OBJECTIVE:To establish a method for the contents determination of 9 components in Guizhi decoction,and com-pare the effects of traditional decoction method and the extracting machine decoction method on these contents in Guizhi decoction. METHODS:HPLC was performed on the column of Agilent Zorbax SB-C18 with mobile phaseof acetonitrile- 0.1% phosphoric ac-id(gradient elution)at a flow rate of 1.0 ml/min,the detection wavelength was 230 nm,254 nm and 280 nm,the column tempera-ture was 25℃,and the injection volume was 10μl. RESULTS:The linear range was 0.410 2-210.0μg/ml for gallic acid(r=0.999 9), 0.994 0-254.5μg/ml for albiflorin(r=0.999 9),1.636 0-1 675.0μg/ml for paeoniflorin(r=0.999 9),0.988 3-506.0μg/ml for liquiri-tin(r=0.999 6),0.987 3-31.59 μg/ml for coumarin(r=0.999 5),0.486 8-124.6 μg/ml for cinnamic acid(r=0.999 5),2.458 0-314.6μg/ml for cinnamaldehyde(r=0.999 5),0.034 3-1.096 μg/ml for 2-methoxy cinnamaldehyde(r=0.999 8),and 1.711 0-219.0 μg/ml for glycyrrdhizic acid (r=0.999 7);RSDs of precision,stability and reproducibility tests were lower than 5%,recoveries were 93.56%-103.19%(RSD=4.00%,n=9)、101.51%-107.32%(RSD=2.21%,n=9)、95.08%-103.76%(RSD=2.87%,n=9)、100.82%-105.73%(RSD=1.85%,n=9)、85.08%-89.12%(RSD=1.40%,n=9)、92.31%-99.12%(RSD=2.71%,n=9)、99.17%-102.32%(RSD=1.24%,n=9)、100.15%-103.98%(RSD=1.18%,n=9)、99.93%-102.61%(RSD=1.03%,n=9). The content of total effective components from the extracting machine decoction method was 4 565μg/g,that from the traditional decoc-tion method was 2 742 μg/g.CONCLUSIONS:The method is simple,stable and reproducible,and can be used for the simultaneous determination of 9 componentsin Guizhi decoction. The contents of gallic acid,albiflorin and 2-methoxy cinnamaldehyde are first re-ported. The total effective components from the extracting machine decoction method are higher than that from the traditional decoc-tion method.

Objective To investigate the effects of 13-hexyl-berbefine hydroehlofide (HB-13) and 13-hexyl-paimatine hydrochloride (HP-13) on the activation of nuclear factor-kappa B (NF-kB) and phosphorylation of p38 mitogen-activated protein kinase (MAPK) in a human keratinocyte cell line, HaCaT stimulated by tumor necrosis factor alpha (TNF-α). Methods HaCaT cells were cultured in the presence of various concentrations (0.39, 0.78, 1.56 μg/mL) of HB-13 or HP-13 for 120 minutes followed by the stimulation with recombinant human TNF-α for 120 minutes (in phosphorylatEd-IkB-α test) or 15 minutes (in phosphorylated-p38 test). Then, HaCaT cells were disrupted, total protein was extracted, and the expressions of phosphorylated I B-α and phosphorylated p38 were detected with Western blot. HaCaT cells receiving neither pretreatment nor stimulation served as blank control, untreated HaCaT cells stimulated by rhTNF-α as stimulator control, and HaCaT cells pretreated with turmeric root tuber and stimulated by rhTNF-α as positive control. Results From 0.39 to 1.56 μg/mL, both HB-13 and HP-13 significantly inhibited the expression of p-IkB-α in HaCaT cells stimulated by rhTNF-α, and a nonsignificant dose-dependent trend was observed for their inhibitory effect, with the ICo value being 0.441 μg/mL for I-IB-13 (r = -0.990, n = 3, P > 0.05) and 0.832 μg/mL for HP-13 (r = -0.992, n = 3, P > 0.05). In contrast, neither 1-113-13 nor HP-13 within the experiment concentration range had a significant effect on the expression of p-p38 in HaCaT cells stimulated by rhTNF-α (P > 0.05). Conclusions Within the experimental concentration range, both HB-13 and HP-13 can inhibit the activation of NF-kB in HaCaT cells induced by TNF-α signal, but neither of them suppress the phosphorylation of p38MAPK induced by TNF-α signal in HaCaT cells.

