The rate of corneal graft rejection is still high for high-risk keratoplasty although immune suppression drug is routinely used.The role of traditional Chinese medicine in corneal transplantation is concerned gradually.Heijingpaichitang on the prevention and treatment of rats with high-risk corneal allograft rejection needs further study.Objective This study was to investigate the inhibitory effect of heijingpaichitang on high-risk corneal transplantation immune rejection in rats.Methods Sixteen female SD rats were used as the donors and 32female Wistar rats were served as recipients.The high-risk corneal trasplantation models were established by corneal suture in 32 Wistar rats,and then homogeneity variant SD-Wistar corneal transplantation was performed.The recipients were randomized into model control group,cyclosporinc A (CsA)group,heijingpaichitang group and CsA +heijingpaichitang group.CsA,heijingpaichitang and CsA + heijingpaichitang was orally administered 4 days after operation once per day for 15 days,and normal saline solution was used at the same way in the model control group.Ocular anterior segment reaction was examined under the slit lamp and corneal opacification,edema and neovasculation were scored based on Larkin' s criteria.Rejection index of the corneal graft was recorded and the graft survival time was calculated.The pathological examination of the corneal graft was carried out in all rats,and the inflammatory cells in the corneas and CD4+ cells in the periphery blood were assayed using flow cytometry.The use of the animals complied with ARVO Statement.Results Corneal graft rejection occurred in 10 days after operation in the model control group,12-13 days in the CsA group and heijingpaichitang group and 22 days in the CsA +heijingpaichitang group.Compared with model control group,the scores of the corneal opacification,corneal edema and neovascularization were significantly lower in the CsA group,heijingpaichitang group and CsA+heijingpaichitang group (P＜0.05),and all the scores were declined in the CsA+ heijingpaichitang group compared with CsA group and heijingpaichitang group(P＜0.01),but no significant differences were seen in the scores between the CsA group and heijingpaichitang group(P＞0.05).The mean survival time of grafts was (10.38 ±1.69)days in the model control group,(22.50 ± 3.07) days in the CsA + heijingpaichitang group,with the significant difference (t =-9.790,P =0.000).The pathological examination of graft showed that the lymphocytes and new blood vessels were less in the CsA+heijingpaichitang group compared with CsA group and heijingpaichitang group 15 days after operation.Flow cytometry verified that the number of lymphocytes in graft,CD4+cells and CD4+/CD8+ in periphery blood were significantly lower in the heijingpaichitang group,CsA group and CsA+heijingpaichitang group compared with model control group (P＜0.05).Conclusions Heijingpaichitang can inhibit immune rejection to certain extent in high-risk corneal transplantation rat and has a similar effect to 0.1％ CsA.Heijingpaichitang and 0.1％ CsA have a synergistic effect.

Objective To explore the influence of coal-arsenic exposure on human T cells proliferation and its mechanism.Methods Blood samples colleoted from individuals which lived in arsenism area of coal-burning type and non-arsenism area in Guizhou Province were divided into exposed group(17),mild(35),moderate(38) and severe arsenism group(19)and control group(35)according to Diagnosis Smndard for Endemic Arsenism (WS/T 211-2001).T cell stimulation index wag determined by methyl thiazolyl tetrazolium(MTT)colorimetric method.The intracellular Ca2+ exponential(IECa2+)in peripheral blood mononuclear cell(PBMC)was analyzed by Fho-3/AM dye and flow cytometry.DNA binding activity of actively T cells nuclear factor(NF-AT)in PBMC was evaluated by electrophoretie mobility shift assay(EMSA).Results Concanavalin A(ConA)stimulation decreased the T cells stimulation indexes in exposed group,mild,moderate and severe arsenism groups(1.315±0.962, 1.611±1.224,1.114±0.545,1.289±0.875)compared with control group(2.322±1.241),all the differences being statistically significant(P＜0.01).After stimulated by anti-CD3 monoclonal antibody(McAb),the T cells stimulation index in exposed group,mild,moderate and severe arsenism group(0.997±0.177,1.103±0.291,1.007±0.221, 0.957±0.205) were lower than that of control group(1.842±0.429,P ＜ 0.01 ). IECa2+ of PBMC after treated by anti-CD3 McAb in mild,moderate and severe arsenism group( 110.130±49.637,92.429±31.191,77.640± 35.372) were lower compared with control group(145.986±59.450,P ＜0.01 ). Moreover,IECa2+ in moderat and severe arsenism group were lower than exposed group(121.337±46.410,P ＜ 0.05). DNA binding activity of PBMC NF-AT in mild,moderate and severe arsenism group(1.354±0.446,1.290±0.291,1.159±0.411 ) were lowered than that of control group(1.722±0.291,P ＜ 0.01) and exposed group(1.611±0.294,P ＜ 0.05). Conclusions The coal-arsenic exposure can reduce the human T cells stimulation indexes,IECa2+ in PBMC and the DNA binding activity of NF-AT. It suggest that arsenic may suppress the proliferation ability of human T cells,which may be partly related to the influence of arsenic on T cell receptor(TCR)/CD3 signal transduetion pathway.

