Rat calcineurin (CaN) A alpha isoform (Ppp3ca) cDNA recombinant adenovirus vector was constructed in order to explore the effect of CaN on the myocardium apoptosis induced by ischemia-reperfusion injury. Total RNA was isolated from the heart of the adult Wistar rat, and Ppp3ca CDS segment of approximate 1.59 kb size was amplified by reverse transcriptional PCR method. Ppp3ca cDNA segment was cloned into pMD18-T Simple vector for sequencing, and the right clone was named T-Ppp3ca. Ppp3ca cDNA segment obtained from T-Ppp3ca was ligated with pShuttle2-IRES-EGFP to construct a recombinant plasmid pShuttle2-Ppp3ca-IRES-EGFP. Ppp3ca-IRES-EGFP expression cassette containing CMV, Ppp3ca-IRES-EGFP and SV40 polyA DNA fragment (3.97 kb) obtained from pShuttle2-Ppp3ca-IRES-EGFP was connected with pAdeno-X backbone sequence to construct a recombinant plasmid pAdeno-Ppp3ca. After being identified by PCR and enzyme digestion, recombinant plasmid pAdeno-Ppp3ca was packaged in HEK293 cells. Supernatant of adenovirus from HEK293 cells was collected after a visible cytopathic effect (CPE) appeared. The DNA of the recombinant adenovirus was extracted with the standard method. The presence of the recombinant adenovirus was verified by PCR. The results showed that sequencing results verified that the PCR product of Ppp3ca gene was identical to GenBank. Agarose electrophoresis showed the bands of recombined plasmid pAdeno-Ppp3ca and the recombinant adenovirus identified by enzyme digestion and PCR were in the right range corresponding with expectation. It was concluded that the recombinant adenovirus carrying rat calcineurin A alpha (Ppp3ca) cDNA as well as a report gene-enhancer green fluorescent protein gene was successfully constructed in this experiment.
Adenoviridae/*genetics ; Calcineurin/*biosynthesis ; Calcineurin/genetics ; Cloning, Molecular ; DNA, Complementary/genetics ; Genetic Vectors/genetics ; Green Fluorescent Proteins/biosynthesis ; Green Fluorescent Proteins/genetics ; Myocardial Reperfusion Injury/*genetics ; Myocardium/chemistry ; Rats, Wistar ; Recombination, Genetic/genetics

Objective To construct rat calcineurin catalytic subunit ?(CnA)/enhanced green fluorescent protein EGFP gene coexpression plasmid for exploring the effect of calcineurin on the myocardium apoptosis induced by ischemia-reperfusion.Methods Total RNA was isolated from the heart of the adult Wistar rat,and CnA CDS segment of approximate 1 590 bp size was amplified by reverse transcription PCR method.CnA cDNA segment was cloned into pMD18-T Simple vector for sequencing,and the right clone was named T-CnA.CnA cDNA segment excised from plasmid T-CnA was ligated with pShuttle2 which had inserted IRES-EGFP segment into before.The DNA of the recombinant plasmid was extracted and was identified by double digesting with Nhe Ⅰ and Not Ⅰ.The right clone was named pShuttle2-CnA-IRES-EGFP.Results Sequencing result verified that the PCR product of CnA gene was identical to GenBank(NM_017041).1% agarose electrophoresis showed the bands of recombinant plasmid pShuttle2-CnA-IRES-EGFP digested by Nhe Ⅰ and Not Ⅰ were in the right range corresponding with expectation(1 590 bp and 5 240 bp).Conclusion Recombinant eukaryotic expression plasmid carrying rat CnA cDNA as well as a report gene-EGFP gene is successfully constructed in this experiment.

Objective: To evaluate the influence of metabolic syndrome (MS) on coronary lesions in elder patients with hypertension.
Methods: A cohort of 210 hypertensive patients at the age of 60 years or elder who received coronary angiography in our hospital from 2012-06 to 2013-01 were studied. The patients were divided into 2 groups, MS group,n=85 and Non-MS group,n=125 patients without MS. The coronary lesion distribution, number and Gensini score were analyzed and compared between 2 groups.
Results: Compared with Non-MS group, MS group showed increased BMI, TG, HDL-C and fasting blood glucose, allP<0.001. There were no real differences for lesion distribution in left main, left anterior descending branch and the number of patients with normal coronary artery between 2 groups, allP>0.05. MS group had higher Gensini score, more lesions at left circumlfex and right coronary artery, more patients with coronary lesions and more patients with 3-branch disease,P<0.05. Logistic regression analysis indicated that MS is the independent risk factor for coronary lesions and the risk is higher than any single components of MS.
Conclusion: MS is related to the severity of coronary lesions in elder patients with hypertension.

