The carboxyl-terminal amino acids 272-299-truncated apoE4 (delta272-299) is the main fragments of apoE4 hydrolysate in neurons. The effects of truncated-ApoE4 (delta272-299) overexpression on tau phosphorylation in cultured N2a cells were investigated. The truncated-apoE4 (delta272-299) cDNA was subcloned into pEGFP-c3 to form recombinant pEGFP-T-apoE4. pEGFP-c3, pEGFP-T-apoE4 and pEGFP-apoE4 were transfected into N2a cells respectively by lipofectamine 2000 method. After 24--48 h, tau phosphorylation was detected by Western blot assay and glycogen synthase kinase-3 (GSK-3) activity by using GSK-3 activity assay. The results showed that the overexpression of both full length-apoE4 and truncated apoE4 fragments in N2a cells induced a dramatic increase in phosphorylation of tau at Ser202 sites and the activation of GSK-3 as compared with untransfected cells, most significantly in the cells transfected with pEGFP-T-apoE4 (P < 0.05). It was concluded that in vitro overexpression of truncated-ApoE4 (delta272-299) can result in tau hyperphosphorylation in N2a cells by activating GSK-3, suggesting truncated-ApoE4 (delta272-299) might contribute the pathogenesis of Alzheimer disease.

BACKGROUND: Puerarin functions to relieve the injury caused by ischemia reperfusion, improve the microcirculation, and inhibit the agglutination of platelet. But it is not clear yet that whether its protection on neurons relates to the apoptosis of cells.OBJECTIVE: To observe the protection of puerarin on neurons injured by mimic ischemia reperfusion in vitro.DESIGN: A random control study. SETTING: Laboratory of Biochemistry and Moleculobiology Department of Preclinical Medicine College of Tongji Medical College of Huazhong University of Science and Technology.PARTICIPANTS: The experiment was done on March 11, 2002. The cultured N2a cells of rat neuromablast were observed.METHODS: Ninety minutes after the cultured N2a cells were put into the 37 ℃ incubator with 0.05 CO2 and 0.95 N2(v/v), puerarin 0.5 mmol/L was added for cuiture for 24 hours. The methyl thiazolyl tetrazolium (MTT)method was used to observe the surviving ability of cells. The Annexin-V staining was adopted to exam the severity of apoptosis in the early stage and the supernate was collected to analyze the activity of lactate dehydrogenase (LDH) to reflect the permeability of cytomembrane. The immunoblotting method was applied to analyze the expression of caspase-3, at the same time, its activity was measured.MAIN OUTCOME MEASURES: The severity of cell apoptosis, the activity of LDH, the expression and activity of caspase-3.RESULTS: Puerarin could improve significantly the surviving rate of N2a cells 24 hours after reperfusion, decrease remarkably the activity of LDH in the culture fluid, lessen obviously the N2a apoptosis (P ＜ 0.01), at the same time, reduce tremendously the activity and expression of caspase-3 induced by ischemia reperfusion (P ＜ 0.01).CONCLUSION: Puerarin can protect nerves, inhibit obviously the N2a apoptosis induced by ischemia reperfusion. The mechanism is that it inhibits greatly the expression and activity of caspase-3.

Alveolar Epithelial Cells ; metabolism ; pathology ; Animals ; Apoptosis ; physiology ; Cell Survival ; physiology ; Cells, Cultured ; Epithelial Cells ; cytology ; physiology ; Genes, bcl-2 ; genetics ; Genes, p53 ; genetics ; Oxidative Stress ; genetics ; Rats

