This study aims to explore the effects and potential mechanisms of Modified Shuyu Pills in ameliorating depressive behaviors in the mouse model of vascular dementia(VaD). Seventy-two three-month-old male C57BL/6 mice were assigned into six groups: sham, model, low-, medium-, and high-dose Modified Shuyu Pills, and fluoxetine. The other five groups except the sham group underwent bilateral common carotid artery stenosis combined with chronic unpredictable stress. Depressive behaviors were assessed by the sucrose preference test and tail suspension test. Cerebral blood flow was measured by laser speckle imaging. Protein levels of low density lipoprotein receptor(LDLR), mitogen-activated protein kinase kinase(MEK), phosphorylated(p)-MEK, extracellular signal-regulated kinase(ERK), and p-ERK in the ventral tegmental area(VTA) and nucleus accumbens(NAc) were determined by Western blot. The fluorescence intensity of myelin basic protein(MBP) in the VTA and NAc were measured by immunofluorescence. Myelin sheath morphology in the VTA and NAc was observed by luxol fast blue staining, and the ultrastructure of myelin sheath in the VTA and NAc was examined by transmission electron microscopy. In the tail suspension test, the immobility time of the model group was longer than that of the sham group(P<0.01). In the sucrose preference test, the sucrose preference rate of the model group was lower than that of the sham group(P<0.01). After intervention with Modified Shuyu Pills, the immobility time in the tail suspension test was shortened(P<0.01), and the sucrose preference rate increased(P<0.01). Laser speckle imaging results showed that compared with the sham group, the model group showed reduced cerebral blood flow(P<0.01), and the reduction was reversed by medium-and high-dose Modified Shuyu Pills(P<0.01). Western blot results indicated that the relative expression levels of LDLR, p-MEK/MEK, and p-ERK/ERK in the VTA and NAc of the model group were lower than those in the sham group(P<0.01). Medium-and high-dose Modified Shuyu Pills reversed this trend(P<0.01). Immunofluorescence results showed that the fluorescence intensity of MBP in the VTA and NAc of the model group was lower than that of the sham group(P<0.01). The medium-and high-dose Modified Shuyu Pills groups showed increased fluorescence intensity of MBP in the VTA compared with the model group(P<0.01). In the NAc, the fluorescence intensity of MBP in all the groups of Modified Shuyu Pills increased to varying degrees compared with that in the model group(P<0.01). Luxol fast blue staining results showed that the model group presented lighter staining intensity and looser arrangement of myelin fibers than the sham group, indicating significant demyelination in the model group. However, after intervention with medium-and high-dose Modified Shuyu Pills, the staining intensity and myelin sheath structure in the VTA and NAc were improved. Transmission electron microscopy results revealed that the myelin sheath in the VTA and NAc of the sham group was intact and dense, while the model group exhibited extensive myelin loss, with myelin sheath degeneration and disintegration. After intervention with Modified Shuyu Pills, the myelin sheath loss in the VTA and NAc of mice was reduced, and the proportion of myelinated tissue increased. In summary, Modified Shuyu Pills may promote myelination via the VTA-NAc circuit by upregulating the LDLR/MEK/ERK signaling pathway, thereby ameliorating depressive-like behaviors in VaD mice.
Animals ; Male ; Drugs, Chinese Herbal/administration & dosage* ; Mice ; Ventral Tegmental Area/metabolism* ; Mice, Inbred C57BL ; Disease Models, Animal ; Depression/genetics* ; Receptors, LDL/genetics* ; Dementia, Vascular/psychology* ; MAP Kinase Signaling System/drug effects* ; Nucleus Accumbens/metabolism* ; Behavior, Animal/drug effects* ; Humans ; Myelin Sheath/drug effects* ; Extracellular Signal-Regulated MAP Kinases/genetics*

