadherin,Snail and p53.(3) Three groups of ceils were set up.The control group was transfected with empty vector ordy.The empty vector group was transfected with empty vector after treated with 2 mg/L harmine for 24 h.The Snail transfection group was transfected with Snail cDNA after treated with 2 mg/L harmine for 24 h.The cell migration and invasion ability were detected after the transfection.Results (1) When the concentration of harmine was above 2 mg/L,the survival rate of A375 cells was significantly lower than that of the control group with the increase of harmine concentrations (P ＜ 0.05).Then,the concentrations of 0,1 and 2 mg/L of harmine were used in the following experiments.(2) With the increase of the harmine concentrations,the number of cells in the scratched area and the number of trans-membrane cells in each group were significantly decreased.The migration and invasion ability of the ceils were decreased gradually.The expression levels of E-cadherin and p53 were increased,while the expression levels of N-cadherin and Snail were decreased.(3) Cell transfection experiments showed that the migration and invasion ability of the cells were increased compared with those of empty vector group after transfection with Snail.Conclusion Harmine can inhibit the proliferation of A375 cells and decrease the abilities of metastasis and invasion,which may be achieved by decreasing the expression of Snail after activating the p53,thereby increasing E-cadherin and down-regulating N-cadherin to inhibit the EMT process.

BACKGROUND:Recently,researches have found that insulin-like growth factor-1(IGF-1)can induce the differentiation of bone marrow-derived mesenchymal stem cells(BMSCs)into chondrocytes,but there are no reports concerning the differentiation of adipose-derived mesenchymal stem cells(ADMSCs)into chondrocytes induced by IGF-1,as well as interaction with transforming growth factor-β1(TGF-β1)during this process.OBJECTIVE:To explore the possibility of inducing ADMSCs chondrogenic differentiation by using IGF-1 and the interaction with TGF-β1 in induction.METHODS:ADMSCs were obtained,and seeded at 2×10~5 cells/cm~2 in culture flask.Insulin-free chondrogenic media containing IGF-1 or(and)TGF-β1 were used to induce ADMSCs.2 weeks later,cells were harvested and stained by using toluidine blue and collagen Ⅱ antibody immunohistochemistry.Intracellular sulfated proteoglycan and collagen Ⅱ coloring were observed.Reverse transcription-polymerase chain reaction(RT-PCR)was used to detect the expression of collagen Ⅱ,aggrecan and Sox9 mRNA.RESULTS AND CONCLUSION:After induced,toluidine blue stain exhibited that the cells in the three induction groups were polygonal,with cytoplasm and cell membrane of blue different dyeing.Immunohistochemistry for type Ⅱ collagen demonstrated that cytoplasm and cell membrane were stained brown in three induction groups.RT-PCR revealed that the expression of collagen Ⅱ,aggrecan,Sox9 mRNA of IGF + TGF group were significantly greater than the IGF and TGF groups,and IGF and TGF groups were significantly stronger than the control group.No significant difference was determined between the IGF and TGF groups.These results indicated that IGF-1 can induce chondrogenic differentiation from ADMSCs,expressing chondrocyte specific cell phenotype.There is synergism of IGF-1 and TGF-01 to induce the differentiation of ADMSCs into chondrocytes.

AIM: To investigate the effect of sulfated cholecystokinin octapeptide (CCK -8 ) on TNF -α induced IL - 6 mRNA expression, NF - κB activation in the rat fibroblast - like synovial cell strain RSC - 364 and its possible receptor mechanisms. METHODS: RSC -364 cells were stimulated with TNF - α( 10 μg/L) in the presence or absence of sCCK- 8( 10-8 - 10-6 mol/L) or/and CCK receptor antagonist proglumide(2 mg/L). IL -6 and CCK receptor A/B (CCK- AR/CCK/BR) mRNA expression were assayed by reverse transcription polymerase chain reaction (RT- PCR) at 3 h after stimulation, and nuclear factor - κB (NF - κB) binding activity was analyzed by electrophoretic mobility shift assay (EMSA) at lh after stimulation. At 30 min of stimulation the IκB protein level in cytoplasma was measured by Western blotting. RESULTS: Both CCK - AR and CCK - BR were constitutively expressed on RSC - 364. sCCK - 8, at concentrations from 10-8 mol/L to 10 -6 mol/L, significantly increased IL - 6 mRNA expression, CCK - AR and CCK - BR mRNA expression, NF - κB binding activity and IκB protein degradation. The effects of sCCK - 8 on NF - κB activity and IκB degradation level were attenuated by CCK receptor antagonist proglumide. CONCLUSION: sCCK - 8 upregulats TNF - α- induced IL - 6 mRNA expression by NF - κB pathway through its receptor on rat synoviocytes, suggesting its possible regulatory role in the pathogenesis of rheumatoid arthritis.

