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.

Polyphyllin Ⅰ(PPⅠ)and polyphyllin Ⅱ(PⅡ)are the main active substances in the Paris polyphylla.However,liver toxicity of these compounds has impeded their clinical application and the potential hepatotoxicity mechanisms remain to be elucidated.In this work,we found that PPⅠ and PⅡ exposure could induce significant hepatotoxicity in human liver cell line L-02 and zebrafish in a dose-dependent manner.The results of the proteomic analysis in L-02 cells and transcriptome in zebrafish indicated that the hepa-totoxicity of PPⅡ and PⅡwas associated with the cholesterol biosynthetic pathway disorders,which were alleviated by the cholesterol biosynthesis inhibitor lovastatin.Additionally,3-hydroxy-3-methy-lglutaryl CoA reductase(HMGCR)and squalene epoxidase(SQLE),the two rate-limiting enzymes in the choles-terol synthesis,selected as the potential targets,were confirmed by the molecular docking,the over-expression,and knockdown of HMGCR or SQLE with siRNA.Finally,the pull-down and surface plasmon resonance technology revealed that PPⅠ could directly bind with SQLE but not with HMGCR.Collectively,these data demonstrated that PPⅠ-induced hepatotoxicity resulted from the direct binding with SQLE protein and impaired the sterol-regulatory element binding protein 2/HMGCR/SQLE/lanosterol synthase pathways,thus disturbing the cholesterol biosynthesis pathway.The findings of this research can contribute to a better understanding of the key role of SQLE as a potential target in drug-induced hepatotoxicity and provide a therapeutic strategy for the prevention of drug toxic effects with similar structures in the future.

Objective To investigate the applicability of zebra fish thrombosis model in antithrombotic activity screening of Chinese materia medica.Methods The living zebra fish thrombosis model was induced by adrenaline hydrochloride. Zebra fish were randomly divided into blank control group, model group, positive medicine group and medication group. Each group was given the corresponding medicine or embryo culture water. O-anisidine staining solution was used to stain and calculate the staining intensity of erythrocytes in zebra fish heart, and quantitative analysis was carried out. The platelet aggregation of transgenic zebra fish was observed and under qualitative analysis. Results Compared with the model group, 100μg/mL salvianolic acid B, 300, 900μg/mL aqueous extract of Salvia miltiorrhiza, 45μg/mL 95% ethanol extract and 400, 1200μg/mL hypothalamus could significantly inhibited the formation of zebra fish thrombosis (P<0.01).ConclusionZebra fish thrombosis model has good applicability in antithrombotic activity screening of Chinese materia medica.

Objective To develop a HPLC method for the simultaneous content determination of three terpenoids inMelastoma dodecandrum Lour. (asiatic acid, betulinic acid, oleanolic acid).Methods The chromatographic column was set with waters SunFire C18 column (4.6 mm×150 mm, 5μm); the moving phase was acetonitrile -0.1%H3PO4; the column temperature was 30℃; the detection wavelength was 200 nm; the flow rate was 0.6 ml/min; and the sample volume was 25μl.Results A good linear relationship was observed in the range of 0.310-6.200μg (r=0.9999), 0.405-8.100μg (r=0.9999), 0.169-3.375μg (r=0.9998) for asiatic acid, betulinic acid, oleanolic acid respectively, with the average recovery rates of 102.08%, 101.81%, 102.22%. Conclusions The established method is convenient and sensitive, repeatability and stability, quality evaluation for Melastoma dodecandrum Lour.

Objective:To investigate and compare the effects of water extracts of Whitmania pigra Whitman and Hirudinaria ma-nillensis Lesson on the angiogenesis of Tg (kdrl:mCherry) zebrafish. Methods:The zebrafish embryos 6-8 hours after fertilization (6-8hpf) were transferred to the culture medium containing Whitmania pigra Whitman or Hirudinaria manillensis Lesson extract at different concentrations, and the culture medium containing the drugs was replaced every 24 h. And then, at 72 hpf, the larvalmorphology and intersegmental vessels were observed under a microscope. The hatchability of 48-and 72-hpf embryos, and the number of intersegmen-tal vessels and the heart rate of 72-hpf juveniles were measured. Results:Compared with the control group, when the concentration of Whitmania pigra Whitmanis was higher than 30μg· ml-1 , and the concentration of Hirudinaria manillensis Lesson was higher than 20μg· ml-1, the number of intersegmental vessels was significantly reduced (P<0. 01). Compared with the control group, at 48 hpf, when the concentration of the drug groups was higher than 40 μg· ml-1 , the hatchability of the two groups significantly decreased ( P<0. 01);at 72 hpf, the hatchability of Whitmania pigra Whitman decreased significantly at the concentration of 100 μg·ml-1 (P<0.01), while the hatchability of Hirudinaria manillensis Lesson decreased significantly at the concentration of 80 μg· ml-1(P <0. 01). There was no obvious yolk sac edema, pericardial edema and spine curvature in the two groups. The heart rate decreased sig-nificantly (P<0. 01), while was still within the normal range. Conclusion:Both Whitmania pigra Whitman and Hirudinaria manillen-sis Lesson have notable anti-angiogenic activity, and the anti-angiogenesis activity of Hirudinaria manillensis Lesson is stronger. They both have effects on the development of zebrafish embryos, while the toxicity is not obvious.