BACKGROUND: Leptin is a kind of polypeptide hormone secreted by fatty tissue, previous studies have shown that leptin plays a certain role in the formation of atherosclerosis.OBJECTIVE: To investigate the effects of leptin on the expression of tumor necrosis factor-alpha (TNF-α) in RAW264.7 cells, and investigate the possible mechanism from the angle of the change of nuclear factor-κB (NF-κB) activity.DESIGN: A controlled observational experiment.SETTING: Department of Biochemistry and Molecular Biology, Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: The experiments were carried out in the Department of Biochemistry and Molecular Biology, Tongji Medical College, and the Department of General Surgery, Union Hospital affiliated to Huazhong University of Science and Technology between April 2005 and February 2006.The cultured RAW264.7 cells were divided into leptin treated groups treated with different concentration (12.5, 25, 50, 100 μg/L), I kappa B kinase inhibitor group and blank control group. Each group had 3 sub-wells,and the experiments were repeated for 3 times.METHODS: Mouse macrophage RAW264.7 cells were incubated into a 6-well plate at the density of 109 cells L-1, cultured in RPMI-1640 culture medium containing bovine serum of 0.1 in volume fraction. When the cells grew to 80%, serum-free culture medium Opti-MEM was changed to culture for another 24 hours, and then the cells were divided into leptin groups treated with different concentration (12.5, 25, 50, 100 μg/L), I kappa B kinase inhibitor group and blank control group. After the cells were incubated with leptin for 4 hours, the expression of TNF-α mRNA expression in RAW264.7 cells was detected with reverse transcription-polymerase chain reaction (RT-PCR). After the RAW264.7 cells were incubated with leptin for 1, 3, 6 and 9 hours, the expression of TNF-c protein expression was detected with double antibody sandwich enzyme-linked immunosorbent assay (ELISA). After the RAW264.7 cells were incubated with leptin for different times, the activity of NF-κB was detected analyzed with electrophoretic mobility shift assay. Another, the RAW264.7 cells were treated with or without 50 μmol/L leptin and/or 100 μmol/L PS1145 (I kappa B kinase specific inhibitor)divided into four groups: blank control group, I kappa B kinase specific inhibitor PS1145 (10 μmol/L) treated group, leptin (50 μmol/L) treated group, leptin (50 μmol/L) + PS1145 (10 μmol/L) group. Aftere incubated for 6 hours, the activity of NF-κB and expression TNF-α mRNA were detected respectively.MAIN OUTCOME MEASURES: ① Effect of leptin of different concentration on the expression of TNF-α mRNA and protein in RAW264.7cells; ② Effect of leptin of different concentration on the activity of NF-κB in RAW264.7 cells; ③ Influence of inhibition I kappa B kinase activity inhibition on expression of TNF-a induced by leptin in RAW264.7cells.RESULTS: ① After the RAW264.7 cells were treated with leptin of different concentration, the TNF-α mRNA level was elevated in a dose-dependent manner, and it reached the peak value emerged in the 50 μg/L leptin treated group. ② The expression of TNF-α protein increased in dose-dependent and time-dependent manners, and it reached the peak val ue at 6 hours in the 50 μg/L leptin treated group. ③ The activity of NF-κB was also positively correlated with the leptin concentration, and it was the highest value at 6 hours treated with 50 μg/L leptin (P ＜ 0.05). ④ I kappa B kinase activity inhibition only partially suppressed the leptin induced elevation of TNF-α expression induced by leptin.CONCLUSION: Leptin can increase the expression and secretion of TNF-α in RAW264.7 cells directly in both dose-dependent and time-dependent manners, and the mechanism may be correlated with the activated NF-κB induced by leptin. It may be one of the mechanisms of atherosclerosis induced by leptin.

Objectives:This paper at measuring the inequity and its influencing factors of medical care utiliza-tion of elderly aged above 60 ( inclusive) . Methods:data comes from 2013 China Health and Retirement Longitudinal Study ( CHARLS) where the population aged 60 and above was selected as the research object. Concentration index ( CI) and its decomposition or centralized curve was used to measure the inequity of medical care services utilization of the elderly, and then the influencing factors of inequity were analyzed by means of the centralized index. Results:The concentration index for outpatient and inpatient service utilization for the elderly was 0 . 0619 and 0 . 1050 , re-spectively, and the concentration curves were below the absolute fair line. The top 2 factors that showed positive con-tribution to the outpatient service utilization included annual per capita consumption expenditure and the pension a-mount. The top 2 factors that showed negative contribution and larger contribution rate to the outpatient service utiliza-tion included New Rural Cooperative Medical Insurance (NRCMI), and Physical Ability in Daily Life (PADL). The top 2 factors that positively and highly contributed to the inpatient service utilization included the household per capita consumption expenditure and the Urban Employees' Basic Medical Insurance (UEBMI). The top 2 factors that nega-tively contributed to the inpatient service utilization included the New Rural Cooperative Medical Insurance ( NRCMI) and the Physical Ability in Daily Life ( PADL) . The horizontal inequity of outpatient and inpatient service utilization was 0. 0739 and 0. 1339, respectively, indicating that there was unfairness in the use of outpatient and inpatient services among elderly. Conclusion:There is inequity of medical care service utilization among the elderly in China. The economic status contributes the largest part of inequity, meaning that it is unfair to the first contribution factor;while the Needs-based fac-tors and New Rural Cooperative Medical Insurance (NRCMI) showed an inequity, narrowing the unfair gap.

