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 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.

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*

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.

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.

Objective:To research the mitochondrial cytochrome c oxidase subunit I (MT-COI) gene methylation levels in patients with occupational chronic benzene poisoning, and to explore effective molec μlar biomarkers in patients with occupational chronic benzene poisoning.Methods:38 confirmed cases of occupational chronic benzene poisoning were selected in the case group. 46 healthy people who underwent physical in our hospital were selected in the control group. Pyrosequencing was used to detect the methylation sites of methylation sites, flow cytometry was used to detect peripheral blood cell count levels, and non-parametric statistical methods were used to analyze the differences in detection results between the two groups.Results:The methylation level of mitochondrial MT-COI site 1 (2.21±0.81) % in the case group was less than that in the control group, and the difference was statistically significant ( P<0.05) . The methylation level of mitochondrial MT-COI site 2 (2.31±0.96%) in the case group was less than that in the control group, and the difference was statistically significant ( P<0.05) . The methylation average level of mitochondrial MT-COI (2.26±0.75) % in the case group was less than that in the control group, and the difference was statistically significant ( P<0.05) . Analysis of the average level of methylation found that the methylation level of mitochondrial MT-COI was correlated with WBC ( P<0.05) . Analysis of the average level of methylation found that the methylation level of mitochondrial MT-COI was correlated with platelets ( r=0.254、0.280, P<0.05) . Conclusion:The level of mitochondrial MT-COI gene methylation in patients with occupational chronic benzene poisoning may be related to the sensitivity to benzene exposure. Mitochondrial MT-COI gene methylation may serve as a potential predictive biomarker for benzene poisoning.

Objective:To research the mitochondrial cytochrome c oxidase subunit I (MT-COI) gene methylation levels in patients with occupational chronic benzene poisoning, and to explore effective molec μlar biomarkers in patients with occupational chronic benzene poisoning.Methods:38 confirmed cases of occupational chronic benzene poisoning were selected in the case group. 46 healthy people who underwent physical in our hospital were selected in the control group. Pyrosequencing was used to detect the methylation sites of methylation sites, flow cytometry was used to detect peripheral blood cell count levels, and non-parametric statistical methods were used to analyze the differences in detection results between the two groups.Results:The methylation level of mitochondrial MT-COI site 1 (2.21±0.81) % in the case group was less than that in the control group, and the difference was statistically significant ( P<0.05) . The methylation level of mitochondrial MT-COI site 2 (2.31±0.96%) in the case group was less than that in the control group, and the difference was statistically significant ( P<0.05) . The methylation average level of mitochondrial MT-COI (2.26±0.75) % in the case group was less than that in the control group, and the difference was statistically significant ( P<0.05) . Analysis of the average level of methylation found that the methylation level of mitochondrial MT-COI was correlated with WBC ( P<0.05) . Analysis of the average level of methylation found that the methylation level of mitochondrial MT-COI was correlated with platelets ( r=0.254、0.280, P<0.05) . Conclusion:The level of mitochondrial MT-COI gene methylation in patients with occupational chronic benzene poisoning may be related to the sensitivity to benzene exposure. Mitochondrial MT-COI gene methylation may serve as a potential predictive biomarker for benzene poisoning.

Objective To investigate the expression types of protease-activated receptors (PAR) in human gingival fibroblasts(HGF) and the functions of PAR in periodontitis.Methods Primary 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.Results HGF 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.Conclusions HGF expressed PAR-1 and PAR-3 and were involved in periodontal inflammation.