Objective To improve the analysis method of the blood components of Yinchenhao decoction （YCHD） in vivo and explore its anti-hepatocellular carcinoma mechanism. Methods Ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry （UPLC-Q-TOF/MS） was used to collect and analyze blood samples from mice. The mice were given a single dose of YCHD with a concentration of 0.1 g/ml and a dose of 25 ml/kg, and then the samples were collected 2 h post–administration, which was to systematically study the chemical components of YCHD in vivo. Network pharmacological methods were used to screen the components and targets of YCHD, and the targets of hepatocellular carcinoma; The common targets of YCHD and hepatocellular carcinoma were identified for GO enrichment and KEGG enrichment. Molecular docking was performed on the main targets to verify the binding ability between the active ingredients and the core targets. The relative mRNA expression levels of serine/threonine-protein kinase（AKT1） and tumor protein p53（TP53） in liver tissues were analyzed via qPCR, including the following mouse groups: mice with concanavalin A（Con-A）-induced acute liver injury without preventive administration, mice with Con-A-induced acute liver injury that received 14 d preventive oral administration of YCHD, and untreated control mice. Results ①The active ingredients of YCHD in the blood were identified by retrieving the data from the in vitro component analysis. They were chrysophanol, herniarin, aloe-emodin, and monotropein. ②The mechanism of action of the blood components against hepatocellular carcinoma （HCC） was further analyzed using network pharmacological methods, and a total of 30 components of YCHD were screened for 213 targets and 215 HCC targets. ③There were 17 intersection targets between YCHD and hepatocellular carcinoma, including AKT1, TP53, receptor tyrosine-protein kinase erbB-2 （ERBB2）, myelocytomatosis oncogene （MYC）, interleukin-1β （IL-1β）, etc. The GO enrichment results indicated that these components were primarily involved in DNA replication，chromosome segregation，leukocyte mediated immunity，leukocyte cell-cell adhesion. The KEGG enrichment results demonstrated that these components were predominantly associated with diverse cancer pathways. Additionally, the results indicated involvement in the citrate cycle （TCA cycle）, pyruvate metabolism, and p53 signaling pathway, ect. ④The results of molecular docking showed that chrysophanol, herniarin, and aloe - emodin had strong binding abilities with AKT1, TP53, ERBB2, MYC, and IL-1β. ⑤The relative expression of AKT1 and TP53 mRNA was significantly higher in the modelling group than in the control group. The relative expression of AKT1 and TP53 mRNA was significantly lower in the drug administration group than in the modelling group. Conclusion There were 4 blood components in YCHD, among which chrysophanol, herniarin, and aloe-emodin may act on AKT1, TP53, ERBB2, MYC, IL-1β and then participated in the regulation of cancer signaling pathways and p53 signaling pathway to play a role in the treatment of HCC.

Sini Decoction (SNT) is a traditional formula recognized for its efficacy in warming the spleen and stomach and dispersing cold. However, elucidating the mechanism of action of SNT remains challenging due to its complex multiple components. This study utilized a synergistic approach combining two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE)-based drug affinity responsive target stability (DARTS) with label-free quantitative proteomics techniques to identify the direct and indirect protein targets of SNT in myocardial infarction. The analysis identified 590 proteins, with 30 proteins showing significant upregulation and 51 proteins showing downregulation when comparing the SNT group with the model group. Through the integration of 2D-DIGE DARTS with proteomics data and pharmacological assessments, the findings indicate that protein disulfide-isomerase A3 (PDIA3) may serve as a potential protein target through which SNT provides protective effects on myocardial cells during myocardial infarction.
Myocardial Infarction/genetics* ; Proteomics/methods* ; Drugs, Chinese Herbal/chemistry* ; Animals ; Protein Disulfide-Isomerases/genetics* ; Male ; Two-Dimensional Difference Gel Electrophoresis/methods* ; Humans ; Rats ; Rats, Sprague-Dawley ; Electrophoresis, Gel, Two-Dimensional

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.