This study was aimed to develop HPLC for determination of vitexin and isovitexin inM. dodecandrum. The HPLC column was SunFireTM C18 (4.6 mm× 150 mm, 5μm). The detection wavelength was 365 nm. The mobile phase was methanol-0.2% formic acid aqueous solution. The column temperature was 40℃. The flow rate was 1.0 mL·min-1. The results showed that the regression equations of vitexin and isovitexin wereY = 1× 106X– 14 396, Y = 1× 106X– 13 900, respectively. The linear ranges were 0.210μg - 1.050μg (r = 0.999) and 0.186μg - 0.930μg (r = 1.000), respectively. The recovery rates were 97.48% and 104.64%, respectively. The RSD were 2.32%and 1.51%, respectively. The sample contents of vitexin and isovitexin were 1.25 and 1.86 mg·g1, respectively. It was concluded that the method was simple, feasible and reproducible for the content determination of vitexin and isovitexin inM. dodecandrum.

Objective:To determine residual sulfite in traditional Chinese medicinal materials or pieces processed by sulfur fumi-gation respectively by iodine titration, acid-base titration and ion chromatography and compare the results. Methods:The three meth-ods were used to determine four kinds of Chinese herbal medicines including Codonopsis radix, Dioscoreae rhizoma, Achyranthis bident-atae Radix and Atractulodis Macrocephalae Rhizoma, the recovery tests were also performed and the results were analyzed and compared to summarize the characteristics and quality control requirements of each method. Results:Iodine titration and acid-base titration had the advantages of simple operation process and low cost. However, there were many interference factors in the two methods, and due to different principles, they were suitable for the determination of different varieties of herbal medicines. Ion chromatography method had the advantages of high sensitivity and strong specificity, while the cost was high. Conclusion: It is suggested that proper methods should be chosen for the determination of sulfur dioxide residues according to actual situations.

This study was aimed to identify and distinguish Metacordyceps liangshanensis recorded by the Sichuan Province Traditional Chinese Medicine Standard from its adulterants and its relative species by combining ITS and COI barcode sequences in order to study the feasibility of this new method. After extracting DNA of 28 species of Cordyceps samples, DNA were amplified and sequenced. And then, ITS and COI sequences were received. Codon-Code Aligner V3.7.1 and Mega 5.0 were used to analyze the variable site and construct the N-J tree. The results showed that the minimum ITS inter-specific K-2P distance was relatively higher than the maximum intra-specific K-2P distance. The inter-specific sequence divergence between M. liangshanensis and its adulterants exhibited high while intra-specific sequence divergence exhibited low. And COI one was the same case. N-J tree of both ITS and COI indicated that same genus belonged together and each species belonged to relatively independent branch. It was concluded that based on the ITS and COI gene, the technology of DNA barcode can be an excellent identification of M. liangshanensis, its adulterants and its relative species. It provided technical support for the further research on species molecular identification and phylogenetics of Cordyceps .

Zebrafish toxicity assessment system is one of the important vertebrate model systems. Zebrafish is playing an increas-ingly important role in the field of toxicology studies because of its small size, short generation cycle, the transparent embryo and high reproductive rate. Now it is widely used in the embryonic derelopmental toxicology, pathological toxicology, environmental toxicology and other areas of toxicology studies with its unique advantages.

Chinese Caterpillar Fungus (CCF) is one of the rare Chinese traditional drugs. As the resource is reducing sharply, the price is rising higher and higher, and there have been much more adulterants in the markets, but until now we don't have a scientific and accurate research on the identification study for this drug. On the basis of resource investigation, during the study of the samples collected by ourselves and the specimens stored in the museum, using the macroscopic and microscopic methods, referring to the literatures of entomology, emphasizing on the characteristics of polypide part, we have studied this species in detail of the macroscopic characters such as the insertion position of the stroma part, the annulations and segments of the caterpillar, the abdominal leg, the pinaculum, and the microscopic characters of the body wall; firstly added the microscopic character of the crotchets on the planta of abdominal leg. The result turned out that the characters which we have studied are regular and stable, and it have laid the foundation for the powder products and patent medicines which have used the crude drug of CCF.