Objective To determine the diversity of Porphytomonas gingivalis lipopolysaccharide (LPS)induced IL-1β,TNF-αand IL-6 levels in THP-1 cells and the associated Toll-like receptors(TLR).Methods P.gingivalis strain ATCC33277 LPS (Pg-LPS)was prepared using phenol-water method and then identified by both infrared spectrometry and limulus test.The levels of IL-1β,TNF-α and IL-6 secreted by THP-1 cells under inducement of Pg-LPS were quantitatively detected using commercial ELISA kits.The bloc-king test using TLR2 or TLR4 monoelonal antibody(McAb)plus the ELISA were used to determine the types of Pg-LPS binding TLR on the surface of target cells.In this study.a commercial LPS from E.coli strain O111:B4(E-LPS)was used as the contr01.Results When 1彬ml ofPg.u,s induced THP.1 ceHs for0.5,6 and 6 h.or l∥rnl ofE-LPs induced for 6,24 and 24 h,respectively,tIIe levels ofTNF.a,IL-1B and IL广6 were in.creased obviously(P<0.01).However,the maximal concentration of tlle t11ree cytokines induced by Pg.LP$ were similar to that induced by E-LPS(P>0.05).tLR2 or TLR4 McAb could block the effects of Pg-LPS in-ducing THP-1 cells to secrete IL-1β and IL-6(P<0.05),where as only TLR2 McAb displayed the inhibition of TNF-α secretion(P<0.05).On the contrast,only TLR4 McAb showed the effects blocking the three cytokines secretion in the THP-1 cells under inducement of E-LPS(P<0.05).Conclusion Pg-LPS shows a slight high-er activity inducing THP-1 cells to secrete IL-1β,TNF-α and IL-6 than E-LPS.TLR2,but not TLR4,is the major receptor of Pg-LPS on the target cells to mediate the secretion of the three cytokines.

According to the Chinese Pharmacopoeia 2015, only processed Aconitum tubers can be clinically applied, and the effect of processing is unclear. This research aimed to explore the effect of processing on cardiac efficacy of alkaloids in Aconitum tubers. First, the chemical ingredients in unprocessed and processed Aconitum tubers were identified and compared by using high performance liquid chromatography time-of-flight mass spectrometry (HPLC-TOF/MS) and multivariate pattern recognition methods. Then the representative alkaloids in Aconitum tubers, aconitine, benzoylaconine, and aconine, which belong to diester-diterpenoid alkaloids, monoester-diterpenoid alkaloids, and amine-diterpenoid alkaloids, respectively, were selected for further validation of attenuated mechanism. Subsequent pharmacological experiments with aconitine, benzoylaconine, and aconine in SD rats were used to validate the effect of processing on cardiac functions. After processing the Aconitum tubers, it was found that the contents of diester-diterpenoid alkaloids were reduced, and those of monoester-diterpenoid alkaloids and amine-diterpenoid alkaloids were increased, suggesting that diesterditerpenoid alkaloids were transformed into monoester-diterpenoid alkaloids and amine-diterpenoid alkaloids. Through further decocting the aconitine in boiling water, it was confirmed that the three alkaloids could be progressively transformed. Pharmacological experiments with aconitine, benzoylaconine, and aconine in SD rats showed that aconitine at a dose of 0.01 mg/kg and aconine at a dose of 10 mg/kg enhanced the cardiac function, while benzoylaconine at a dose of 2 mg/kg weakened the cardiac function. The effect of processing is attributed to the transformation of the most toxic diester-diterpenoid alkaloids into less toxic monoesterditerpenoid alkaloids and amine-diterpenoid alkaloids.

A comparison of the volatile compounds in Rhizomes Curcumae （Ezhu） and Radix Curcumae （Yujin） was undertaken using gas chromatography mass spectrometi-y （GC-MS）. Ultrasonic extraction and GC-MS methods were developed for the simultaneous determination of five sesquiterpenes, namely, α-pinene, β-elemene, curcumol, germacrone and curdione, in Ezhu and Yunjin. Good linearity （r〉0.999） and high inter-day precision were observed over the investigated concentration ranges. The validated method was successfully used for the simultaneous determination of five sesquiterpenes in Ezhu and Yujin. The quantitative method can be effectively used to evaluate and monitor the quality of Chinese curcuma in clinical use.