Objective To explore the association between serum levels of homocysteine (Hcy) and relation factors in the patients with manic episodes of bipolar Ⅰ disorder(BD).Methods The case-control study was used for this study.A total of 73 inpatients with manic episodes of BD(patient group) and 39 healthy individuals (control group) were enrolled.Diagnosis was made according to the Diagnostic and Statistical Manual of Mental Disorders Fourth Edition(DSM-Ⅳ) criteria.Serum levels of Hcy was measured by enzymatic cycling assay.Young Mania Rating Scale(YMRS) were used to assess the severity of clinical symptoms of patients.Results There was a statistical difference in serum Hcy levels between patient group ((16.89± 14.67)μmol/L) and control group ((10.61±6.46) μmol/L) (P＜0.01),and serum Hcy levels was higher in men((20.42± 16.52) μmol/L) than that in women((10.87±8.02) μmol/L) in patients group(P＜0.05).The prevalence rate of high Hcy was 35.6％ in the patient group and 13.5％ in the control group(P＜0.05).Correlation analysis showed body mass index(BMI) had positive correlation with serum levels of Hcy (r=0.317,P＜0.05).Stepwise multiple regression analysis showed male,BMI were associated with serum levels of Hcy among the patients (all P＜ 0.05).Conclusion Serum Hcy levels in the patients with manic episodes of bipolar Ⅰ disorder increase,and it is associated with gender and BMI.

Objective To investigate the status of cognitive impairment and its associated factors among the elderly in community in Beijing. Methods 2055 elderly people aged 60 or more from communities in Beijing were investigated and assessed with Mini-Mental State Examination (MMSE). Results 1985 from 2055 met the requirements of the questionnaire. Univariate analysis showed that age (F=37.068,P<0.001), gender (β=-0.187), educational level (F=21.875, P<0.001), marriage (β=0.328), social communication (β=0.124), and frequency of physical exercise (F=21.58, P=0.01, β=0.126) were related to the scores of MMSE (P<0.001). While multivariate analysis showed that educational level (β=0.421), age (β= -0.226), gender (β=-0.226), and frequency of physical exercise (β=0.128) were possibly the influential factors for the scores of MMSE (P<0.001). Conclusion Age, educational level, and frequency of physical exercise are important factors affecting cognitive functions of the elderly, in which age is negative, and the others are positive.

Rat calcineurin (CaN) A α isoform (Ppp3ca) cDNA recombinant adenovirus vector was constructed in order to explore the effect of CaN on the myocardium apoptosis induced by ischemiareperfusion injury. Total RNA was isolated from the heart of the adult Wistar rat, and Ppp3ca CDS segment of approximate 1.59 kb size was amplified by reverse transcriptional PCR method. Ppp3ca cDNA segment was cloned into pMD18-T Simple vector for sequencing, and the right clone was named T-Ppp3ca. Ppp3ca cDNA segment obtained from T-Ppp3ca was ligated with pShuttle2-IRES-EGFP to construct a recombinant plasmid pShuttle2-Ppp3ca-IRES-EGFP. Ppp3ca-IRES-EG-FP expression cassette containing CMV, Ppp3ca-IRES-EGFP and SV40 polyA DNA fragment (3.97 kb) obtained from pShuttle2-Ppp3ca-IRES-EGFP was connected with pAdeno-X backbone sequence to construct a recombinant plasmid pAdeno-Ppp3ca. After being identified by PCR and enzyme digestion, recombinant plasmid pAdeno-Ppp3ca was packaged in HEK293 cells. Supernatant of adenovirus from HEK293 cells was collected after a visible cytopathic effect (CPE) appeared.The DNA of the recombinant adenovirus was extracted with the standard method. The presence of the recombinant adenovirus was verified by PCR. The results showed that sequencing results veri fied that the PCR product of Ppp3ca gene was identical to GenBank. Agarose electrophoresis showed the bands of recombined plasmid pAdeno-Ppp3ca and the recombinant adenovirus identified by enzyme digestion and PCR were in the right range corresponding with expectation. It was concluded that the recombinant adenovirus carrying rat calcineurin A α (Ppp3ca) cDNA as well as a report gene-enhancer green fluorescent protein gene was successfully constructed in this experiment.

