This study investigated the effects and underlying mechanisms of vanillic acid(VA) against cardiac fibrosis(CF) induced by isoproterenol(ISO) in mice. Male C57BL/6J mice were randomly divided into control group, VA group(100 mg·kg~(-1), ig), ISO group(10 mg·kg~(-1), sc), ISO + VA group(10 mg·kg~(-1), sc + 100 mg·kg~(-1), ig), ISO + dynamin-related protein 1(Drp1) inhibitor(Mdivi-1) group(10 mg·kg~(-1), sc + 50 mg·kg~(-1), ip), and ISO + VA + Mdivi-1 group(10 mg·kg~(-1), sc + 100 mg·kg~(-1), ig + 50 mg·kg~(-1), ip). The treatment groups received the corresponding medications once daily for 14 consecutive days. On the day after the last administration, cardiac functions were evaluated, and serum and cardiac tissue samples were collected. These samples were analyzed for serum aspartate aminotransferase(AST), lactate dehydrogenase(LDH), creatine kinase-MB(CK-MB), cardiac troponin I(cTnI), reactive oxygen species(ROS), interleukin(IL)-1β, IL-4, IL-6, IL-10, IL-18, and tumor necrosis factor-α(TNF-α) levels, as well as cardiac tissue catalase(CAT), glutathione(GSH), malondialdehyde(MDA), myeloperoxidase(MPO), superoxide dismutase(SOD), total antioxidant capacity(T-AOC) activities, and cytochrome C levels in mitochondria and cytoplasm. Hematoxylin-eosin, Masson, uranium acetate and lead citrate staining were used to observe morphological and mitochondrial ultrastructural changes in the cardiac tissues, and myocardial injury area and collagen volume fraction were calculated. Flow cytometry was applied to detect the relative content and M1/M2 polarization of cardiac macrophages. The mRNA expression levels of macrophage polarization markers [CD86, CD206, arginase 1(Arg-1), inducible nitric oxide synthase(iNOS)], CF markers [type Ⅰ collagen(Coll Ⅰ), Coll Ⅲ, α-smooth muscle actin(α-SMA)], and cytokines(IL-1β, IL-4, IL-6, IL-10, IL-18, TNF-α) in cardiac tissues were determined by quantitative real-time PCR. Western blot was used to detect the protein expression levels of Coll Ⅰ, Coll Ⅲ, α-SMA, Drp1, p-Drp1, voltage-dependent anion channel(VDAC), hexokinase 1(HK1), NOD-like receptor protein 3(NLRP3), apoptosis-associated speck-like protein(ASC), caspase-1, cleaved-caspase-1, gasdermin D(GSDMD), cleaved N-terminal gasdermin D(GSDMD-N), IL-1β, IL-18, B-cell lymphoma-2(Bcl-2), B-cell lymphoma-xl(Bcl-xl), Bcl-2-associated death promoter(Bad), Bcl-2-associated X protein(Bax), apoptotic protease activating factor-1(Apaf-1), pro-caspase-3, cleaved-caspase-3, pro-caspase-9, cleaved-caspase-9, poly(ADP-ribose) polymerase-1(PARP-1), and cleaved-PARP-1 in cardiac tissues. The results showed that VA significantly improved cardiac function in mice with CF, reduced myocardial injury area and cardiac index, and decreased serum levels of AST, CK-MB, cTnI, LDH, ROS, IL-1β, IL-6, IL-18, and TNF-α. VA also lowered MDA and MPO levels, mRNA expressions of IL-1β, IL-6, IL-18, and TNF-α, and mRNA and protein expressions of Coll Ⅰ, Coll Ⅲ, and α-SMA in cardiac tissues, and increased serum levels of IL-4 and IL-10, cardiac tissue levels of CAT, GSH, SOD, and T-AOC, and mRNA expressions of IL-4 and IL-10. Additionally, VA ameliorated cardiac pathological damage, inhibited myocardial cell apoptosis, inflammatory infiltration, and collagen fiber deposition, reduced collagen volume fraction, and alleviated mitochondrial damage. VA decreased the ratio of F4/80~+CD86~+ M1 cells and the mRNA expressions of CD86 and iNOS in cardiac tissue, and increased the ratio of F4/80~+CD206~+ M2 cells and the mRNA expressions of CD206 and Arg-1. VA also reduced protein expressions of p-Drp1, VDAC, NLRP3, ASC, caspase-1, cleaved-caspase-1, GSDMD, GSDMD-N, IL-1β, IL-18, Bad, Bax, Apaf-1, cleaved-caspase-3, cleaved-caspase-9, cleaved-PARP-1, and cytoplasmic cytochrome C, and increased the expressions of HK1, Bcl-2, Bcl-xl, pro-caspase-3, pro-caspase-9 proteins, as well as the Bcl-2/Bax and Bcl-xl/Bad ratios and mitochondrial cytochrome C content. These results indicate that VA has a significant ameliorative effect on ISO-induced CF in mice, alleviates ISO-induced oxidative damage and inflammatory response, and its mechanism may be closely related to the inhibition of Drp1/HK1/NLRP3 and mitochondrial apoptosis signaling pathways, suppression of myocardial cell inflammatory infiltration and collagen fiber deposition, reduction of collagen volume fraction and CollⅠ, Coll Ⅲ, and α-SMA expressions, thus mitigating CF.
Animals ; Isoproterenol/adverse effects* ; Male ; Mice ; Signal Transduction/drug effects* ; Vanillic Acid/administration & dosage* ; Dynamins/genetics* ; Mice, Inbred C57BL ; Fibrosis/genetics* ; Apoptosis/drug effects* ; Mitochondria/metabolism* ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics* ; Myocardium/metabolism* ; Humans

Ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS/MS) was used to rapidly identify the chemical components in Dracocephalum moldavica, and UPLC was employed to determine the content of its main components. MS analysis was performed using an electrospray ionization(ESI) source and data were collected in the negative ion mode. By comparing the retention time and mass spectra of reference compounds, and using a self-built compound database and the PubChem database, 68 compounds were identified from D. moldavica, including 36 flavonoids, 22 phenylpropanoids, 4 phenols, and 6 other compounds. On this basis, a UPLC quantitative method was established to simultaneously determine 8 main components, i.e., luteolin-7-O-glucuronide, apigenin-7-O-glucuronide, rosmarinic acid, diosmetin-7-O-glucuronide, tilianin, acacetin-7-O-glucuronide, acacetin-7-O-(6″-O-malonyl)-glucoside, and acacetin. A Waters ACQUITY BEH C_(18) column(2.1 mm × 100 mm, 1.7 μm) was used, with acetonitrile and a water solution containing 0.1% formic acid and 0.1% phosphoric acid as the mobile phase for gradient elution. The detection wavelength was set at 330 nm, with a flow rate of 0.4 mL·min~(-1), and the column temperature was maintained at 35 ℃. The 8 components demonstrated good linearity(r≥0.999 9) over a wide mass concentration range(50 or 100 times). The average recovery rate ranged from 97.5% to 105.1%, and the relative standard deviations(RSDs) were 0.90% to 3.4%(n= 6), indicating that the method was simple, accurate, and reliable. In 17 batches of D. moldavica samples, the content of these 8 components ranged from 0.405 to 2.10, 0.063 to 0.342, 0.446 to 2.43, 0.415 to 1.47, 1.57 to 4.34, 0.173 to 0.386, 1.00 to 5.40, and 0.069 to 0.207 mg·g~(-1), respectively. These results indicate significant differences in the internal quality of the samples, highlighting the need for strict quality control to ensure their pharmacodynamic efficacy. This study provides a scientific basis for the rapid discovery of pharmacodynamic substances, comprehensive quality control, and the formulation or revision of quality standards for D. moldavica.
Tandem Mass Spectrometry/methods* ; Chromatography, High Pressure Liquid/methods* ; Drugs, Chinese Herbal/chemistry* ; Lamiaceae/chemistry* ; Flavonoids/chemistry*

The ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS~E) technology was employed to compare the chemical components between the aerial and underground parts of Coptis chinensis samples from different batches. According to the retention time, molecular ion peak, and LC-MS~E fragment information of the reference substances and available literature, we identified a total of 40 components. Thirty-three and 31 compounds were respectively identified in the underground part(taproots) and the aerial part(stems and leaves) of C. chinensis. Among them, 24 compounds, including alkaloids(e.g., berberine and jatrorrhizine) and phenolic acids(e.g., chlorogenic acid, quinic acid, and tanshinol), were common in the two parts. In addition, differential components were also identified, such as magnoline glucoside in the underground part and(±) lariciresionol-4-β-D-glucopyranoside in the aerial part. The analysis of fragmentation pathways based on spectra of reference substances indicated the differences among samples of different batches. Furthermore, we performed the principal component analysis(PCA) for the peak areas of C. chinensis in different batches. The results showed that the underground part and the aerial part were clearly clustered into two groups, indicating that the chemical components contained in the two parts were different. Furthermore, the results of partial least squares discriminant analysis(PLS-DA) identified 31 differential compounds(VIP value>1) between the underground part and the aerial part, mainly including alkaloids, phenolic acids, lignans, and flavonoids. This study proves that C. chinensis possesses great development potential with multiple available compounds in stems and leaves. Moreover, it sheds light on for the development and utilization of non-medicinal organs of C. chinensis and other Chinese medicinal herbs.
Chromatography, High Pressure Liquid/methods* ; Coptis chinensis ; Spectrometry, Mass, Electrospray Ionization/methods* ; Tandem Mass Spectrometry/methods* ; Technology

【Objective】To investigate whether bone marrow mesenchymal stem cells（BMSC）over-expressing FoxM1genecanattenuatelipopolysaccharide（LPS）-inducedapoptosisofalveolarepithelialcells，andexploreitspossi⁃ blemechanism.【Methods】SDratBMSCwereisolatedandculturedbywholebonemarrowadherencemethod.FoxM1 genewasoverexpressedinBMSCbylentiviraltransfection.TheexpressionofthetargetgeneFoxM1wasverifiedbyWestern blot.ApoptosisofA549cellswasmeasuredbyTUNELandflowcytometry.Andthemulti-factorlevelofsupernatantin BMSC-A549co-culturesystemwasdetectedbyMilliplexmethod.【Results】TUNELandflowcytometryconfirmedthat theapoptosisrateofA549inducedbyLPSdecreasedafterco-culturewithBMSCoverexpressingFoxM1，andthediffer⁃ encewasstatisticallysignificant（P <0.05）.MilliplexassayshowedthatthelevelsofIL-13，IL-21，IL-23，MIP-1a， MIP-1bandinBMSCoverexpressing FoxM1 geneandA549co-culturesystemweresignificantlyincreased，whilethe MIP-3alevelissignificantlyreduced.【Conclusion】BMSCoverexpressingFoxM1genecanattenuateLPS-inducedapop⁃ tosisofalveolarepithelialcells.BMSCmayplayananti-apoptoticrolebychangingthelevelsofinflammation-related cytokinesreleasedbyA549cells.

Pelvic inflammatory disease is an infectious disease. At present, Western medicine is mainly treated with antibiotics. However, the situation of antibiotics abuse is so grim that the potential risks such as the imbalance of bacteria, the resistance of bacteria, the production of super bacteria and the increase of adverse reactions are becoming more and more serious. Therefore, it is urgent to find a way to supplement or substitute antibiotics for the treatment of this disease. Traditional Chinese medicine treatment of the disease is effective and has its unique advantages. This paper mainly discusses the advantages and evidences of traditional Chinese medicine (TCM) treatment of pelvic inflammatory disease, to further prove the effectiveness and safety of TCM treatment and to provide medical evidence of reducing antibiotics use.

Objective To investigate the effect of povidone - iodine diluent on proliferation and apoptosis of cervical cancer cell HeLa and to provide the theoretical basis for its clinical application. Methods Human cervical cancer cell line HeLa in logarithmic growth phase were treated with different dilutions of povidone - iodine and the cells treated with physiological saline were set as the control group. The cells viability,morphological change,formation of apoptotic bodies,cell apoptosis and the apoptosis - related protein expression in HeLa cells were assessed by MTT assay,Hoechst33342 staining, AnnexinV / PI flow cytometry and Western blotting. Results Povidone - iodine diluent remarkably inhibited human cervical cancer cell line HeLa growth in a concentration - dependent manner. The inhibitory rates of HeLa cells were 25. 3% , 30. 8% ,33. 4% ,60. 3% ,71. 2% ,85. 3% ,89. 1% and 91. 2% when the concentration of povidone - iodine solution were 0. 001% ,0. 005% ,0. 01% ,0. 05% ,0. 1% ,0. 5% ,1% and 2% ,respectively. The nuclear chromatin of HeLa cells treated with povidone - iodine dilution was agglutinated and contracted,and the nucleus was fragile and appeared apoptotic body,with dense and dense stain or fragment dense staining. With the increase of the concentration of povidone -iodine dilution,the apoptotic rate of HeLa cells increased,so were Caspase - 8 ,Caspase - 3 and cleaved PARP. Conclusion Diluted povidone - iodine can strongly inhibit the proliferation of human cervical cancer cell line HeLa and the possible mechanism was the promotion of apoptosis.

