Objective To establish a new RP-HPLC method for simultaneous determination of chlorogenic acid,forsythin, and baicalin in shuanghuanglian powder for injection after ultrasonic atomization. Methods Hypersil ODS2 C18(250 mmí4. 6 mm,5 μm) was used as the chromatographic column. The mobile phase was methanol-0. 2% phosphate acid solution (4060). Flow rate was 1. 0 mL·min-1 . Sample volume was 5μL. Column temperature was 30℃. Detection wavelength was 324 nm at 0-10 min and 277 nm at 10-25 min. Results Contents of chlorogenic acid,forsythin, and baicalin had good linear relationship with the respective peak area (r≥0. 999 7) within the scope of the sample volume. The RSD was <2% for precision, reproducibility, and stability. Recovery rate was 98. 50%-101. 12% (n=6). Conclusion The method is rapid, accurate and reproducible, with high resolution. It can determine the content of three kinds of components at the same time. The three components in shuanghuanglian powder for injection did not change significantly before and after ultrasonic atomization.

Objective To investigate the mechanism that BC047440 gene regulates nuclear fac-tor κB sigal passway and analyze the differential expression gene between HepG2 cells and HepG2 cells BC047440 gene silenced by RNAi using 35K Human Genome Array. Methods The differential expres-sion gene between HepG2 cells and HepG2 cells with BC047440 gene silenced was analyzed by 35K Human Genome Array, and the data were submitted to the database and MAS system of Capitalbio Corporation.Then TRAF6 was confirmed by RT-PCR test. Results Among the total 35000 probe sets, the expression of 59 genes was down-regulated for more than 50% and 130 genes were up-regulated more than 2 fold in the silencing group when compared with normal controls. TRAF6 mRNA was decreased for 29.5% in silicening HepG2 compared with that of wild HepG2 by RT-PCR, which is similar to human genome array(23.06%).Conclusion The high throughput and effective oligomicroarray can analyze the differential expression gene and BC047440 gene might regulate NF-κB signal pathway inderectly by TRAF6.

Objective To investigate the distribution of the obstructive lesion of hepatic vein and inferior cava vein in patients with Budd-Chiari syndrome in Henan Province of China.Methods A total of 231 cases with Budd-Chiari syndrome were diagnosed by combination of three imaging techniques which either included colour Dopple ultrasound,multislice CT and vasography or colour Dopple ultrasound,MR angiography and vasography.All the hepatic veins,accessory hepatic veins and inferior vena cavae were imaged and their obstructions were detected and analysed.Results Out of the 231 patients there were 5 cases(2.2%)with simple obstruction of inferior vena cava with normal hepatic branches.Thirty-three cases(14.3%)had simple hepatic vein obstruction with normal inferior vena cava.The remaining 193(83.5%)cases had vein obstruction both in hepatic vein and inferior vena cava.Conclusion The most frequent form of Budd-Chiari symdrome in Henan province of China is the complex obstruction of inferior vena cava and heptic vein,and simple obstruction of inferior vena cava occurs the least.

Objective To investigate whether the proliferation of HepG2 ceils is influenced by interfering BC047440 gene with small hairpin RNA (shRNA) expression plasmid. Methods According to the sequence of BC047440 gene, 2 pairs of BC047440 gene-specific shRNA (shRNA1 and shRNA2) were designed and synthesized. After primer annealing, they were inserted into plasmid pGenesil-1 to construct the shRNA expression plasmids. The recombinant plasmids were transfected into HepG2 cells. The expression of BC047440 gene was detected by quantitative fluorescent PCR, the proliferation of HepG2 cells by MTT assay and the changes of cell cycle by flow cytometry. Results Two shRNA expression plasmids were constructed successfully and were confirmed by restriction enzyme digestion and sequencing. Quantitative fluorescent PCR analysis showed that shRNA1 and shRNA2 could specifically inhibit the expression of BC047440 gene in HepG2 cells, with the inhibition rate of 80.22% and 58.63%, respectively. The shRNA effectively inhibited the proliferation of HepG2 cells, and arrested the cell cycle in S phase. Conclusions The shRNA significantly inhibits the expression of BC047440 gene and the proliferation of HepG2 cells. The expression of BC047440 may be correlated with the proliferation of HepG2 cells.