OBJECTIVETo investigate the cystathione beta synthase (CBS) gene T833C, G919A, 844ins68 polymorphisms and plasma homocysteine (Hcy) levels in ethnic Uyghur and Han patients with essential hypertension (EH) in Xinjiang.

METHODSFour hundred twenty nine cases including 211 Uyghur and 218 Han EH patients were recruited, whilst 410 healthy individuals including 210 Uyghurs and 200 Hans were used as the controls. Amplification refractory mutation system (ARMS) was adopted to analyze the CBS gene polymorphisms including T833C, G919A and 844ins68. Enzymoimmunoassay was applied to determine the plasma level of Hcy. Chemiluminescence was applied to determine the plasma folic acid and vitamin B12.

RESULTSCompared with the controls, the plasma Hcy level was significantly higher in the EH group in both ethnic Uyghurs and Hans (P < 0.05). Plasma levels of Hcy in T833C, G919A genotypes (for both heterozygotes and homozygotes) were statistically higher than wild types (P < 0.05). A significant difference was detected in G919A polymorphism between the EH patients and controls in both Uyghur and [CM(144.5mm] Han ethnics (Uyghur: x² = 10.264, P < 0.01; Han: x² = 23.075, P < 0.01), and in T833C between the EH patients and controls in ethnic Uyghurs (x² = 40.254, P < 0.01). Logistic regression analysis indicated that age (OR=1.151, P=0.047, 95% CI = 1.002-1.323), T833C (CC) (OR = 1.078, P = 0.003, 95% CI = 1.043-1.114), obesity (OR = 1.284, P = 0.021, 95% CI = 1.038-1.590), hyperhomocysteine (OR = 3.296, P = 0.016, 95% CI = 1.244-8.733) were independent risk factors for EH among ethnic Uygurs, while age (OR = 1.162, P = 0.007, 95% CI = 1.042-1.297), obesity (OR = 3.501, P = 0.003, 95% CI = 1.521-8.060), hyperhomocysteine (OR = 1.046, P = 0.031, 95% CI = 1.011-1.459) were independent risk factors for EH in ethnic Hans after adjusting for confounding factors.

CONCLUSIONPlasma level of Hcy is associated with ethnic Uyghur and Han patients with EH in Xinjiang. CBS gene T833C CC genotype may be associated with the EH among Uyghur ethnics.

Adult ; Aged ; China ; ethnology ; Cystathionine beta-Synthase ; genetics ; Essential Hypertension ; Female ; Gene-Environment Interaction ; Homocysteine ; blood ; Humans ; Hypertension ; genetics ; Logistic Models ; Male ; Middle Aged ; Polymorphism, Genetic

OBJECTIVETo assess the association of plasma homocysteine (Hcy) level and 66A/G and 524C/T polymorphisms of methionine synthase reductase (MSR) gene with essential hypertension (EH) in ethnic Uygurs and Hans from Xinjiang.

METHODSFrom September 2011 to July 2014, 199 Uyghur and 216 Han patients were collected, while 195 healthy Uyghur ethnics and 217 healthy Han ethnics were recruited as the controls. Polymerase chain reaction and restriction fragment length polymorphism (PCR-RELP) was adopted to detect the above polymorphisms. Enzyme immunological assay was applied to measure the levels of plasma Hcy.

RESULTSCompared with the control, plasma Hcy levels were significantly higher in EH group in both Uyghur and Han ethnics (P<0.05). In both ethnic groups, there were also significant differences in MSR 524C/T polymorphism between the patients and controls (Uyghur: chi-square=6.559, P=0.038; Han: chi-square=12.684, P=0.002). No significant difference was found in MSR 66A/G polymorphism between the patients and controls in both ethnic groups (P>0.05). Plasma Hcy level in those with a 66G/524C genotype was statistically higher than that with 66A/524T (P<0.05). After adjusting confounding factors such as gender and age, Logistic regression analysis indicated that age (OR=1.924, 95% CI:1.177- 3.164, P=0.009), obesity (OR=2.491, 95% CI: 1.584-3.920, P<0.01), hyperhomocysteine (OR=1.609, 95% CI: 1.016-2.548, P=0.043) were independent risk factors for EH in Uygurs, while age (OR=1.133, 95% CI: 1.010-81.272, P=0.033), hyperhomocysteine level (OR=3.894, 95% CI: 2.432-6.237, P<0.01), and obesity (OR=1.864, 95% CI: 1.141-3.046, P=0.013) were independent risk factors for EH in Han ethnics. No association was found between the polymorphisms and EH in Uygurs and Hans.