Objective To study the effect of carboxyamidotriazole-orotate(CTO)on the proliferation and fatty acid anabolism regulation of human pancreatic cancer cells.Methods Human pancreatic cancer cell lines AsPC-1,AsPC-1/GEM(AR),PANC-1 and MiaPaCa-2 were used as the study subjects;cell survival rate was detected by sulfo-nylrhodamine B(SRB);the mRNA level of key genes for fatty acid synthesis was detected by qPCR;the protein level of the AMPK/ACC pathway was detected by Western blot;intracellular lipid metabolites were examined by liquid chromatography-mass spectrometry(LC-MS).Results Comparing to control group,CTO significantly de-creased the cell viability of AsPC-1,AR,PANC-1,and MiaPaCa-2(P＜0.05).CTO down-regulated the mRNA level of key fatty acid synthesis genes(P＜0.05).CTO significantly reduced the protein expression of AMPK,ACC and c-Myc(P＜0.05),while increasing the protein expression of p-AMPK and p-ACC(P＜0.05).CTO decreased lipid metabolite content in AR cells(P＜0.05).Conclusions CTO attenuates cellular fatty acid anabolism by inhibition of oncogene c-Myc expression and AMPK/ACC pathway,down-regulates the expression of fatty acid synthesis-related genes,and then inhibits proliferation of the human pancreatic cancer cell lines AsPC-1,AR,PANC-1 and MiaPaCa-2.

Objective To explore and analyze the feasibility of using indocyanine green(ICG)near-infrared fluores-cence(NIF)imaging technology for the early diagnosis of oral potential malignant disorders and oral squamous cell car-cinoma.Methods 7,12-Dimethylbenz[a]anthracene in acetone solution was used to induce various pathological models of buccal mucosal lesions(mild/moderate dysplasia,severe dysplasia,squamous cell carcinoma)in golden hamster.ICG-NIF was conducted for the quantitative analysis of the fluorescence signal of lesion tissue,and evaluation of the diagnos-tic and discriminative capabilities of the ICG-NIF technology for mucosal lesions in various pathological states.Immuno-histochemical staining was perform to examine the mi-crovessel density(MVD)and microlymphatic vessel den-sity(MLVD)of mucosa in various pathological states and explore the histological reasons underlying the differ-ences in fluorescence signals.Results The results of ICG-NIF fluorescence quantitative analysis reveal the higher fluorescence intensity of mucosal lesions in the experimental group compared with that of the normal mucosa on the control side,with statistical differences(P＜0.05).Moreover,the more severe the malignancy of mucosal lesions in the experimental group,the higher the fluorescence intensity.According to histopathological analysis,the malignant pro-gression of mucosal lesions in golden hamsters was accompanied with an increase in MVD(P＜0.05)and a decrease in MLVD(P＜0.05).Conclusion The abnormal proliferation of mucosal lesions in golden hamsters exhibits a difference in ICG-NIF fluorescence signal compared with normal mucosal tissue.Fluorescence quantitative analysis methods can provide assistance in differentiation and show potential for clinical applications.

Objective To analyze three-dimensional(3D)radiographic characterizations of bone island(BI)in jaw using cone-beam computed tomography(CBCT).Methods CBCT data from four thousand patients were selected,reconstructed and analyzed using NNT 10.0 software.The sagittal,coronal and axial planes were used to analyze the 3D radiographic characteristics of BIs,including the localization,shape,density,boundary,the relationship between BIs and tooth and bone cortex,diameter and anatomical structures and complications involved.Their relationship with gender were analyzed.Results A total of 803 people had BIs,with the prevalence rate of 20.08%,including 338 males with 389 BIs and 465 females with 526 BIs.Both males and females had a dominant BI,and the ratio between male and female was 1∶1.38,but the difference was not statistically significant(P＞0.05).The BIs of both male and female mostly occurred in the mandibular premolars and molars area,and appeared irregular in shape,dense and contact with lingual bone cortex.Mostly BIs were apical type and with unclear boundary.The mean maximum diameter of mesial/distal direction was greater than buccal/lingual direction(P＜0.05).The most commonly involved anatomy structure was the inferior alveolar neural canal,cortical infil-tration and mental foramen.Conclusion There are no significant differences between males and females in the three-dimensional image characteristics of BIs in Chinese populations.CBCT can accurately and comprehensively analyze the 3D radiographic characteris-tics of BI and its relationship with the surrounding teeth and bone.

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.

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*

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.