OBJECTIVETo observe the efficacy of acupuncture on pain after replantation of severed finger.

METHODSA total of 80 patients who underwent replantation of severed finger were randomly divided into an observation group and a control group, 40 cases in each one. The patients in the control group were treated with postoperative routine care of hand surgery, while patients in the observation group, based on the regular treatment, were treated with acupuncture within first 72 h of surgery. The health side of Yanglingquan (GB 34), Xuehai (SP 10), Hegu (LI 4), Houxi (SI 3) were selected and the needles were retained for 30 min. The acupuncture was given for 6 times. The evaluation was performed by using visual analogue scale (VAS) 2 h, 4 h, 6 h, 12 h, 24 h, 48 h and 72 h after surgery. The use of analgesics after surgery was recorded in the two groups, and the blood supply and survival rate of severed finger were evaluated.

RESULTSCompared between the two groups, the VAS 4 h, 6 h, 12 h, 24 h and 48 h after surgery in the observation group was lower than that in the control group (all P<0. 05); the use frequency of analgesics in the observation group was lower than that in the control group (P<0. 05); the abnormality rate of blood supply in the observation group was lower than that in the control group (P<0. 05).

CONCLUSIONAcupuncture can significantly relieve postoperative pain of replantation of severed finger, and reduce the occurrence rate of abnormal blood supply, which is worthy of clinical promotion.

Acupuncture Points ; Acupuncture Therapy ; Adult ; Female ; Finger Injuries ; complications ; surgery ; Humans ; Male ; Middle Aged ; Pain, Postoperative ; etiology ; therapy ; Replantation

BACKGROUND: The degree of neurofilament (NF) phosphorylation is closely correlated with the occurrence of Alzheimer disease (AD), and apolipoprotein E4 (apoE4) is a generally acknowledged liability factor for AD, but the effect and mechanism of apoE4 on the NF phosphorylation in neurons are not very clear. It has been reported that in the neurons cultured in vitro and in brain tissue of AD patients, the amino acid residues of apoE4 protein C terminal (272-299) could be truncated by hydrolysis,and produce truncated-apoE4 fragment. The latter interacts with the NF phosphorylation in neurofibrillary tangles (NFTs), which are the characteristic pathological changes of AD.OBJECTIVE: To observe the effect of truncated-apoE4 overexpression on the NF phosphorylation in the cultured neurons.DESIGN: A non-randomized controlled observation.SETTING: Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: The experiment was carried out in the Laboratory of Biochemistry and Molecular Biology, Tongji Medical College, Huazhong University of Science and Technology in December 2005. The mice neuroma cell strain N2a was provided by Dr. Xu.METHODS: The truncated-apoE4(△272-299) cDNA was subcloned into pEGFP-c3 to form pEGFP-T-apoE4 recombinant. Then pEGFP-c3, pEGFP-apoE4 and pEGFP-T-apoE4 were transfected into N2a cells by lipofectamine2000 respectively. NF phosphorylation was detected by Western blot assay. The activities of glycogen synthase kinase-3 (GSK-3) and cyclin-dependent kinase 5(CDK-5) were measured.MAIN OUTCOME MEASURES: The degree of NF phosphorylation and the activities of GSK-3 and CDK-5 were mainly observed.RESULTS: In the transfected groups, the contents of phosphorylated NF were significantly increased, the GSK-3 activities were significantly increased, which were the most significant in the pEGFP-T-apoE4 transfected group (P＜0.05), but the CDK-5 activities were not significantly different from that in th e control group (P＞0.05).CONCLUSION: These results indicate that in vitro overexpression of truncated-apoE4(△272-299) can lead to NF hyperphosphorylation by activating of GSK-3 but not CDK-5, which may be the underline mechanism of AD induced by truncated-apoE4(△272-299).

