Objective To establish the quality standard of Qingshen Granules. Methods Thin-layer chrmatography was used to identify Rheum officinale Baill., Hedyotis Diffusa Wild. and Coptis chinensis Franch.. The contents of rhein, emodin and chrysophanol were determined by HPLC. The HPLC consisted of Kromasil C18 (4.6 mm×250 mm, 5 μm), methanol-0.1% phosphonic acid (70∶30) as mobile phase, flow rate of 1.0 mL/min, detection wavelength at 245 nm, and the column temperature was 30 ℃. Results The results of TLC showed that relevant spots were clear without interference against the negative sample. The calibration curves for rhein, emodin and chrysophanol were found to be linear within the range of 0.003 3-0.42 μg (r =0.999 9), 0.008 0-0.51 μg (r=1.000 0), 0.009 9-0.32 μg (r=1.000 0), respectively. The average recoveries were 98.84%, 99.04% and 100.35% with RSD of 1.30% (n=6), 1.34% (n=6) and 1.89% (n=6), respectively. Conclusion The methods are accurate and quick in qualitative identification and quantitative assay, and can be used for the quality control of Qingshen Granules.

Objective:To investigate mechanisms underlying the signal crosstalk of VEGF-IL-6-STAT3 between cutaneous melanoma cells and vascular endothelial cells.Methods:EC-304 vascular endothelial cells were divided into 3 groups: control group cultured in conventional endothelial cell-conditioned medium, vascular endothelial growth factor (VEGF) group cultured in endothelial cell-conditioned medium containing 50 μg/L VEGF 165, A375 co-culture group co-cultured with a melanoma cell line A375. After 24-, 48- and 72-hour treatment, the culture medium was collected, and enzyme-linked immunosorbent assay was performed to detect the level of interleukin-6 (IL-6) . Cultured A375 cells were divided into 4 groups: control group receiving conventional culture in Dulbecco′s modified Eagle′s medium (DMEM) , A375+ EC-304 group co-cultured with EC-304 cells, A375+ EC-304+ IL-6 group co-cultured with EC-304 cells in DMEM containing 50 μg/L IL-6 (an agonist of the signal transducer and activator of transcription-3 [STAT3] pathway) , A375+ EC-304+ JSI-124 group co-cultured with EC-304 cells in DMEM containing 1 μmol/L JSI-124 (a STAT3 pathway inhibitor) . After 24-, 48- and 72-hour treatment, cells were collected, and Western blot analysis, cell counting kit-8 (CCK8) assay and Transwell invasion assay were performed to determine the protein expression of STAT3 and phosphorylated (p) -STAT3, cellular proliferative activity and invasive activity, respectively. Two-way analysis of variance and t test were used for statistical analysis. Results:The level of IL-6 significantly increased in the culture medium of EC-304 cells in the VEGF group and A375 co-culture group compared with the control group ( FVEGF = 29.63, P < 0.001; FA375 = 11.09, P = 0.020) . Compared with the control group, the A375+ EC-304 group showed significantly enhanced protein expression of p-STAT3 in A375 cells ( P < 0.001) , increased cell activity ( P < 0.001) , and increased number of invasive cells (152.66 ± 16.04 vs. 86.13 ± 7.24, t= 4.43, P < 0.001) ; compared with the A375+ EC-304 group, the A375+ EC-304+ IL-6 group showed significantly increased protein expression of p-STAT3 ( P < 0.001) , enhanced cell activity ( P < 0.001) , and increased number of invasive cells (187.34 ± 14.38, t= 2.17, P < 0.001) ; compared with the A375+ EC-304 group, the A375+ EC-304+ JSI-124 group showed significantly decreased protein expression of p-STAT3 ( P < 0.001) , decreased cell activity ( P < 0.001) , and decreased number of invasive cells (124.92 ± 8.72, t=-1.86, P < 0.001) . Conclusion:There is a signal crosstalk of VEGF-IL-6-STAT3 between cutaneous melanoma cells and vascular endothelial cells, which may play an important role in the proliferation and invasion of A375 cells.