Objective To investigate the effects of triptolide on the expression of a series of proteins associated with interferon-γ (IFN-γ)signaling in HaCaT keratinocytes.Methods After pretreatment with difrerent dosages of triptolide(10-10-10-7 mol/L),HaCaT cells were stimulated by recombinant human IFN-γ(rhIFN-γ,500 U/mL)for various periods followed by the collection of cells.Then,total protein was extracted from these cells and subjected to Western blotting for the detection of expression of interferon-γ receptor α(IFN-γRα),phosphorylated Janus kinase 2(pJAK2)and suppressor of cytokine signaling (SOCS1).Results Triptolide at the concentrations of 10-8 mol/L and 10-7 mol/L significantly inhibited the IFN-γRα expression upregulated by rhIFN-γ(both P＜0.05).The expression of pJAK2 induced bv rhIFN-γ was also suppressed by triptolide at the concentrations of 10-9 moI/L and 10-8 mol/L(both P＜0.05).The inhibition of triptolide on IFN-γRα and pJAK2 expression was dose-dependent and the 50%inhibitory concentrations(IC50 value)were 1.37×10-8 mol/L and 2.83×10-9 mol/L,respectively.On the contrary,triptolide upregulated the expression of SOCS1 stimulated by rhIFN-γ at the concentrations of 10-10,10-9 and 10-8 mol/L(P＜0.05,0.05,0.01,respectively)with the 50%effective dosage(ED50 value)at 3.32 × 10-11 mol/L.Conclusions By inhibiting the expression of IFN-γRα as well as phosphorylation of JAK2 and upregulating the expression of SOCS1,triptolide inhibits the phosphorylation of STAT-1,resulting in the inhibition of genetic transcription of multiple inflammatory factors induced by IFN-γ signaling in HaCaT keratinocytes,and the inhibition probably contributes to the efficacy of triptolide in the treatment of IFN-γ-dependent inflammatory skin disorders,such as psoriasis.

Objective Analysis and adscription of volatiles from Guizhi Tang and study on its improvement of the learning and memory in dementia mice induced by scopolamine.Methods The volatile oil from Guizhi Tang(GZT),Guizhi and Shengjiang was extracted using steam distillation method and was analyzed by GC-MS. Morris water maze and step-down test were carried out for obtain the difference of the learning and memory improvement in 40 ICR mice from randomized groups, such as the control group, the model group, the donepezil group (2 mg/kg), the low dose of volatile oil of GZT (5 mg/kg), and the high dose of volatile oil of GZT (20 mg/kg), and ACh, AchE, BchE and chE in serum were detected by ELISA. Results Among 38 identyfied volatile ingredients from GZT, 18(44% in weight) was from Guizhi, and 9 was from Shengjiang. Compared with the model group, the low and high dose of GZT volatile oil significantly increased swimming distance ratio in destination quadrant (26.74% ± 16.42%vs.9.42% ± 8.50%, P<0.05); goal quadrant time scale (43.51% ± 25.12%vs. 14.50% ± 12.23%,P<0.05)) increased significantly than the model group ; the number of errors in the experiment platform (1.63 ± 1.19vs. 0.25 ± 0.46, P<0.05) obviously increased than model group ; platform test in the made errors times (0.57 ± 0.98vs. 4.43 ± 2.4, P<0.05) significantly reduced. The GZT total volatile oil groups significantly reduced cognitive obstacles small rat serum in the cholinester enzyme (chE) (140.90 ± 3.27, 144.79 ± 6.71vs. 134.49 ± 3.36,P<0.05); acetylcholinesterase (AchE) (3.30 ± 1.31, 3.94 ± 0.78 vs.8.52 ± 3.39,P<0.05); butyrylcholinesterase (BchE) (3.22 ± 0.45, 3.66 ± 0.53vs. 7.99 ± 0.79,P<0.05); and acetylcholine (Ach) (4.10 ± 0.38, 3.03 ± 0.25vs.1.72 ± 0.50, P<0.05) significantly increased.Conclusions The GZT volatile oil mainly from Guizhi and Shengjiang can improve the learning and memory ability in dementia mice induced by scopolamine via a cholinergic mechnism.