Based on the focal adhesion kinase(FAK)/steroid receptor coactivator(Src)/extracellular regulated protein kinase(ERK) pathway, this study explored the effects of Xihuang Pills on angiogenesis, invasion, and metastasis in prostate cancer. Liquid chromatography-tandem mass spectrometry(LC-MS/MS) was used to analyze and identify the active ingredients of Xihuang Pills. Bioinformatics techniques, including R language and Perl programs, were employed to analyze the interactions between prostate cancer-related targets and the potential targets of Xihuang Pills. A subcutaneous transplantation tumor model of prostate cancer was established in nude mice using PC3 cells to verify the efficacy and molecular mechanisms of Xihuang Pills. In vitro cellular experiments, including cell proliferation assays(CCK-8), Transwell assays, scratch assays, real-time quantitative reverse transcription PCR, and Western blot, were used to detect the effects of Xihuang Pills on the proliferation, invasion, and migration of prostate cancer cells, as well as on FAK/Src/ERK pathway-related targets. LC-MS/MS identified 99 active ingredients in Xihuang Pills, including gallic acid, gentisic acid, artemisinin, corilagin, phenylbutazone-glucoside, thujic acid, and arecoic acid B. Network pharmacological analysis of the active ingredients in Xihuang Pills revealed that the FAK/Src/ERK signaling pathway was a key pathway in its anti-prostate cancer effects. In vivo and in vitro experiments confirmed that Xihuang Pills significantly inhibited the proliferation, invasion, and migration of PC3 and LNCaP cells, suppressed the growth of PC3 subcutaneous tumors, and reduced the protein expression levels related to the FAK/Src/ERK signaling pathway. In conclusion, the inhibition of angiogenesis, invasion, and metastasis by regulating the FAK/Src/ERK pathway is one of the mechanisms by which Xihuang Pills exert anti-prostate cancer effects.
Humans ; Male ; Prostatic Neoplasms/enzymology* ; Drugs, Chinese Herbal/chemistry* ; Animals ; Mice ; Cell Proliferation/drug effects* ; Mice, Nude ; Cell Movement/drug effects* ; Cell Line, Tumor ; src-Family Kinases/genetics* ; Neovascularization, Pathologic/metabolism* ; Neoplasm Metastasis ; Neoplasm Invasiveness ; Focal Adhesion Kinase 1/genetics* ; Extracellular Signal-Regulated MAP Kinases/genetics* ; MAP Kinase Signaling System/drug effects* ; Focal Adhesion Protein-Tyrosine Kinases/genetics* ; Signal Transduction/drug effects* ; Angiogenesis

OBJECTIVE: To investigate the hemostatic effect of modified Sijunzi Granules (MSG) in primary immune thrombocytopenia (ITP) zebrafish model and explore the potential mechanism. METHODS: AB strain wild type zebrafish were treated with simvastatin (6 µmol/L) for 24 h to establish the hemorrhage model (model control group). The zebrafish were treated with MSG at different doses (55.6, 167, and 500 µg/mL), respectively. The hemostatic effect was assessed by examining the intestinal bleeding and hemostatic rate. 5-Hydroxytryptamine (5-HT) content was determined using enzyme-linked immunosorbent assay (ELISA) assay. The expressions of 5-HT2aR, 5-HT2bR, and SERT genes were detected by quantitative real-time polymerase chain reaction(PCR). The protein expressions of protein kinase B (Akt), p-Akt, extracellular regulated protein kinases (Erk), and p-Erk were examined using Western blot analysis. RESULTS: The intestinal bleeding rate was 37%, 40%, and 80% in the 55.6, 167, and 500 µg/mL dose of MSG, respectively, in which 55.6 and 167 µg/mL MSG dose groups were associated with significantly decreased intestinal bleeding rate when compared with the model control group (70%, P<0.05). Significantly higher hemostatic rates were also observed in the 55.6 (54%) and 167 (52%) µg/mL MSG dose groups (P<0.05). MSG increased the 5-HT content and mRNA expression levels of 5-HT2aR, 5-HT2bR, and SERT (P<0.05). In addition, caspase3/7 activity was inhibited (P<0.05). Significant increase in p-Akt and p-Erk was also detected after treatment with MSG (P<0.05). CONCLUSIONS MSG could reduce the incidence and severity of intestinal bleeding in zebrafish by activating MAPK/Erk and PI3K/Akt signal pathways through regulating the levels of 5-HT and its receptors, which may provide evidence for the treatment of ITP.
Animals ; Zebrafish ; Serotonin/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Drugs, Chinese Herbal/pharmacology* ; Hemostatics/pharmacology* ; MAP Kinase Signaling System/drug effects* ; Receptors, Serotonin/genetics* ; Extracellular Signal-Regulated MAP Kinases/metabolism* ; Signal Transduction/drug effects* ; Hemostasis/drug effects*

OBJECTIVETo investigate the effects of evodiamine (Evo), a component of Evodiaminedia rutaecarpa (Juss.) Benth, on cardiomyocyte hypertrophy induced by angiotensin II (Ang II) and further explore the potential mechanisms.