BACKGROUNDThe diagnosis of myelodysplastic syndrome (MDS), especially hypoplastic MDS, and MDS with low blast counts or normal karyotype may be problematic. This study characterized ID4 gene methylation in patients with MDS and aplastic anemia (AA).

METHODSThe methylation status of ID4 was analyzed by bisulfite sequencing polymerase chain reaction (PCR) and quantitative real-time methylation-specific PCR (MethyLight PCR) in 100 patients with MDS and 31 patients with AA.

RESULTSThe MDS group had a higher ID4 gene methylation positivity rate (22.22%) and higher methylation levels (0.21 [0-3.79]) than the AA group (P < 0.05). Furthermore, there were significant differences between the hypoplastic MDS and AA groups, the MDS with low blast count and the AA groups, and the MDS with normal karyotype and the AA groups. The combination of genetic and epigenetic markers was used in much more patients with MDS (62.5% [35/56]) than the use of genetic markers only (51.79% [29/56]).

CONCLUSIONSThese results showed that the detection of ID4 methylation positivity rates and levels could be a useful biomarker for MDS diagnosis.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Anemia, Aplastic ; genetics ; Child ; CpG Islands ; genetics ; DNA Methylation ; genetics ; Female ; Humans ; Inhibitor of Differentiation Proteins ; genetics ; Male ; Middle Aged ; Myelodysplastic Syndromes ; genetics ; Young Adult

OBJECTIVETo study the molecular mechanism of epidermal growth factor receptor (EGFR) signaling pathway in mediating paclitaxel-resistance and improving paclitaxel sensitivity in human melanoma A375 cells.

METHODSHuman melanoma cell line A375 cells were treated with different concentrations of paclitaxel with or without 20 µmol/L AG1478 (EGFR inhibitor), 40 µmol/L PD98059 (extracellular signal conditioning kinase (ERK) 1/2 blockers) or 10 µmol/L LY294002 (PI3K inhibitor). MTT method was used to measure the proliferation of A375 cells. Flow cytometry was used to detect cell cycle and apoptosis in the A375 cells. The expressions of P-EGFR, P-ERK and P-AKT proteins were determined by Western blot analysis.

RESULTSPaclitaxel (0.001 µmol/L to 0.1 µmol/L) inhibited the growth of A375 cells (P < 0.01) and induced apoptosis (P < 0.05) in a dose- and time-dependent manner. AG1478 (20 µmol/L) increased the 0.01 µmol/L paclitaxel-induced inhibition rate from 38.5% to 62.6% at 72 h. Different doses of paclitaxel induced apoptosis in A375 cells by different ways, in which G0/G1 phase cells were decreased and mitotic phase was prolonged at 0.01 µmol/L, and cell cycle arrest at G2/M phase by 0.1 µmol/L paclitaxel. When DNA damage occurred in A375 cells exposed to paclitaxel, expression of P-EGFR, P-ERK and P-AKT proteins was increased. When EGFR signaling pathway was blocked, paclitaxel did not activate MAPK signaling pathway or PI3K/AKT signaling pathway and did not change its effect on cell cycle in vitro. When EGFR was inhibited by 20 µmol/L tyrophostin AG1478, the 0.001 and 0.01 µmol/L paclitaxel-induced early apoptosis rate in A375 cells was increased by 1.73- and 1.80-fold, respectively. When the ERK signaling was blocked by 40 µmol/L PD98059, the 0.001 and 0.01 µmol/L paclitaxel-induced early apoptosis rate in A375 cells was increased by 2.73- and 2.25-fold, respectively. When the AKT signaling was blocked by 10 µmol/L LY294002, the 0.001 and 0.01 µmol/L paclitaxel-induced early apoptosis rate in A375 cells was increased by 2.02- and 1.46-fold, respectively.

CONCLUSIONSHuman melanoma A375 cells produce resistance to paclitaxel (0.001 to 0.1 µmol/L) by activating MAPK signaling and PI3K/AKT signaling pathways. Targeting EGFR, ERK and AKT signaling pathways significantly enhances the cytotoxic effect of paclitaxel on human melanoma cells.