Objective To explore the feasibility of fluorescence quantitative PCR(FQ-PCR)in prenatal diagnosis of the fetal RhD genotyping using free DNA from RhD-negative pregnant women.Methods The fetal RhD gene was amplified from 78 RhD-negative pregnant women with single fetus maternal plasma (gestation from 11 to 40 weeks).Rhe existence of fetal DNA was confirmed by amplification ofnine different polymorphic short tandem repeat loci(STR)and sex-determining region Y chromosome(SRY)gene.Exon5,7,10 and intron 4 were amplified by real-time polymerase chain reaction with TaqMan probe.The results of fetal RhD genotyping were evaluated retrospectively by the serologic analysis of infant cord blood.Results Among the 78 specimens,the SRY positive signals were detected from samples of 41 and were all identified male fetal through 8ex observation after newborn infants delivered from the women enrolled.The mean concentration of SRY gene reached(214.7±120.9)eopies/ml.RhD genotyping results of 70 cases were in complete concordance with the resets through serological detection of fetal cord blood after delivery.In addition,5 cases were false-positive.3 cages were considered inconclusive.The coincidence rate was 90%(70/78).From 5 false-positive cases,4 cases were identified as RhDel phenotype by detecting RHDl227A allele gene.The final accuracy rate of FQ-PCR was 95%(74/78)in the fetal RhD genotyping.Conclusion FQ-PCR analysis for noninvasive prenatal of fetal RhD genotyping could be useful in prevention and diagnosis of hemolytic disease of newborn.

Objective To establish a simple and effective method for isolation and culture of mouse adipose-derived stem cells (mASCs)in vitro,in order to provide the sufficient sources of seed cells for the research of mesenchymal stem cells.Methods The mouse inguinal fat tissues were isolated in vitro and performed a digestion with 0.1% collagenase type NB4,then adipose-derived stem cells(ASCs)were seeded and adhered to the culture dishes in low glucose DMEM containing 10% fetal calf serum.The cellu-lar morphology,in vitro proliferation capacity,multidifferentiation potential and immunophenotype were assessed.Results The mASCs showed good cell morphology,extremely strong proliferation capacity and potential of adipogenesis,osteogenesis and chon-drogenesis via in vitro three-dimensional induction.The cellular surface antigen phenotype was consistent with that reported by lit-erature,and the expression of CD34 and CD105 was positive,Sca-1 was highly expressed,CD45 and SSEA-1 were not expressed. Conclusion Using the experimental methods in this research can culture the high purity of mASCs with the excellent stem cell properties and extremely strong proliferative ability.

Cinnamaldehyde was shown to have significant anti-inflammatory and anti-pyretic actions in studies from both others' and our lab. Prostaglandin E2 (PGE2) plays a key role in generation of these pathological states, while PGE, synthase-1 (mPGES-1) is one of crucial biological elements in the process of PGE2 production. And as a downstream inducible terminal prostaglandin synthase of COX-2, mPGES-1 is now regarded as a more promising novel drug target than COX-2 and is attracting more and more attention from both academia and pharmaceutical industry. The purpose of present study was to further investigate the anti-inflammatory and antipyretic molecular mechanisms of cinnamaldehyde based on the mouse macrophage cell line RAW264. 7 in vitro. The PGE2 was identified by using the method of enzyme-linked immunosorbent assay (ELISA) and the expression of COX-2 and mPGES-1 at mRNA and protein levels was detected by the Real-time PCR and Western blotting methods respectively. The experimental results suggested that cinnamaldehyde could evidently reverse the increased production of PGE2induced by IL-1beta. Moreover, the up-regulated expression levels of mPGES-1 and COX-2 were significatly decreased. Together, these results provide compelling evidence that the down-regulated actions to both the production of PGE2 as well as the expression of mPGES-I might account for, at least in part, the anti-inflammatory and anti-pyretic effects of cinnamaldehyde.
Acrolein ; analogs & derivatives ; pharmacology ; Animals ; Blotting, Western ; Cell Line ; Dinoprostone ; metabolism ; Interleukin-1beta ; pharmacology ; Intramolecular Oxidoreductases ; metabolism ; Macrophages ; drug effects ; metabolism ; Mice ; Prostaglandin-E Synthases ; Real-Time Polymerase Chain Reaction