METHODSCardiomyocytes from neonatal Sprague Dawley rats were isolated and characterized, and then the cadiomyocyte cultures were randomly divided into control, model (Ang II 0.1 μmol/L), and Evo (0.03, 0.3, 3 μmol/L) groups. The cardiomyocyte surface area, protein level, intracellular free calcium ([Ca]) concentration, activity of nitric oxide synthase (NOS) and content of nitric oxide (NO) were measured, respectively. The mRNA expressions of atrial natriuretic factor (ANF), calcineurin (CaN), extracellular signal-regulated kinase-2 (ERK-2), and endothelial nitric oxide synthase (eNOS) of cardiomyocytes were analyzed by real-time reverse transcriptionpolymerase chain reaction. The protein expressions of calcineurin catalytic subunit (CnA) and mitogen-activated protein kinase phosphatase-1 (MKP-1) were detected by Western blot analysis.

RESULTSCompared with the control group, Ang II induced cardiomyocytes hypertrophy, as evidenced by increased cardiomyocyte surface area, protein content, and ANF mRNA expression; increased intracellular free calcium ([Ca]) concentration and expressions of CaN mRNA, CnA protein, and ERK-2 mRNA, but decreased MKP-1 protein expression (P<0.05 or P<0.01). Compared with Ang II, Evo (0.3, 3 μmol/L) significantly attenuated Ang II-induced cardiomyocyte hypertrophy, decreased the [Ca] concentration and expressions of CaN mRNA, CnA protein, and ERK-2 mRNA, but increased MKP-1 protein expression (P<0.05 or P<0.01). Most interestingly, Evo increased the NOS activity and NO production, and upregulated the eNOS mRNA expression (P<0.05).

CONCLUSIONEvo signifificantly attenuated Ang II-induced cardiomyocyte hypertrophy, and this effect was partly due to promotion of NO production, reduction of [Ca]i concentration, and inhibition of CaN and ERK-2 signal transduction pathways.

Angiotensin II ; Animals ; Atrial Natriuretic Factor ; metabolism ; Calcineurin ; genetics ; metabolism ; Calcium ; metabolism ; Dual Specificity Phosphatase 1 ; genetics ; metabolism ; Extracellular Signal-Regulated MAP Kinases ; genetics ; metabolism ; Hypertrophy ; Myocytes, Cardiac ; drug effects ; metabolism ; pathology ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type III ; metabolism ; Quinazolines ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Rats, Sprague-Dawley

OBJECTIVESTo investigate the hair growth-promoting effect of Miscanthus sinensis var. purpurascens (MSP) flower extracton on in vitro and in vivo models.

METHODSMSP flower extract was extracted in 99.9% methanol and applied to examine the proliferation of human dermal papilla cells (hDPCs) in vitro at the dose of 3.92-62.50 μg/mL and hair growth of C57BL/6 mice in vivo at the dose of 1000 μg/mL. The expression of transforming growth factor β1 (TGF-β1), hepatocyte growth factor (HGF), β-catenin, substance P was measured by relative quantitative realtime polymerase chain reaction. Histopathological and immunohistochemical analysis were performed.

RESULTSMSP (7.81 μg/mL) down-regulated TGF-β1 and up-regulated HGF and β-catenin in hDPCs (P<0.01). MSP (1000 μg/mL)-treated mice showed the earlier transition of hair follicles from the telogen to the anagen phase. The number of mast cells was lower in the MSP-treated mice than in other groups (P<0.05 vs. NCS group). Substance P and TGF-β1 were expressed in hair follicles and skin of the MSP group lower than that in negative control. Stem cell factor in hair follicles was up-regulated in the MSP-treated mice (P<0.01).

CONCLUSIONSThe MSP flower extract may have hair growth-promotion activities.

Animals ; Antioxidants ; pharmacology ; Cell Count ; Cell Proliferation ; drug effects ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Female ; Flowers ; chemistry ; Hair Follicle ; cytology ; drug effects ; growth & development ; Hepatocyte Growth Factor ; metabolism ; Humans ; Mast Cells ; cytology ; Mice, Inbred C57BL ; Phosphorylation ; drug effects ; Plant Extracts ; pharmacology ; Poaceae ; chemistry ; RNA, Messenger ; genetics ; metabolism ; Skin ; metabolism ; Stem Cell Factor ; metabolism ; Stress, Psychological ; pathology ; Substance P ; metabolism ; Transforming Growth Factor beta ; genetics ; metabolism ; Vascular Endothelial Growth Factor A ; genetics ; metabolism ; beta Catenin ; metabolism