Objective To investigate the activation of T lymphocytes in human peripheral blood and the signaling molecules in protein kinase C/nuclear factor KB(PKC/NF-κB) pathway expressivity or activity changes in human peripheral blood mononuclear cells(PBMCs) exposed to coal-arsenic,to explore the role of PKC/NF-κB signal pathway in activation of T cells in human exposed to coal-arsenic. Methods Blood samples were collected from individuals who lived in arsenism area of coal-burning in Guizhou province, and were divided into asymptomatically exposed group (12),mild arsenism group (33),moderate arsenism group (34) and severe arscnism group (15) according to Diagnosis Standard for Endemic Arsenism (WS/T 211-2001). The individuals who lived in non-arsenism area were control group(27). The ratio of activated T ceils was analyzed by flow cytometry. DNA binding activity of NF-κB in PBMCs was evaluated by electrophoretic mobility shift assay(EMSA). The expression of PKCθ and phospho-PKCθ(pPKCθ) in PBMCs were detected with western blotting analysis. Results The ratio of activating T cells in asymptomatically exposed group[(21.76±15.31)%],mild arsenism group[(18.41±11.36)%],moderate arsenism group[(17.78±11.93)%]and severe arsenism group[(18.79±13.38)%]were all higher than that of control group[(3.19±2.12)%],the difference among all groups being statistically significant(F = 7.893,P < 0.05). DNA binding activity of PBMCs NF-κB in asymptomatically exposed group,mild arsenism group,moderate arsenism group and severe arsenism group(1.49±0.24,1.58±0.30,1.57±0.34,1.51±0.16) were higher than that of the control group(1.30±0.17),the difference being statistically sign/ficant(P < 0.05 or < 0.01). The expression of PBMCs pPKCθ in mild arsenism group,moderate arsenism group and severe arsenism group(0.64± 0.14,0.64±0.27,0.62±0.12) were all lower than that of the control group(0.93±0.20),the difference being statistically significant(P < 0.05). There were significant negative correlations between the expression of pPKCθ and the activity of NF-κB(r =-0.565,P < 0.01). There were significant positive correlations between the activity of NF-κB and the ratio of activating T cells(r = 0.546,P < 0.01). Conclusion Coal-arsenic enhances the DNA binding activity of NF-κB,reduces the expression of PBMCs pPKCθ in human PBMCs and up-regulates the activity of T cells. It suggests that the PKC/NF-κB signal might be one of transduction pathway via activating of T cells by coal-arsenic.