A comparison of the volatile compounds in Rhizomes Curcumae (Ezhu) and Radix Curcumae (Yujin) was undertaken using gas chromatography-mass spectrometry (GC-MS).Ultrasonic extraction and GC-MS methods were developed for the simultaneous determination of five sesquiterpenes,namely,α-pinene,β-elemene,curcumol,germacrone and curdione,in Ezhu and Yunjin.Good linearity (r＞0.999) and high inter-day precision were observed over the investigated concentration ranges.The validated method was successfully used for the simultaneous determination of five sesquiterpenes in Ezhu and Yujin.The quantitative method can be effectively used to evaluate and monitor the quality of Chinese curcuma in clinical use.

An offline two-dimensional system combining a rat cardiac muscle cell membrane chromatography time-of-flight mass spectrometry (CMC-TOF/MS) with a high performance liquid chromatography time-of-flight mass spectrometry (HPLC-TOF/MS) was established for investigating the parent components and metabolites in rat urine samples after administration of the roots of Aconitum carmichaeli.On the basis of the analysis of the first dimension,retention components of the mine sample were collected into 30 fractions (one fraction per minute).Then offline analysis of the second dimension was carried out.34 compounds including 24 parent alkaloids and 10 potential metabolites were identified from the dosed rat urine,and then binding affinities of different compounds on cell membranes were compared and influences of some functional groups on activity were estimated with the semi-quantification and curve fitting method.As a result,binding affinities decreased along with the process of deacylation,debenzoylation and demethylation,which may be related to the alleviation of toxicity in the procedure of herb processing or metabolism.Moreover,some minor components in rat urine (Songorine,14-benzoylneoline,Deoxyaconitine,etc.) exerted relatively strong affinity on cell membranes are worth exploring.The results delivered by the system suggest that the CMC can be applied to in vivo study.

An offline two-dimensional system combining a rat cardiac mascle cell membrane chromatography time-of-flight mass spectrometry （CMC-TOF/MS） with a high performance liquid chromatography time-of-flight mass spectrometry （HPLC-TOF/MS） was established for investigating the parent components and metabolites in rat urine samples after administration of the roots of Aconitum carmichaeli. On the basis of the analysis of the first dimension, retention components of the urine sample were collected into 30 fractions （one fraction per minute）. Then offline analysis of the second dimension was carried out. 34 compounds including 24 parent alkaloids and 10 potential metabolites were identified from the dosed rat urine, and then binding affinities of different compounds on cell membranes were compared and influences of some functional groups on activity were estimated with the semi-quantification and curve fitting method. As a result, binding affinities decreased along with the process of deacylation, debenzoylation and demethylation, which may be related to the alleviation of toxicity in the procedure of herb processing or metabolism. Moreover, some minor components in rat urine （Songorine, 14-benzoylneoline, Deoxyaconitine, etc. ） exerted relatively strong affinity on cell membranes are worth exploring. The results delivered by the system suggest that the CMC can be applied to in vivo study.

: To assess the () recombinant gingivalis gingipain R2 (rRgpB)-induced Ca mobilization in human gingival fibroblast (HGF) mediated by protease-activated receptor (PAR) and its downstream signal transduction pathways. : Flow cytometry was used to detect the expression of PAR in HGF. The proliferation of HGF was measured by CCK-8. The dynamic changes of intracellular Ca concentration in HGF induced by rRgpB and the blocking effect of PAR-1 antagonist were observed by laser confocal microscopy. Western blot was performed to determine the phosphorylation levels of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) 1/2, p38 mitogen-activated protein kinase (p38 MAPK) and p65 in HGF. : PAR-1 and PAR-3 were expressed in HGF, and the rRgpB could promote the proliferation of HGF. rRgpB caused a transient increase in [Ca], which could be completely suppressed by vorapaxar, a PAR-1 antagonist. The phosphorylation levels of JNK, ERK1/2 and p65 were significantly up-regulated after the induction of rRgpB for and (all <0.05), which was completely inhibited by vorapaxar. However, the phosphorylation level of p38 MAPK had no significant change after rRgpB stimulation. : rRgpB causes an increase in [Ca] in HGF mediated by PAR-1. JNK, ERK1/2 and nuclear factor-κB may be involved in intracellular signal transduction after PAR-1 activation.
Fibroblasts ; Humans ; JNK Mitogen-Activated Protein Kinases/metabolism* ; MAP Kinase Signaling System ; Phosphorylation ; Signal Transduction ; p38 Mitogen-Activated Protein Kinases/metabolism*