OBJECTIVETo investigate the role of nuclear factor kappa B (NF-kappaB) pathway inhibition in lipopolysaccharide (LPS)-stimulated apoptosis of polymorphonuclear neutrophils (PMNs).

METHODSRats with acute lung injury induced by LPS intratracheal instillation and cultured human venous PMNs were studied. Pyrrolidine dithiocarbamate (PDTC) and gliotoxin were used as NF-kappaB inhibitors. Additionally, to explore the role of extracellularly regulated protein kinase as an upstream signal in NF-kappaB pathway on regulating LPS-stimulated PMN apoptosis, PD098059, the specific inhibitor of extracellularly regulated protein kinase, was also applied. The lung injury was determined by protein content and PMN numbers in bronchoalveolar lavage fluid. PMN apoptosis was measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) end labeling and DNA fragmentation. IkappaBalpha degradation was analyzed by Western blot. NF-kappaB DNA binding activity was detected by an electrophoretic mobility shift assay.

RESULTS(1) The increase of protein content and PMN numbers in bronchoalveolar lavage fluid induced by LPS (100 micro g per rat) intratracheal instillation were alleviated by PDTC (50, 100, or 200 mg/kg, i.p.) in a dose-dependent manner. (2) PMNs apoptosis in vivo or in vitro was delayed by LPS, and accelerated by PDTC, gliotoxin or PD098059 pretreatment. (3) IkappaBalpha degradation and increased NF-kappaB DNA binding activity mediated by LPS were inhibited by PDTC, gliotoxin or PD098059 pretreatment.

CONCLUSIONInhibition of either NF-kappaB itself or the upstream signals in NF-kappaB pathway such as extracellularly regulated protein kinases has therapeutic effect on LPS-induced acute lung injury, in which the dysregulation of PMN apoptosis plays an important role.

Animals ; Apoptosis ; Cells, Cultured ; Humans ; Lipopolysaccharides ; pharmacology ; NF-kappa B ; physiology ; Neutrophils ; drug effects ; physiology ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; physiology

AIM:To investigate the effect of sulfated cholecystokinin octapeptide(CCK-8) on TNF-? induced IL-6 mRNA expression,NF-?B activation in the rat fibroblast-like synovial cell strain RSC-364 and its possible receptor mechanisms.METHODS:RSC-364 cells were stimulated with TNF-?(10 ?g/L) in the presence or absence of sCCK-8(10-8-10-6 mol/L) or/and CCK receptor antagonist proglumide(2 mg/L).IL-6 and CCK receptor A/B(CCK-AR/CCK/BR) mRNA expression were assayed by reverse transcription polymerase chain reaction(RT-PCR) at 3 h after stimulation,and nuclear factor-?B(NF-?B) binding activity was analyzed by electrophoretic mobility shift assay(EMSA) at 1h after stimulation.At 30 min of stimulation the I?B protein level in cytoplasma was measured by Western blotting.RESULTS:Both CCK-AR and CCK-BR were constitutively expressed on RSC-364.sCCK-8,at concentrations from 10-8 mol/L to 10-6 mol/L,significantly increased IL-6 mRNA expression,CCK-AR and CCK-BR mRNA expression,NF-?B binding activity and I?B protein degradation.The effects of sCCK-8 on NF-?B activity and I?B degradation level were attenuated by CCK receptor antagonist proglumide.CONCLUSION:sCCK-8 upregulats TNF-?-induced IL-6 mRNA expression by NF-?B pathway through its receptor on rat synoviocytes,suggesting its possible regulatory role in the pathogenesis of rheumatoid arthritis.