Objective To explore the changes of plasma melatonin(MT)level in hypoxic-ischemic encephalopathy(HIE)neonates,and elucidate the function of rnelatonin in the pathogenesis and the prognosis of HIE.Methods Fourty HIE neonates were divided into 2 groups,20 mild HIE neonates and 20 moderate or severe HIE ones.The femoral vein blood were collected in 48 h and on 7 d after birth in mild HIE group,and in 48 h,on 7 d and(14±4)d after birth in moderate on severe HIE group.Twenty normal term infants served as control group.The level of plasma MT was determined with enzyme-labeled immunosorbent assay.Results Compared with control group[(8.003±1.840)ng/L],The MT level in mild HIE group in 48 h after birth[(13.311±4.025)ng/L]was higher(P＜0.01),but there was no difference on 7 d[(6.605±1.269)ng/L](P＞0.05);The MT level in moderate or severe HIE group in 48 h after birth[(5.487±1.997)ng/L]was lower(P＜0.01),but it was higher on 7 d[(16.201±5.594)ng/L](P＜0.01),there was no difference on(14±4)d[(6.799±1.765)ng/L](P＞0.05).Conclusion MT may have protective action on HIE.The prognosis of HIE with rising MT level in 48 h after birth is better than that with lower MT level.

Several putative transcription factor binding sites (TFBSs) exist in the PCV2 rep gene promoter. To explore if porcine circovirus type 2 (PCV2) could regulate the viral replication by using these TFBSs, we conducted electrophoretic mobility shift assay (EMSA), DNA-pull down and liquid chromatography-tandem mass spectrometric (LC-MS/MS) assays. EMSA confirmed the binding activity of the rep gene promoter with nuclear proteins of host cells. DNA-pull down and LC-MS/MS identified the porcine transcription factor AP-2δ (poTFAP2δ) could bind the PCV2 rep gene promoter. Dual-luciferase reporter assay, quantitative real-time PCR, Western blotting and indirect immunofluorescent assay demonstrated that poTFAP2δ could not only promote the activity of the rep gene promoter, but also enhance the transcription/translation activity of the rep/cap gene and the virus titer of PCV2 during the entire life cycle of PCV2 infection. This study revealed the molecular mechanism of PCV2 using host proteins to enhance the viral replication, provided a new perspective for studying the pathogenic mechanism of PCV2 from virus and host interactions, and provided a theoretical basis for developing highly effective PCV2 vaccines.
Animals ; Cell Line ; Chromatography, Liquid ; Circoviridae Infections ; Circovirus ; DNA Helicases ; Diabetes Mellitus, Type 2 ; Promoter Regions, Genetic ; Swine ; Tandem Mass Spectrometry ; Transcription Factor AP-2 ; Virus Replication

Background: TAM receptors (Tyro3, Axl and Mertk), as subfamily of receptor tyrosine kinases, are intracellular negative feedback regulators and play a crucial role in regulating inflammation and immune response. Aims: To study the expressions and clinical value of TAM receptors in serum and intestinal mucosa of patients with ulcerative colitis (UC). Methods: Forty⁃five patients who were initially diagnosed as active UC from June, 2020 to May, 2021 at the First Affiliated Hospital of Kunming Medical University were enrolled prospectively. Fifteen healthy subjects were served as the control group. Eight cases each in moderate UC group and healthy control group were selected randomly for detection of TAM receptors in serum and intestinal mucosa by ELISA, real⁃time PCR and Western blotting. The correlations of serum Tyro3 with routine clinical indicators of UC were analyzed by Pearson correlation coefficient. Furthermore, immunohistochemistry was used to detect the expression level of TAM receptors in intestinal mucosa in all UC patients and the healthy controls. Results: Expressions of Axl and Mertk were not fully consistent in serum and intestinal mucosa in UC patients. While the serum Tyro3 level, as well as the intestinal Tyro3 mRNA and protein expressions were consistently increased in moderate UC patients than in controls (all P<0.05). Serum level of Tyro3 was positively correlated with platelet count and C⁃reactive protein, and negatively correlated with albumin in moderate UC patients (r=0.97, r=0.96, r=-0.86, all P<0.05). Positivity rate of Tyro3 in intestinal mucosa of UC patients was positively correlated with the disease severity (all P<0.05). Conclusions: Tyro3 is closely related to UC and positively correlated with the disease severity. It might be a promising novel molecular marker and therapeutic target of UC.