CONCLUSIONAge, hyperhomocysteine and obesity were common independent risk factors for EH in both Uygur and Han ethnics from Xinjiang. The MSR 66G genotype can increase the plasma concentration of Hcy, while MSR 524T genotype may reduce it. MSR 524C/T TT genotype may be a protective factor for EH. MSR polymorphisms 66A/G and 524C/T are not independent risk factors for EH in Uygur and Han ethnics from Xinjiang.

Adult ; Aged ; Asian Continental Ancestry Group ; ethnology ; genetics ; China ; ethnology ; Essential Hypertension ; Female ; Ferredoxin-NADP Reductase ; genetics ; metabolism ; Homocysteine ; blood ; Humans ; Hypertension ; blood ; enzymology ; ethnology ; genetics ; Male ; Middle Aged ; Polymorphism, Single Nucleotide

OBJECTIVE：To develop a method for simultaneous determination of 5 components in classical formula Huaihua san，including rutin ，naringin，neohesperidin，quercetin and pulegone. METHODS ：HPLC wavelength switching method was adopted. The determination was performed on Cosmosil C 18 column with mobile phase consisted of acetonitrile- 0.05％ phosphoric acid solution （gradient elution ）at the flow rate of 1.0 mL/min. The detection wavelengths were set at 257 nm for rutin ，283 nm for naringin and neohesperidin ，254 nm for quercetin ，252 nm for pulegone ，respectively. The column temperature was set at 30 ℃， and sample size was 10 μL. RESULTS：The linear range was 21.7-2 170 μg/mL for rutin，46-4 600 μg/mL for naringin，22.3- 2 230 μg/mL for neohesperidin，0.96-96 μg/mL for quercetin，2.7-270 μg/mL for pulegone（all r＞0.999），respectively. RSDs of precision，stability（24 h）and reproducibility tests were all lower than 2％（n＝6）. Average recoveries were 100.70％，99.31％， 101.10％，100.03％ and 99.63％（all RSD ＜2％，n＝9）. Among 3 batches of Huaihua san samples ，the contents of above 5 components were 20.055-22.615，25.557-27.806，11.428-13.250，0.350-0.478，2.372-4.011 mg/g，respectively. CONCLUSIONS ： Established method is simple ，accurate and reproducible ，and could be used for the simultaneous determination of 5 components in Huaihua san.

Total glucosides of Rhizoma Smilacis Glabrae (RSG) are selective immunosuppressants that exhibit primary efficacy in the treatment of rheumatoid arthritis through targeted inhibition of activated T cells. In this study, we aimed to investigate the potential application of RSG in the treatment of psoriasis and elucidate its mechanism of action and material basis. Our findings revealed significant improvements upon administration of RSG in an imiquimod (IMQ)-induced psoriasis model. These improvements were characterized by a remarkable increase in the number of tail scales in mice and a substantial amelioration of skin erythema, ulceration, and flaking. By transcriptome sequencing and T-cell flow sorting assay, we identified notable effects of RSG on the modulation of various cellular processes. Specifically, RSG prominently down-regulated the Th17/Treg ratio in damaged skin tissues and reduced the proportion of G₂ phase cells. Furthermore, RSG exhibited a stimulatory effect on the proliferation and differentiation of epithelial cells. Of particular interest, we discovered that β-sitosterol, sitostenone, stigmasterol, smiglanin, and cinchonain Ib displayed potent inhibitory effects on the IL-17-mediated inflammatory response in HaCaT cells. In summary, our study highlights the therapeutic potential of RSG in the treatment of psoriasis, attributed to its ability to regulate the Th17/Treg balance. These findings contribute to the development of new indications for RSG and provide a solid theoretical foundation for further exploration in this field.
Animals ; Mice ; T-Lymphocytes, Regulatory ; Psoriasis/drug therapy* ; Arthritis, Rheumatoid ; Biological Assay ; Glucosides/pharmacology*