Objective To detect genetic polymorphism of myeloperoxidase (MPO) gene and catalase (CAT) gene and their activities, and to analyze their relationship with arsenic poisoning caused by coal-burning. Methods One hundred and thirty arsenic poisoning patients were chosen as case group in Jiaole Village, Xingren County, Guizhou Province(an endemic area). One hundred and forty healthy residents living in 13 km away were chosen as control group. Their blood was collected. Polymerase chain reaction-restriction fragment length polymorphism technique(PCR-RFLP) was used to detect polymorphism of MPO-463G/A and CAT-262C/T. Ultraviolet spectmphotometer method was used to detect myeloperoxidase activity. Chromatometry method was used to detect catalase activity. Results The genotype frequency of MPO-463G/A at GG, GA, AA site was 47.24%(60/127), 44.09%(56/127),8.67% (11/127) in case group and 42.34% (58/137),48.17% (66/137)1,9.49% (13/137) in control group, respectively. The difference between the two groups was not significant(χ2 = 0.642, P > 0.05). The genotype frequency of CAT-262C/T, at CC, CT, TT site was 65.60%(82/125),28.80%(36/125),5.60%(7/125) in case group and 76.51%(101/132), 18.94% (25/132) ,4.55% (6/132) in control group, respectively, without significant difference (χ2 =3.845, P>0.05). The relationship between polymorphism of MPO-463G/A and CAT-262C/T and the risk of arsenic poisoning was not found in this study(ORadj= 1.36, 95%CI: 0.74-2.50 for MPO; ORadj=1.35, 95%CI: 0.69-2.63 for CAT). The activities of MPO and CAT were (25.30±8.70)U/L and (2.80± 1.09)×103 U/L in case group, while (22.76±7.59)U/L and (3.90±1.01)×103U/L in control group with a significant difference(F=0.760 for MPO, F=0.855 for CAT, all P < 0.05). The genotype of MPO-463G/A and CAT-262C/T was not found to have relationship with the activities of MPO, CAT(F=1.312,2.822 for MPO; F= 0.151,0.036 for CAT, P>0.05). Conclusions Genetic polymorphism of MPO-463G/A and CAT-262C/T is not found to have relationship with arsenic poisoning. Arsenic can lead to the change of MPO and CAT activity, which, however, may not be affected by MPO-463G/A and CAT-262C/T polymorphism.

Objective: To observe the effects of electroacupuncture (EA) on the protein and gene expressions of Bax, Caspase-3 and Bcl-2 in cerebral cortex of type 2 diabetic rats with cognitive impairment (CI), and to explore the mechanism of EA in improving the learning and memory abilities. Methods: A total of 100 Sprague-Dawley (SD) rats were divided into a normal group (n=10) and a model group (n=90) by random number table method. Rats in the model group were intraperitoneally injected with a small dose of streptozotocin (STZ) to establish the type 2 diabetic models, after being fed with high-fat and high-sugar diet for 1 month. Twenty CI rats were selected from the 50 successful model rats by the Morris water maze (MWM) test and randomly divided into a model group and an EA group according to the blood glucose level and MWM data (n=10). Rats in the EA group received acupuncture at Zusanli (ST 36), Neiting (ST 44) and Yishu (Extra), of which Zusanli (ST 36) and Neiting (ST 44) were stimulated by EA apparatus, 20 min/time, once a day for 6 d a week and 4 consecutive weeks. The rats in the model and the normal groups were fixed without treatment. After 4-week treatment, the random blood glucose level of the rats was measured; the learning and memory abilities of rats were measured by MWM; terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was used to detect apoptotic cells; Western blot (WB) and real-time quantitative polymerase chain reaction (RT-qPCR) were used to detect the protein and gene expressions of Bax, Caspase-3 and Bcl-2 in cerebral cortex. Results: After modeling, the random blood glucose level and the escape latency tested by MWM were significantly increased, and the number of crossing the platform tested by the MWM was decreased in the EA and model groups, and were significantly different from those in the normal group (P<0.05 or P<0.01), while the differences between the model group and the EA group were not statistically significant (all P>0.05). After 4-week treatment, the random glucose level and the escape latency tested by MWM were significantly increased (both P<0.05), and the number of crossing the original platform tested by the MWM was significantly reduced (P<0.01), the protein and gene expressions of Bax and Caspase-3 were significantly increased (all P<0.001), the protein and gene expressions of Bcl-2 were significantly reduced (both P<0.001), and the number of neuron apoptosis was significantly increased (P<0.001) in the model group than in the normal group; the random blood glucose level was significantly reduced (P<0.05), the escape latency tested by MWM was significantly shortened (P<0.05), and the number of crossing the original platform tested by MWM was significantly increased (P<0.05), the protein and gene expressions of Bax and Caspase-3 were significantly reduced (all P<0.001), the protein and gene expressions of Bcl-2 were significantly increased (both P<0.001), and the number of neuron apoptosis was significantly reduced (P<0.001) in the EA group than in the model group. Conclusion: EA can improve the learning and memory damages induced by type 2 diabetic model rats with CI; the action mechanism may be achieved via anti-apoptosis.