Antineoplastic Agents, Phytogenic ; administration & dosage ; pharmacology ; Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Chromones ; pharmacology ; Dose-Response Relationship, Drug ; Drug Resistance, Neoplasm ; Extracellular Signal-Regulated MAP Kinases ; antagonists & inhibitors ; metabolism ; Flavonoids ; pharmacology ; Humans ; Melanoma ; metabolism ; pathology ; Morpholines ; pharmacology ; Paclitaxel ; administration & dosage ; pharmacology ; Phosphatidylinositol 3-Kinases ; antagonists & inhibitors ; metabolism ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; metabolism ; Quinazolines ; pharmacology ; Receptor, Epidermal Growth Factor ; antagonists & inhibitors ; metabolism ; Signal Transduction ; drug effects ; Tyrphostins ; pharmacology

OBJECTIVESTo evaluate the value of the metastatic to examined lymph nodes (rN) ratio in gastric cancer patients who underwent radical resection.

METHODSIn this retrospective study, data were collected from the medical records of 710 patients who underwent radical gastrectomy (R0) for gastric cancer from 1980 to 2006 in the Department of Surgical Oncology at the First Affiliated Hospital of China Medical University. The patients were divided into 2 groups according to the number of examined lymph nodes: Group 1 consisted of 327 patients with <15 examined lymph nodes and Group 2 consisted of 383 patients with ≥15 lymph nodes. rN categories staging and pN categories were divided separately according to the metastatic lymph node ratio and the examined lymph nodes. The prognostic factors were analyzed by univariate (Log-rank) and multivariate (Cox model) analysis methods.

RESULTSThe median survival time was 74 months (95% CI:55.6-92.4 months) in Group 1 and 96 months (95% CI:77.8-119.2 months) in Group 2, and the difference was not statistically significant (P>0.05). On multivariate analysis, the N ratio remained as an independent prognostic factor in both Group 1 (P<0.01, RR=1.225, 95% CI:1.102-1.362) and Group 2 (P<0.01, RR=1.421, 95% CI:1.269-1.592). However, pN stage was an independent prognostic factor only in Group 1. When the rN ratio classification was applied, there were no significant differences between each categories (P>0.05). However, the overall survival of patients with pN1 disease in Group 1 was significantly shorter than that in Group 2 according to the pN stage classification (P<0.01).

CONCLUSIONSThe metastatic lymph node ratio is an independent prognostic factor of the prognosis of gastric cancer. The staging system based on metastatic lymph node ratio (rN) is more reliable than the system based on the number of metastatic lymph nodes in the prediction of the prognosis of gastric cancer.

Adult ; Aged ; Aged, 80 and over ; Female ; Follow-Up Studies ; Humans ; Lymph Nodes ; pathology ; Lymphatic Metastasis ; Male ; Middle Aged ; Prognosis ; Retrospective Studies ; Stomach Neoplasms ; diagnosis ; pathology ; surgery

OBJECTIVETo investigate the clinicopathological characteristics and prognostic factors of stage III gastric cancer.

METHODSA retrospectively study of 1007 patients with Stage III gastric cancer in a single institute in China was performed. The patients underwent surgical resection from January 1991 to December 2005. Univariate and multivariate analyses were performed using log-rank test and Cox proportional hazards model to access the prognostic factors in stage III gastric cancer patients who received curative (R0) gastric resection.

RESULTSThe mean age of the 1007 patients was 58.7 years and the male-to-female ratio was 2.6:1.0. There were 242 patients with stage IIIA disease, 403 patients with stage IIIB, and 362 patients with stage IIIC. R0, R1, and R2 resection were performed in 754 patients (74.9%), 56 patients (5.5%), and 197 patients (19.6%), respectively. The 5-year survival rate (37.8%) of patients who received R0 resection was significant higher than that of patients who received R1(21.2%) and R2(8.9%) resection (P<0.05). Multivariate analysis revealed that pN stage, pT stage, and Borrmann type were independent prognostic factors (all P<0.01).

CONCLUSIONSStage III gastric cancer patients have certain clinicopathological characteristics and R0 resection should be performed if possible. Lymph node count, depth of tumor invasion, and Borrmann type are independent prognostic factors in stage III gastric cancer patients undergoing R0 resection.

Adult ; Aged ; Aged, 80 and over ; Female ; Follow-Up Studies ; Humans ; Male ; Middle Aged ; Prognosis ; Retrospective Studies ; Stomach Neoplasms ; pathology ; surgery ; Young Adult