Seventy-three patients with type 2 diabetes mellitus were divided into atheroselerosis(AS) group and non-AS group according to the intima-media thickness(IMT)of the carotid artery.The plasma visfatin level in AS group was higher than that in non-As group[(44.95±10.14 vs 34.52±9.08)μg/L,P<0.05],and both of them were higher than that of the control [(24.46±7.18)μg/L,both P<0.05 ].The visfatin level Was positively correlated with IMT,waist-to-hip ratio,visceral fat thickness,fasting insulin,and HOMA insulin resistance index.Age,duration of diabetes,HbA_(1C),and visfatin level were the major risk factors for IMT of the carotid artery.

Objective To investigate the possible role of plasma leptin and total cholesterol in the pathophysiology of attempted suicide in depressive episode patients. Methods The subjects were 25 depressive episode patients who had recently attempted suicide and 30 depressive episode patients without suicide attempt. The Hamilton Depression Rating Scale-24 items ( HAMD24 ), Beck Helpless Rating Scale (BHS) and Self-rating Idea of Suicide Scale (SIOSS) were used to evaluate the severity of symptoms. In addition, 32 individuals who took part in health examination were selected as health control group. Body height and weight were measured to get body mass index (BMI) of all objects, and plasma leptin and total cholesterol were measured by venous blood before taking pill. Results (1)HAMD score , BHS score and SIOSS score in patients with attempted suicide were higher than patients without suicide (P ＜ 0.01 ). (2) The total cholesterol and plasma leptin in patients with attempted suicide ( (3.3 ±0.9)mmol/L, (6.1 ±3.7)μg/L)were lower than that in patients without suicide( (3.6± 1.2)mmol/L, (9.4 ± 4.4)μg/L; P ＜ 0.05 ～ 0. 0l ), while total cholesterol and leptin in patients without suicide attempt ( (3.6 ± 1.2 ) mmol/L, ( 9.4 ± 4.4 ) μg/L) were lower than that in normal controls ( ( 4.8 ± 0.9 )mmol/L, ( 13.4 ± 6.7 ) μg/L; P ＜ 0.05 ～ 0.01 ). (3) Plasma leptin and total cholesterol of all patients were positively correlated with BMI(P＜0.01 ). Moreover, plasma leptin and total cholesterol in patients with attempted suicide and patients without attempted suicide were inversely positively correlated with HAMD score , BHS score and SIOSS score. Plasma leptin was significantly positively correlated with total cholesterol in patients with attempted suicide and patients without suicide, but there was no significant difference in normal controls(P＞ 0. 05). Conclusion The results suggest that decreased plasma leptin is related to the pathophysiology of attempted suicide in depressive episode patients.

Objective:To analyze the focuses and shortcomings of the long-term care policy for disabled elderlies in China, for references to optimize such policies.Methods:The data came from the policy documents on long-term care for disabled elderlies published on respective official websites of various ministries and commissions from January 1, 2013 to April 20, 2022. Text quantitative analysis was used to extract policy clauses, and three types of policy tools(including 13 types of sub-tools) were used to analyze these provisions.Results:132 clauses were extracted from a total of 34 policy documents, of which the number of clauses of supply, demand and environmental policy tools were 28(21.21%), 25(18.94%) and 79(59.85%) respectively. There were few clauses on scientific and technological information support(5) and capital investment(3) in the supply-type policy tools; In terms of demand-based policy tools, there were only a few clauses for government purchase(5), pilot projects(3) and service outsourcing(2); In terms of environmental policy tools, there were only a few clauses for standard design(9) and supervision and management(7).Conclusions:The long-term care policy structure of the disabled elderly in China was not balanced, the supply-based policy tools had less scientific and technological information support, the demand-based policy tools were short and single, and the environment-based policy tools were widely used but lack of standard design. Relevant policy-making departments should further improve the accuracy of policy targets, optimize the internal structure of policy tools, pay attention to the application of scientific and technological information policies and clarify the relevant standards of care services.