The aims of the present study were to evaluate the effects of puerarin on angiotensin II-induced cardiac fibroblast proliferation and to explore the molecular mechanisms of action. Considering the role of HO in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation, we hypothesized that modulating catalase activity would be a potential target in regulating the redox-sensitive pathways. Our results showed that the activation of Rac1 was dependent on the levels of intracellular HO. Puerarin blocked the phosphorylation of extracellular regulated protein kinases (ERK)1/2, abolished activator protein (AP)-1 binding activity, and eventually attenuated cardiac fibroblast proliferation through the inhibition of HO-dependent Rac1 activation. Further studies revealed that angiotensin II treatment resulted in decreased catalase protein expression and enzyme activity, which was disrupted by puerarin via the upregulation of catalase protein expression at the transcriptional level and the prolonged protein degradation. These findings indicated that the anti-proliferation mechanism of puerarin was mainly through blocking angiontensin II-triggered downregulation of catalase expression and HO-dependent Rac1 activation.
Angiotensin II ; pharmacology ; Angiotensin II Type 1 Receptor Blockers ; pharmacology ; Animals ; Animals, Newborn ; Catalase ; genetics ; metabolism ; Cell Proliferation ; drug effects ; Cells, Cultured ; Extracellular Signal-Regulated MAP Kinases ; antagonists & inhibitors ; metabolism ; Fibroblasts ; Gene Expression Regulation ; drug effects ; Heart ; drug effects ; Hydrogen Peroxide ; metabolism ; pharmacology ; Isoflavones ; pharmacology ; Mice ; Myocardium ; cytology ; enzymology ; metabolism ; NADPH Oxidases ; antagonists & inhibitors ; metabolism ; Neuropeptides ; metabolism ; Signal Transduction ; drug effects ; Transcription Factor AP-1 ; antagonists & inhibitors ; metabolism ; Transcriptional Activation ; drug effects ; rac1 GTP-Binding Protein ; metabolism

Animals ; Cell Nucleus ; enzymology ; Drosophila Proteins ; genetics ; metabolism ; Drosophila melanogaster ; Extracellular Signal-Regulated MAP Kinases ; genetics ; metabolism ; Long-Term Potentiation ; physiology ; MAP Kinase Signaling System ; physiology ; Memory Consolidation ; physiology ; Neurons ; cytology ; enzymology

BACKGROUNDCathepsin L (CatL) is a cysteine protease with strong matrix degradation activity that contributes to photoaging. Mannose phosphate-independent sorting pathways mediate ultraviolet A (UVA)-induced alternate trafficking of CatL. Little is known about signaling pathways involved in the regulation of UVA-induced CatL expression and activity. This study aims to investigate whether a single UVA irradiation affects CatL expression and activity and whether mitogen-activated protein kinase (MAPK)/activator protein-1 (AP-1) pathway is involved in the regulation of UVA-induced CatL expression and activity in human dermal fibroblasts (HDFs).

METHODSPrimary HDFs were exposed to UVA. Cell proliferation was determined by a cell counting kit. UVA-induced CatL production and activity were studied with quantitative real-time reverse transcription polymerase chain reaction (RT-PCR), Western blotting, and fluorimetric assay in cell lysates collected on three consecutive days after irradiation. Time courses of UVA-activated JNK and p38MAPK signaling were examined by Western blotting. Effects of MAPK inhibitors and knockdown of Jun and Fos on UVA-induced CatL expression and activity were investigated by RT-PCR, Western blotting, and fluorimetric assay. Data were analyzed by one-way analysis of variance.

RESULTSUVA significantly increased CatL gene expression, protein abundance, and enzymatic activity for three consecutive days after irradiation (F = 83.11, 56.14, and 71.19, respectively; all P < 0.05). Further investigation demonstrated phosphorylation of JNK and p38MAPK activated by UVA. Importantly, inactivation of JNK pathway significantly decreased UVA-induced CatL expression and activity, which were not affected by p38MAPK inhibition. Moreover, knockdown of Jun and Fos significantly attenuated basal and UVA-induced CatL expression and activity.

CONCLUSIONSUVA enhances CatL production and activity in HDFs, probably by activating JNK and downstreaming AP-1. These findings provide a new possible molecular approach for antiphotoaging therapy.