Therapeutic drug monitoring(TDM)has played an important role in clinical medicine for precise dosing.Currently,chromatographic technology and immunoassay detection are widely used in TDM and have met most of the needs of clinical drug therapy.However,some problems still exist in practical appli-cations,such as complicated operation and the influence of endogenous substances.Surface plasmon resonance(SPR)has been applied to detect the concentrations of small molecules,including pesticide residues in crops and antibiotics in milk,which indicates its potential for in vivo drug detection.In this study,a new SPR-based biosensor for detecting chloramphenicol(CAP)in blood samples was developed and validated using methodological verification,including precision,accuracy,matrix effect,and extraction recovery rate,and compared with the classic ultra-performance liquid chromatography-ultraviolet(UPLC-UV)method.The detection range of SPR was 0.1-50 ng/mL and the limit of detec-tion was 0.099±0.023 ng/mL,which was lower than that of UPLC-UV.The intra-day and inter-day ac-curacies of SPR were 98％-114％and 110％-122％,which met the analysis requirement.The results show that the SPR biosensor is identical to UPLC-UV in the detection of CAP in rat blood samples;moreover,the SPR biosensor has better sensitivity.Therefore,the present study shows that SPR technology can be used for the detection of small molecules in the blood samples and has the potential to become a method for therapeutic drug monitoring.

OBJECTIVETo investigate the expression types of protease-activated receptors(PAR) in human gingival fibroblasts(HGF) and the functions of PAR in periodontitis.

METHODSPrimary HGF were cultured.Reverse transcription PCR(RT-PCR) was used to detect the expression of PAR in HGF. Recombinant gingipain R (rRgp) was applied to HGF. The change of PAR expression on the cell surface was analyzed by real-time quantitative RT-PCR, and enzyme-linked immunosorbent assay (ELISA) was used to detect the change of the interleukin (IL)-6 production from HGF. The results of RT-PCR and ELISA were statistically analyzed using the two independent samples t-test of SPSS10.0 software.

RESULTSHGF expressed PAR-1 and PAR-3. The expression of PAR-1 and PAR-3 changed after two rRgp treatment with HGF cells. The relative expression of PAR-1 was decreased from 1.04 ± 0.31 to 0.67 ± 0.11 and 0.31 ± 0.11. The relative expression of PAR-3 was decreased from 1.01 ± 0.44 to 0.79 ± 0.13 and 0.44 ± 0.12(P < 0.05). The level of IL-6 was increased after rRgp treatment for 8 h. The control group was (18.77 ± 4.09) µg/L, the rRgp treatment groups were (179.36 ± 15.81) and (320.56 ± 26.19) µg/L respectively.

CONCLUSIONSHGF expressed PAR-1 and PAR-3 and were involved in periodontal inflammation.

Adhesins, Bacterial ; Cell Membrane ; Cysteine Endopeptidases ; Enzyme-Linked Immunosorbent Assay ; Fibroblasts ; Gingiva ; metabolism ; Humans ; Interleukin-6 ; Periodontitis ; metabolism ; Receptors, Proteinase-Activated ; biosynthesis

Objective To cultivate glioblastoma U87 stem-like cells(SLCs)and to detect the level of stemness bio-markers,mitochondrial respiratory capacity and the capacity of in vivo tumorigenesis.Methods B-27,growth factors EGF and bFGF was added into DMEM/F-12 culture in serum-free stem cell culture medium for U87 SLCs.Suspended culture of U87 SLCs was suspended using the neuro-sphere formation assay,while adherent culture of U87 SLCs was achieved by coating Matrigel matrix on the culture surface.The mRNA and protein level of stemness biomarkers in culture were detected using real-time quantitative PCR and Western blot.The proportion of CD133+cells in culture was detected by flow cytometry.The changes of cell oxygen consumption rate were detected by Seahorse cell metabo-lism analysis.Cell tumorigenesis ability was verified by subcutaneous tumor transplantation in animals.Results U87 SLCs in stem cell culture medium would grow into typical sphere morphology within one week,and the spheres would continue to grow as the culture process prolongs.At the appropriate concentration of adhesive,U87 SLCs adhered to and grow well in stem cell culture medium.The mRNA transcription of stemness biomarkers such as CD133,nes-tin,OLIG2,CD44,CD15,and integrin α6(ITGA6)was significantly increased as found in both culture methods,and the protein levels of CD133 and nestin were also increased under both methods(P＜0.05).U87 SLCs showed higher mitochondrial reserve respiratory capacity(P＜0.05).U87 SLCs could form larger subcutaneous tumors with fewer inoculated cells(P＜0.05),and grew faster in vivo with stronger tumorigenic ability.Conclusions U87 SLCs have typical stemness characteristics and may function as tumor cell model with higher stemness properties.