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ObjectiveTo screen out the transcriptomes related to the intervention of Wuzi Yanzongwan on the spermatogenic function of semi-castrated male mice， and to explore its potential mechanism in the intervention of the progress of low spermatogenic function. MethodBalb/c mice were randomly divided into sham-operated group， model group， testosterone propionate group（0.2 mg·kg-1·d-1， intramuscular injection） and Wuzi Yanzongwan group（1.56 g·kg-1·d-1， intragastric administration） according to body weight， with 12 mice in each group. The right testicle and epididymis were extracted from the model group and the drug administration group to construct the semi-castrated model of low spermatogenic function， while the fur and the right scrotum of the sham-operated group were only cut and immediately sterilized and sutured. At the end of the intervention， hematoxylin-eosin（HE） staining was used to observe the histopathology of testis， enzyme-linked immunosorbent assay（ELISA） was used to detect the levels of serum testosterone（T）， luteinizing hormone（LH） and follicle stimulating hormone（FSH）. The sperm count and motility of epididymis were measured by automatic sperm detector of small animal. Transcriptomic microarray technology was used to detect the mRNA expression level of testicular tissue in each group， the transcriptome of genes related to the regulation of Wuzi Yanzongwan was screened， and three mRNAs were selected for Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） to verify the transcriptome data. Through the annotation analysis of Gene Ontology（GO） and the signaling pathway analysis of Kyoto Encyclopedia of Genes and Genomes（KEGG）， the related functions of drugs regulating transcriptome were analyzed. ResultCompared with the sham-operated group， the testicular tissue of mice in the model group showed spermatogenic injury， contraction and vacuolization of the seminiferous tubules， reduction of spermatogenic cells at all levels， widening of the interstitial space， obstruction of spermatogonial cell development and other morphological abnormalities， and serum T significantly decreased， LH significantly increased（P<0.01）， and FSH elevated but no statistically significant difference， the count and vitality of epididymal sperm significantly decreased（P<0.01）. There were 882 differentially expressed mRNAs in the testicular tissues， of which 565 were up-regulated and 317 were down-regulated. Cluster analysis showed that these differentially expressed mRNA could effectively distinguish between the sham-operated group and the model group. Compared with the model group， the damage to testicular tissue in the Wuzi Yanzongwan group was reduced， the structure of the seminiferous tubules was intact， vacuolization was reduced， and the number of spermatogenic cells at all levels was significantly increased and arranged tightly. The serum T significantly increased， LH significantly decreased（P<0.01）， and FSH decreased but the difference was not statistically significant. The count and vitality of sperm in the epididymis were significantly increased（P<0.01）. Moreover， Wuzi Yanzongwan could regulate 159 mRNA levels in the testes of semi-castrated mice， of which 32 were up-regulated and 127 were down-regulated， and the data of the transcriptome assay was verified to be reliable by Real-time PCR. GO and KEGG analysis showed that the transcriptome functions regulated by Wuzi Yanzongwan were involved in the whole cell cycle process of sperm development such as sex hormone production of interstitial cells in testis， renewal， differentiation， metabolism， apoptosis and signal transduction of spermatogenic cells， and were closely related to the biological behaviors of signaling pathways such as spermatogenic stem cell function， endoplasmic reticulum protein processing and metabolic program. ConclusionWuzi Yanzongwan can effectively improve the low spermatogenic function of semi-castrated male mice， and its mechanism may be related to the regulation of testicular transcriptional regulatory network， the synthesis of sex hormones in testicular interstitial cells， the function of spermatogenic stem cells， the whole cell cycle process of spermatogenesis， as well as the expression of endoplasmic reticulum protein processing and metabolic program related genes transcription.
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Objective To establish a pre-column derivation HPLC-UV method for the content determination of hyodeoxycholic acid in artificial calculus bovis. Methods The hyodeoxycholic acid was derived by 2-bromo-2’-acetonaphthoneat using triethylamine as the catalyst in 60 ℃ water bath. After that, a HPLC-UV method was established to determine the content of hyodeoxycholic acid in artificial calculus bovis. Results When the derivatising time at 60 ℃ water bath was 50 min, the radio of the molar amount of derived reagents and hyodeoxycholic was over 20:1 and the radio of catalyst and hyodeoxycholic was over 15:1; the reaction was completed. The calibration curve was linear within the range of 0.1–2 μg for hyodeoxycholic acid (r=0.999 7), and the average recovery was 97.85% (RSD=1.6%). In this sample, the content of hyodeoxycholic is 4.12%. Conclusion The method is with high sensitivity, highly reproducible, reliable and accurate for the content determination of hyodeoxycholic acid in artificial calculus bovis.