Anthracenes ; pharmacology ; Cathepsin L ; metabolism ; Cells, Cultured ; Child ; Child, Preschool ; Enzyme Inhibitors ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; antagonists & inhibitors ; Fibroblasts ; cytology ; drug effects ; metabolism ; radiation effects ; Humans ; Imidazoles ; pharmacology ; MAP Kinase Signaling System ; drug effects ; radiation effects ; Oncogene Proteins v-fos ; genetics ; metabolism ; Proto-Oncogene Proteins c-jun ; genetics ; metabolism ; Pyridines ; pharmacology ; Skin ; cytology ; Ultraviolet Rays

PURPOSE: Our previous studies have shown that oncostatin M (OSM) promotes trophoblast invasion activity through increased enzyme activity of matrix metalloproteinase (MMP)-2 and -9. We further investigated OSM-induced intracellular signaling mechanisms associated with these events in the immortalized human trophoblast cell line HTR8/SVneo. MATERIALS AND METHODS: We investigated the effects of OSM on RNA and protein expression of MMP-2 and -9 in the first-trimester extravillous trophoblast cell line (HTR8/SVneo) via Western blot. The selective signal transducer and activator of transcription (STAT)3 inhibitor, stattic, STAT3 siRNA, and extracellular signal-regulated kinase (ERK) siRNA were used to investigate STAT3 and ERK activation by OSM. The effects of STAT3 and ERK inhibitors on OSM-induced enzymatic activities of MMP-2 and -9 and invasion activity were further determined via Western blot and gelatin zymography. RESULTS: OSM-induced MMP-2 and -9 protein expression was significantly suppressed by STAT3 inhibition with stattic and STAT3 siRNA silencing, whereas the ERK1/2 inhibitor (U0126) and ERK silencing significantly suppressed OSM-induced MMP-2 protein expression. OSM-induced MMP-2 and MMP-9 enzymatic activities were significantly decreased by stattic pretreatment. The increased invasion activity induced by OSM was significantly suppressed by STAT3 and ERK1/2 inhibition, though to a greater extent by STAT3 inhibition. CONCLUSION: Both STAT3 and ERK signaling pathways are involved in OSM-induced invasion activity of HTR8/SVneo cells. Activation of STAT3 appears to be critical for the OSM-mediated increase in invasiveness of HTR8/SVneo cells.
Blotting, Western ; Cell Movement/*drug effects ; Cell Proliferation/*drug effects ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Humans ; Matrix Metalloproteinase 2/genetics/*metabolism ; Matrix Metalloproteinase 9/genetics/*metabolism ; Oncostatin M/genetics/*metabolism ; Phosphorylation/drug effects ; RNA, Messenger/metabolism ; RNA, Small Interfering ; STAT3 Transcription Factor/*metabolism ; Signal Transduction/*drug effects

BACKGROUNDPreviously, we reported that dual-specificity phosphatase 1 (DUSP1) was differentially expressed in endometrioid adenocarcinoma (EEA). However, the role of DUSP1 in EEA progression and the relationship between DUSP1 and medroxyprogesterone (MPA) are still unclear.

METHODSThe expression of DUSP1 in EEA specimens was detected by immunohistochemical analysis. The effect of DUSP1 on cell proliferation was analyzed by Cell Counting Kit 8 and colony formation assay, and cell migration was analyzed by transwell assay. MPA-induced DUSP1 expression in EEA cells was measured by Western blot.

RESULTSDUSP1 expression was deficient in advanced International Federation of Gynecology and Obstetrics stage, high-grade and myometrial invasive EEA. In EEA cell lines (Hec1A, Hec1B, RL952, and Ishikawa), the DUSP1 expression was substantially higher in Ishikawa cells than in other cell lines (P < 0.05). Knockdown of DUSP1 promoted Ishikawa cells proliferation, migration, and activation of mitogen-activated protein kinases/extracellular signal-regulated kinase (MAPK/Erk) pathway. MPA-induced DUSP1 expression and inhibited MAPK/Erk pathway in Ishikawa cells.

CONCLUSIONSOur data suggest that DUSP1 deficiency promotes EEA progression via MAPK/Erk pathway, which may be reversed by MPA, suggesting that DUSP1 may serve as a potential therapeutic target for the treatment of EEA.

Carcinoma, Endometrioid ; metabolism ; Cell Culture Techniques ; Cell Proliferation ; genetics ; physiology ; Dual-Specificity Phosphatases ; genetics ; metabolism ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Female ; Humans ; Mitogen-Activated Protein Kinases ; metabolism