BACKGROUND: One of the key neuropathological changes in Alzheimer disease is that neurofibrils over phosphorylated cytoskeletal protein (such as r and neurofilaments) composed of entwist together, and the phosphorylation of τ protein can be catalyzed by cyclin dependent kinase 5 (CDK5),however whether the phosphorylation of neurofilaments can be catalyzed by CDK5, as well as its role in the pathogenesis of Alzheimer diseases is less acknowledged.OBJECTIVE: To explore the role of over-expression of intracellular CDK5 in the phosphorylation of neurofilamentsDESIGN: Randomized controlled study.SETTING: Biochemical and Molecular Biological Department of Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: This study was conduced at Biochemical and Molecular Biological Department of Tongji Medical College, Huazhong University of Science and Technology between February and May 2001. In vitro cultured rat neuroblastoma cell strain (N2a) was adopted as subjects.METHODS: In vitro cultured N2a cells were divided into 2 groups, namely transfection group and non-transfection group. In transfection group,CDK5 gene was transfected into N2a cell line by using liposome transfection technique so as to obtain N2a/CDK5 cell line stably expressing CDK5, immune-precipitation and enzyme activity assay was used to detect the CDK5 activity, meanwhile immunofluorescence technique and immuneblot assay was used to detect CDK5 expression and phosphorylation of neurofilaments.MAIN OUTCOME MEASURES: Phosphorylation of neurofilaments in both groups.RESULTS: In transfection group of N2a cell line, CDK5 expression increased presented by deep coloration of SMI31 antibody and weak coloration of SMI32 antibody, implying hyper-phosphorylation of neurofilaments. Meanwhile, the activity of CDK5 was 3.5 times higher than that in non-transfection group.CONCLUSION: Intracellular over-expressison of CDK5 would lead to hyperactivity of CDK5 and hyper-phosphorylation of neurofilaments, however the hyper-phosphorylation of neurofilamentsmight invlove in the pathological development of AD.

OBJECTIVE:To systematically review the efficacy and safety of Moxifloxacin(MFX)in the treatment of acute ex-acerbation of chronic bronchitis (AECB),and provide evidence-based reference for Tational use of MFX. METHODS:Retrieved from PubMed,EMBase,Cochrane Library,Medline,CBM,CJFD,VIP and Wanfang Database,the relevant conference proceed-ings and grey literature were also hand-searched,randomized controlled trials(RCT)about MFX(test group)versus other antibiot-ics (control group) in the treatment of AECB were collected. Meta-analysis was performed by using Rev Man 5.2 software with ITT analysis and PP analysis after literature selection,data extraction and quality evaluation. RESULTS:Totally 14 RCTs were in-cluded,involving 6 058 patients. Results of PP analysis showed,the clinical effective rate in test group was similar to that of con-trol group [RR=1.02,95%CI(1.00,1.04),P=0.06],while bacteria clearance rate was significantly higher than control group [RR=1.07,95%CI(1.04,1.11),P<0.001]. Results of ITT analysis showed,the clinical effective rate in test group was significantly high-er than control group [RR=1.03,95%CI(1.00,1.06),P=0.03],while there was no significant difference in the bacteria clearance rate [RR=1.02,95%CI(0.92,1.12),P=0.73] and the incidence of adverse reactions [RR=0.97,95%CI(0.87,1.08),P=0.52] be-tween 2 groups. CONCLUSIONS:The efficacy of MFX is not inferior to other antibiotics in the treatment of AECB,safety is simi-lar to other antibiotics.

OBJECTIVE:To study the effect of catalpol on the activity and apoptosis of osteoclasts (OC) in the osteoblasts (OB)-OC co-culture system and its mechanism. METHODS:OB and OC were isolated respectively from the SD rats of 1-3 days and 5-7 days old to establish OB-OC co-culture system. After treated with 0(blank control),0.05,0.5,5,50 and 100 mg/L catal-pol for 48,72 and 96 h,the number of bone absorption lacuna for OC was observed by inverted microscope to reflect OC activity. After treated with 0(blank control)and 0.05 mg/L catalpol for 48,72 and 96 h,the activity of tartrate resistant acid phosphatase (TRACP)in OC was detected,and the apoptosis rate of OC was calculated. After treated with 0(blank control)and 0.05 mg/L ca-talpol,mRNA expression of osteoprotegerin(OPG)in OB was detected. RESULTS:In OB-OC co-culture system,the number of bone absorption lacuna in 0.05-50 mg/L catalpol groups was significantly lower than blank control group(P<0.01),indicating ca-talpol could inhibit OC activity,especially 0.05 mg/L catapol. Compared with blank control,0.05 mg/L catapol lowered the activity of TRACP but increased the apoptosis rate of OC(P<0.05);mRNA expression of OPG was up-regulated in OB(P<0.01). CON-CLUSIONS:In OB-OC co-culture system,catalpol can inhibit the activity of OC and induce the apoptosis of OC,and its mecha-nism may be associated with the mRNA expression up-regulation of OPG in OB.