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Objective To study the relationship among cardiac function,prognosis and different levels of blood pressure in elderly patients with acute attack of chronic heart failure.Methods A total of 204 elderly patients with acute attack of chronic heart failure were enrolled in this study.According to systolic blood pressure (SBP) level,all the patients were divided into group A (37 cases,SBP ≤100 mmHg,1mmHg =0.133 kPa),group B(73 cases,SBP 101-140 mmHg),group C (56 cases,SBP 141-180 mmHg)and group D (38 cases,SBP ＞ 180 mmHg).The left ventricularcar end-diastolic volume (LVEDV),left ventricular ejection fraction (LVEF),cardiothoracic ratio (C/T),N terminal pro-brain natriuretic peptide (NT-proBNP) and creatinine were measured.According to cardiogenic death or not after 1 year's follow-up,patients were divided into death group (53 cases) and survival group (151 cases),and the indicators were compared.Results The proportion of hypertension in group C and group D was significantly higher than that in group A and group B [82.1％(46/56),89.5％(34/38) vs.35.1％(13/37),37.0％ (27/73)],the proportion of ischemic heart disease and cardiac function in New York heart association (NYHA) class Ⅳ in group C and group D was significantly higher than that in group A [69.6％ (39/56),73.7％ (28/38) vs.29.7％ (11/37); 53.6％(30/56),57.9％(22/38) vs.21.6％(8/37)],and the differences were statistically significant (P ＜0.05).There were significant differences in lgNT-proBNP,C/T,LVEF and LVEDV among the four groups [group A:3.95 ±0.34,0.73 ±0.05,(36.60 ±5.21)％,(125.36 ±44.38) ml;group B:3.65 ±0.29,0.66 ±0.07,(41.34 ±6.19)％,(110.72 ±50.39) ml;group C:3.32 ±0.25,0.60 ±0.05,(48.25 ±5.68)％,(90.47 ±33.64) ml;group D:3.07 ±0.26,0.52 ±0.04,(56.47 ±7.12)％,(72.49 ±38.36) ml](P＜ 0.05).The incidence rate of cardiogenic death in group A was significantly higher than that in the other three groups [56.8％ (21/37) vs.23.3％ (17/73),17.9％ (10/56),13.2％ (5/38)] (P ＜ 0.05).The smoking rate,NYHA cardiac function class Ⅳ,triglyceride (TG),uric acid,fasting blood glucose (FBG),2 h postprandial blood glucose (PBG),glycosylated hemoglobin (HbA1c),creatinine,lgNT-proBNP,cardiothoracic ratio,LVEDV in death group were significantly higher than those in survival group,high-density lipoprotein cholesterol (HDL-C),LVEF and SBP were lower than those in survival group [(0.85 ± 0.29) mmol/L vs.(1.12 ±0.36) mmol/L,(40.28 ±5.62)％ vs.(51.75 ±8.96)％,(124.38 ±22.67) mmHg vs.(141.57 ±24.39) mmHg],and the difference had statistical significance (P ＜0.05).Pearson correlation analysis showed that SBP was negatively correlated with lgNT-proBNP,C/T,LVEDV (r =-0.379,-0.498,-0.413,P ＜0.05),and positively correlated with LVEF(r =0.625,P ＜ 0.05).Multiple linear regression analysis results showed that the LVEF,creatinine,SBP (OR =0.942,0.829,1.033) and the prognosis of acute attack of chronic heart failure had obvious relationship.Conclusion Level of SBP is an important index reflecting cardiac function and prognosis in elderly patients with acute attack of chronic heart failure.
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[ ABSTRACT] AIM:To construct a lentiviral vector for stable delivery of the ER-α36 gene and to detect its effect on SGC7901 cell growth.METHODS: The efficient RNAi targeting sequences identified for the ER-α36 gene were screened.The Oligo DNA was synthesized with target sequences and annealed to form double-stranded DNA.Then it was digested by Xho I and EcoR I and connected with GV307 vector to produce LV-ER-α36-RNAi lentiviral vector.PCR was used to screen the positive clones and sequence.The LV-ER-α36-RNAi, pHelper 1.0 and pHelper 2.0 plasmids were co-transfected into 293T cells for producing lentiviral vector and infecting SGC7901 cell line.Fluorescence microscopy, real-time PCR and Western blotting were used to detect the transfection efficiency and gene silencing effect.17β-estrodial at concentration of 1 ×10 -10 mol/L was used to stimulate the recombinant cell line, and the action on the growth of gastric cancer cells and the expression of Src, ERK1/2 and cyclin D1 were determined.RESULTS: DNA sequencing analysis confirmed the identity of recombinant shRNA expression vectors.Immunofluorescence assay demonstrated that transfection efficiency was above 80%.Transfection of LV-ER-α36-RNAi significantly knocked down the expression of ER-α36 at mR-NA and protein levels with tetracycline ( TeT) simulating as revealed by real-time PCR and Western blotting.Compared with control group, the growth of the recombinant cell line declined and the expression of Src, ERK1/2 and cyclin D1 and the activation of Src decreased (P<0.05).CONCLUSION: Lentiviral vectors that silence ER-α36 expression are con-structed successfully and can be used to study the role of ER-α36 in gastric cancer.The ER-α36 is related with many kinds of cancer cell growth, including gastric cancer cells.