OBJECTIVETo establish a recombinant plasmid of CFP32 of Mycobacterium tuberculosis in E. coli, and to analyze its antigenicity.

METHODSRv0577 gene was amplified by polymerase chain reaction from genome of Mycobacterium tuberculosis, and then cloned into vector pMD18-T followed by the subclone into the expression vector pET21a. Recombinant CFP32 was expressed and purified. The antigenicity of the recombinant protein was analyzed by using Western-blot. The purified recombinant CFP32 protein was used as an antigen to screen the sera of 7 pulmonary TB patients (n = 97), as well as the other pulmonary disease patients (n = 25), and the clinically healthy controls (n = 38) by ELISA.

RESULTSRecombinant plasmid of CFP32 was established, and be expressed efficiently in E. coli BL21 (DE3). The relative molecular mass of the protein was about 300,000 by SDS-PAGE analysis. The protein purified by Ni-NTA was in a purity over 90%, which was confirmed by Western-blot analysis. ELISA analysis showed its sensitivity and specificity were 63.9% (62/97) and 96.8% (2/63) respectively.

CONCLUSIONThe recombinant expression plasmid pET21a CFP32 has been constructed and CFP32 proteins has been successfully expressed and be purified in E. coli and, ELISA analysis has identified the recombinant CFP32 as a candidate antigen for TB serodiagnosis.

Antigens, Bacterial ; blood ; Bacterial Proteins ; genetics ; immunology ; Cloning, Molecular ; Escherichia coli ; Gene Expression ; Humans ; Mycobacterium tuberculosis ; genetics ; isolation & purification ; Plasmids ; Recombinant Proteins ; Serologic Tests ; Tuberculosis, Pulmonary ; diagnosis ; microbiology

The distribution information of Lomatogonium rotatum. was collected by interview investigation and field survey, and 55 related environmental factors were collected, the habitat suitability study was conducted based on geographic information system (GIS) and maximum entropy model. The AUCs of ROC curve were both above 0.99, indicating that the predictive results with the maximum model were highly precise. The results showed that 13 major environmental factors have obvious influence on ecology suitability distributions of L. rotatum, including month average temperature of February et al., the suitable distribution areas are mainly concentrated in the east-central of Inner Mongolia, including Hexigten banner, Duolun county, Zhenglan banner et al., The zoning results basically coincide with the genuine producing areas, and further afford new suitable distribution areas, which can provide reference for L. rotatum's wild nursery and the siting of introduction and cultivation.
China ; Ecosystem ; Environment ; Gentianaceae ; growth & development ; Geographic Information Systems ; Rain ; Temperature

Objective To explore the biological exposure limit of liver dysfunction induced by arsenic-coal burning, and screen sensitive biornarkers for its' liver dysfunction monitoring. Methods One hundred and eighteen subjects from the exposed area and 50 control from non-pollution area were studied. Their urinary and hair contents of arsenic were tested as exposure biomarkers by Ag-DDC assay. Total bile acid(TBA, detected by enzymatic cycling method), glutathione S-transferase (GSTs, detected by chemical colorimetry) and γ-glutamyl transpeptidase (γ-GT, detected by colorimetry of diazotization reagent) were used as biomarkers indicating liver cell damage. were used as liver fibrosis biomarkers. The benchmark dose (BMD) and the lower confidence limit of benchmark dose(BMDL) of urinary and hair arsenic were calculated. Sensitivity of each biomarker was estimated according to the BMD and BMDL value. Results The geometric mean of urinary and hair arsenic(98.50 mg/kg Cr, 7.42 mg/kg) μg/L) in the exposed group were significantly higher than urinary and hair arsenic (22.98 mg/kg Cr, 1.28 mg/kg) and each biomarker in the control group(4.63 μmol/L, 13.76 U/L,36.45 U/L,54.62 μg/L,74.45 μg/L,54.81 μg/L, P<0.01). Significant dose-effect relationship existed between urinary and hair arsenic contents and each biomarker. BMD and BMDL value of urinary arsenic was 49.53-101.96 mg/kg Cr and 39.02-70.15 mg/kg Cr, respectively. Those of hair arsenic were 3.04-5.02 mg/kg and 2.36-3.25 mg/kg, respectively. According to BMD and BMDL value of urinary and hair arsenic, the sensitivity of biomarkers decreased in the order of GSTs, TBA and Conclusions According to the lowest BMD and BMDL of urinary and hair arsenic, averaged reference value of urinary and hair arsenic in the local normal population, we suggest urinary 35.0 mg/kg Cr and hair 2.5 mg/kg as their biological exposure limits for those with liver dysfunction induced by arsenic-coal burning. GSTs, TBA, γ-GT and HA, Ⅳ. C, PC-Ⅲ can respectively reflect liver cell damage and liver fibrosis caused by arsenic-coal burning in different degrees, among which, GSTs and HA are the most sensitive biomarkers respectively for liver cell damage and liver fibrosis.