This study was aimed to investigate the effect of Nan-She-Teng (Celastrus orbiculatus) extract on epithe-lial-mesenchymal transition(EMT) in HepG2 cells. Except the control group, human hepatocellular carcinoma HepG2 cells in other groups were treated with Celastrus Orbiculatus extract in different concentrations (10, 20, 40, 80, and 160 μg·mL-1). The protein expression levels related to EMT were detected by western blotting. At 48 h after fertiliza-tion, the zebrafish embryos were randomly assigned to 7 groups as follows: untreated control group (E3 buffer), DMSO group (E3 buffer with 1% DMSO), and different dosages treatment of C.orbiculatus extract (10, 20, 40, 80, and 160μg·mL-1) for 24 h. The protein expressions of mTOR signaling pathways were detected by western blotting. The re-sults showed that compared with the control group, C.orbiculatus extract significantly increased E-cadherin protein expression. Simultaneously, C.orbiculatus extract inhibited vimentin and mTOR signaling pathways at protein levels. It was concluded that to a certain extent, C.orbiculatus extract prevented EMT in HepG2 cells by modulating the mTOR signaling pathway. Therefore, it suggested that mTOR can be chosen as a new therapeutic target for clinical treatment of hepatic carcinoma.

Aim To investigate the effect of non-T cell binding peptide(FNS007)on collagen type Ⅱ-induced arthritis(CIA)in mice and the possible mechanisms.Methods The CIA model was induced by intradermal injection of bovine CⅡ+Freunds adjuvant.At the clinical onset of CIA,mice were randomly divided into 6 groups: blank control group(Control),model group,ORENCIA(abatacept)group,FNS007 low dose(1.2 mg·kg-1)group,FNS007 middle dose(2.4 mg·kg-1)group and FNS007 high dose(4.8 mg·kg-1)group.FNS007 was given by intravenous injection on the first day of arthritis and every other day until the study was terminated on d 28 after injection of the drug.The paw thickness and the ankle joint width were measured,and the arthritis scores were recorded.At termination,interferon-γ(IFN-γ),tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and level of anti-CⅡ antibody in serum were examined by enzyme-linked immunosorbent assay(ELISA).Bone injury was analyzed by X-ray imaging,and HE staining was conducted to observe the histopathologic changes and pathological score of ankle tissues.Results CIA models were successfully induced.Compared with CIA group,FNS007 high dose significantly reduced the paw thickness and the ankle joint left-right diameter,lowered arthritis scores in CIA mice,reduced serum concentrations of IFN-γ,IL-6 and anti-CⅡ antibodies,and lowered the radiographic and histologic scores.Compared with CIA group,FNS007 middle dose group showed marked reduction in the arthritis scores,IL-6 content in serum,and inhibion in the radiographic and histologic scores.The arthritis scores,concentration of IFN-γ,the radiographic and histologic scores were significantly reduced in FNS007 low dose group compared with those in model group.Conclusion FNS007 can effectively inhibit the progression of CIA through inhibiting T-cell activation and reducing inflammatory cytokines,anti-CⅡ antibodies,and histoclasia and bone destruction.

This study was aimed to investigate whether the extracts of Celastrus orbiculatus enhanced the invasion function of maspin tumor inhibitor gene through the construction of maspin overexpression human gastric carcinoma MGC803 cell line. Maspin was cloned into plasmid GV208-EGFP eukaryotic expression vector. And then, the recombinant plasmid GV208-maspin-EGFP was transfected into human gastric carcinoma MGC803 cells. After the maspin overexpression MGC803 cell were treated with Celastrus orbiculatus extracts in different concentrations (10, 20, 40 μg·mL-1), the invasion effects were detected by Transwell chamber assay. The results showed that after the successful construction of maspin overexpression cell line, the number of cells invading through Matrigel was obviously decreased in the Transwell chamber assay. It also showed drug concentration dependency. It was concluded that maspin gene can inhibit invasion of gastric carcinoma MGC803 cells. Simultaneously, the extracts of Celastrus orbiculatus can enhance the function of maspin gene.