Objective:To verify the clinical safety and efficacy of new intelligent ventilation mode adaptive minute ventilation (AMV)+IntelliCycle ventilation in patients with mild-to-moderate acute respiratory distress syndrome (ARDS).Methods:The patients with mild-to-moderate ARDS, admitted to intensive care unit (ICU) of the First Affiliated Hospital of Jinzhou Medical University from February 2018 to February 2019, were enrolled in the study. The patients were divided into synchronous intermittent mandatory ventilation+pressure support ventilation (SIMV+PSV) group and AMV+IntelliCycle group according to the random number table method. All patients were given mechanical ventilation, anti-infection, analgesia and sedation, nutritional support and symptomatic treatment of primary disease after admission. SV800 ventilator was used for mechanical ventilation. In the AMV+IntelliCycle group, after setting the minute ventilation volume (VE), inhaled oxygen concentration (FiO 2) and positive end expiratory pressure (PEEP), the ventilator was turned on the full-automatic mode, and the preset value of VE percentage was 120%. In the SIMV+PSV group, the ventilator parameters were set as follows: the ventilation frequency was 12-20 times/min, the inspiratory expiratory ratio was 1∶1-2, the peak inspiratory pressure (PIP) limit level was 35-45 cmH 2O (1 cmH 2O = 0.098 kPa), and the setting of FiO 2 and PEEP was as the same as that of AMV+IntelliCycle group, the triggering flow was set to 2 L/min. All of the clinical parameters between the two groups were compared. The main outcomes were duration of mechanical ventilation, ventilator alarm times, manual operation times, and the mechanical power; the secondary outcomes were respiratory rate (RR), VE, tidal volume (VT), PIP, mouth occlusion pressure (P0.1), static compliance (Cst), work of breathing (WOB), and time constant at 0, 6, 12, 24, 48, 72, and 120 hours; and the blood gas analysis parameters of patients before and after ventilation were recorded. Results:A total of 92 patients with mild-to-moderate ARDS were admitted during the study period, excluding those who quit the study due to death, abandonment of treatment, accidental extubation of tracheal intubation and so on. Eighty patients were finally enrolled in the analysis, with 40 patients in SIMV+PSV group and AMV+IntelliCycle group respectively. ① Results of main outcomes: compared with the SIMV+PSV mode, AMV+IntelliCycle ventilation mode could shorten the duration of mechanical ventilation (hours: 106.35±55.03 vs. 136.50±73.78), reduce ventilator alarm times (times: 10.35±5.87 vs. 13.93±6.87) and the manual operations times (times: 4.25±2.01 vs. 6.83±3.75), and decrease the mechanical power (J/min: 12.88±4.67 vs. 16.35±5.04, all P < 0.05). But the arterial partial pressure of carbon dioxide (PaCO 2) of AMV+IntelliCycle group was significantly higher than that of SIMV+PSV group [mmHg (1 mmHg = 0.133 kPa): 41.58±6.81 vs. 38.45±5.77, P < 0.05]. ② Results of secondary outcomes: the RR of both groups was improved significantly with the prolongation of ventilation time which showed a time effect ( F = 4.131, P = 0.005). Moreover, compared with SIMV+PSV mode, AMV+IntelliCycle mode could maintain a better level of RR, with intervention effect ( F = 5.008, P = 0.031), but no interaction effect was found ( F = 2.489, P = 0.055). There was no significant difference in VE, PIP, P0.1 or Cst between the two groups, without intervention effect ( F values were 3.343, 2.047, 0.496, 1.456, respectively, all P > 0.05), but they were significantly improved with the prolongation of ventilation time in both groups, with time effect ( F values were 2.923, 12.870, 23.120, 7.851, respectively, all P < 0.05), but no interaction effect was found ( F values were 1.571, 1.291, 0.300, 0.354, respectively, all P > 0.05). The VT, WOB or time constant in both groups showed no significant changes with the prolongation of ventilation time, and no significant difference was found between the two groups, there was neither time effect ( F values were 0.613, 1.049, 2.087, respectively, all P > 0.05) nor intervention effect ( F values were 1.459, 0.514, 0.923, respectively, all P > 0.05). Conclusion:AMV+IntelliCycle ventilation mode can shorten the ventilation time of patients with mild-to-moderate ARDS, reduce mechanical power, and reduce the workload of medical care, but PaCO 2 in the patients with AMV+IntelliCycle mode is higher than that in the patients with SIMV+PSV mode.