AIM To establish the UPLC fingerprints of Jasminum elongatum (Bergium) Wild.and to determine the contents of isochlorogenic acid B and ethylcaffeate.METHODS The analysis of methanol extract of J.elongatum was developed on a 25 ℃ thermostatic Agilent Ecplise XDB-C18 column (2.1 mm × 100 mm,1.7 μm),with the mobile phase comprising of acetonitrile-0.1％ methanoic acid flowing at 0.5 mL/min in a gradient elution manner,and the detection wavelength was set at 260 nm.RESULTS There were eighteen common peaks in the fingerprints of ten batches of samples,with the similarities of more than 0.85.Isochlorogenic acid B and ethylcaffeate showed good linear relationships within the ranges of 7.67-38.35 μg/mL (R2 =0.999 4),9.60-96.0 μg/mL (R2 =0.999 7),whose average recoveries were 98.61％,99.09％ with the RSDs of 0.84％,1.25％,respectively.CONCLUSION This stable and reliable method can be used for the quality control of J.elongatum.

OBJECTIVETo investigate the interaction of deficiency in thrombosis-related gene in a mouse model.

METHODSTo generate mice carrying mutations in alpha-galactosidase A (Gla) and factor V Leiden (Fvl) and analyze the phenotypes, namely, tissue fibrin deposition and thrombus formation in organs.

RESULTSFibrin deposition in organs of mice carrying both mutations in Gla and Fvl was significantly increased compared with that in mice with single mutaton: [Gla(-/0) Fv(Q/Q)+Gla(-/-)Fv(Q/Q)] vs.[Gla(-/0)Fv(+/+)]=(0.28+/-0.03)% vs.(0.07+/-0.007)%, P<0.01; [Gla(-/0)Fv(Q/Q)+Gla(-/-)Fv(Q/Q)] vs.[Gla(+/0)Fv(Q/Q)+Gla(+/+)Fv(Q/Q)]=(0.28+/-0.03)% vs.(0.11+/-0.02)%, P< 0.01. Meanwhile, the number of thrombi on organ sections of mice carrying both mutations in Gla and Fvl was significantly increased compared with the single mutation carrier: [Gla(-/0)Fv(Q/Q)+Gla(-/-)Fv(Q/Q)] vs.[Gla(-/0)Fv(+/+)]=1.9+/-0.7 vs. 0.0+/-0.0, P<0.05; [Gla(-/0)Fv(Q/Q)+Gla(-/-)Fv(Q/Q)] vs. [Gla(+/0)Fv(Q/Q)+Gla(+/+)Fv(Q/Q)]=1.9+/-0.7 vs. 0.3+/-0.1, P<0.05.

CONCLUSIONThese observations demonstrated that there was synergistic effect in Gla and Fvl deficiency in mice. It suggested that there could be a combination of GLA deficiency and FVL or other thrombosis-related gene defect in patients with genetic severe early-onset thrombosis.

Animals ; Factor V ; genetics ; Fibrin ; metabolism ; Immunohistochemistry ; Mice ; Mutation ; Thrombosis ; genetics ; metabolism ; alpha-Galactosidase ; genetics