Objective Based on TLR4/NF-κB/MLCK pathway,the therapeutic effect and mechanism of Baihe Dihuang Decoction on insomnia with intestinal flora disturbance in mice induced by p-chlorophenlalanine(PCPA)and multi-factor stimulation were studied.The aim is to provide theoretical basis for clinical use.Methods Eighty-four KM mice were randomly divided into normal group,model group,positive group(Diazepam,1.38 mg·kg-1),Baihe group(2.25 g·kg-1),Dihuang group(2.25 g·kg-1)and Baihe Dihuang Decoction group(4.5 g·kg-1).Insomnia mouse model was established by intraperitoneal injection of PCPA for 2 days combined with 4 weeks of multi-factor stimulation,including stimulating the tail with forceps clip for 2 minutes,reversing day and night for 24 hours,wetting the padding for 24 hours,tilting the cage at 45° for 24 hours,alternating the cages for 24 hours,fasting food for 24 hours,and getting cold bath for 3 minutes,etc.After successfully modeling,corresponding drug treatment was given.The anxiety-like behavior of mice was observed by elevated cross maze system.The latency and duration of sleep were observed by righting reflex experiment.16sRNA sequencing was used to analyze the composition and structure of intestinal flora in mice.The concentration changes of γ-aminobutyric acid(GABA),glutamic acid(Glu),tryptophan(Trp),5-hydroxytryptophan(5-HTP)and 5-hydroxytryptamine(5-HT)in brain and colon were detected by liquid chromatography-mass spectrometry(LC-MS).Immunohistochemical method was used to detect the expressions of zona atresia protein 1(ZO-1)and Occludin in colon.Real-time fluorescence quantitative PCR(qRT-PCR)was used to detect the genes including interleukin-1β(IL-1β),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),ZO-1,Occludin,Toll-like receptor 4(TLR4),nuclear factor κB(NF-κB)and myosin light chain kinase(MLCK)in colonic tissue.Western Blot was used to detect the expressions of TLR4,NF-κB,phosphorylated nuclear factor κB(p-NF-κB),MLCK,myosin light chain(MLC)and phosphorylated myosin light chain(p-MLC)in colonic epithelial tissue.Results Compared with the normal group,the distance of entering the open arm and the duration of stay in the open arm of model group in the elevated cross maze were significantly shortened(P＜0.05).The sleep latency was significantly prolonged,and the sleep duration was significantly shortened(P＜0.05).The richness and uniformity of intestinal flora were decreased(P＜0.05,P＜0.01).The concentration of neurotransmitters GABA,Trp,5-HTP and 5-HT in the brain decreased significantly(P＜0.01),while the concentration of Glu increased significantly(P＜0.01).The concentration of GABA and Glu in colon decreased significantly(P＜0.01),while the concentration of Trp,5-HTP and 5-HT increased significantly(P＜0.01).The expression levels of inflammatory factors IL-1β,IL-6 and TNF-α in colonic tissue were significantly increased(P＜0.01),and the expression levels of ZO-1 and Occludin genes and proteins were significantly decreased(P＜0.01).The gene expression levels of TLR4,NF-κB and MLCK were significantly increased(P＜0.01),and the protein expression levels of TLR4,p-NF-κB,MLCK and p-MLC were significantly increased(P＜0.01).Compared with the model group,Baihe Dihuang Decoction could significantly prolong the distance of entering the open arm and the duration of stay in the open arm of insomnia mice in the elevated cross maze(P＜0.05).The sleep latency was significantly shortened,and the sleep duration was significantly increased(P＜0.05).The richness and uniformity of intestinal flora were increased(P＜0.05,P＜0.01).The concentration of neurotransmitters GABA,Trp,5-HTP and 5-HT in brain was increased(P＜0.05,P＜0.01),and the concentration of Glu was decreased(P＜0.01).The concentration of GABA and Glu in colon was increased(P＜0.01),while the concentration of Trp,5-HTP and 5-HT was decreased(P＜0.05,P＜0.01).The expression levels of IL-1β,IL-6 and TNF-α genes were down-regulated(P＜0.01),the expression levels of ZO-1 and Occludin genes and proteins were up-regulated(P＜0.01),TLR4,NF-κB,MLCK gene expression levels and TLR4,p-NF-κB,MLCK,p-MLC protein expression levels were down-regulated in the pathway(P＜0.01).Conclusion Baihe Dihuang Decoction can effectively treat insomnia with intestinal flora disorders.Its mechanism of action may be related to the regulation of brain and intestinal neurotransmitter disorders,down-regulating TLR4/NF-κB/MLCK signaling pathway,and up-regulating tight junction proteins expression,reducing inflammatory responses,and then repairing the mechanical barrier of intestinal mucosa.

With the rapid development of gene editing technology, the study of spermatogonial stem cells (SSCs) holds great significance in understanding spermatogenesis and its regulatory mechanism, developing transgenic animals, gene therapy, infertility treatment and protecting rare species. Biogenesis of lysosome-related organelles complex 1 subunit 1 (BLOC1S1) is believed to have anti-brucella potential. Exploring the impack of BLOC1S1 on goat SSCs not only helps investigate the ability of BLOC1S1 to promote SSCs proliferation, but also provides a cytological basis for disease-resistant breeding research. In this study, a BLOC1S1 overexpression vector was constructed by homologous recombination. The BLOC1S1 overexpression cell line of goat spermatogonial stem cells was successfully constructed by lentivirus packaging, transfection and puromycin screening. The overexpression efficiency of BLOC1S1 was found to be 18 times higher using real time quantitative PCR (RT-qPCR). Furthermore, the results from cell growth curve analysis, flow cytometry for cell cycle detection, and 5-ethynyl-2'-deoxyuridine (EdU) staining showed that BLOC1S1 significantly increased the proliferation activity of goat SSCs. The results of RT-qPCR, immunofluorescence staining and Western blotting analyses revealed up-regulation of proliferation-related genes (PCNA, CDK2, CCND1), and EIF2S3Y, a key gene regulating the proliferation of spermatogonial stem cells. These findings strongly suggest that the proliferative ability of goat SSCs can be enhanced through the EIF2S3Y/ERK pathway. In summary, this study successfully created a goat spermatogonial stem cell BLOC1S1 overexpression cell line, which exhibited improved proliferation ability. This research laid the groundwork for exploring the regulatory role of BLOC1S1 in goat spermatogonia and provided a cell platform for further study into the biological function of BLOC1S1. These findings also establish a foundation for breeding BLOC1S1 overexpressing goats.
Animals ; Male ; Goats ; Stem Cells ; Spermatogonia/metabolism* ; Cell Proliferation ; Flow Cytometry ; Testis/metabolism*