Journal: PLoS ONE

Article Title: Tetramethylpyrazine (TMP), an Active Ingredient of Chinese Herb Medicine Chuanxiong, Attenuates the Degeneration of Trabecular Meshwork through SDF-1/CXCR4 Axis

doi: 10.1371/journal.pone.0133055

Figure Lengend Snippet: A , Immunohistochemical staining analysis indicated that the primary cultured trabecular meshwork (PHTM) cells are positive for CD44, FN and LN. B, RT-PCR analysis indicated that myocilin expression in PHTM cells was strongly up-regulated after a 7-day incubation with dexamethasone (DEX; 0.392±0.134 vs. 1.461±0.307; p<0.01). C, The cell cycle analysis demonstrated that PHTM cells were dramatically arrested in G1 (G1: 94.37±3.45% and S: 3.43±0.06%), indicative of a non-dividing cell status. D, The RT-PCR and western blot analyses indicated that both the mRNA and protein levels of CXCR4 were markedly increased in PHTM cells after a 48-h incubation with TGF-β1, TGF-β2 or DEX (CXCR4 protein expression: Con, 0.076±0.014%; TGF-β1, 0.222±0.036%; TGF-β2, 0.164±0.031%; DEX, 0.155±0.025%; p<0.01. CXCR4 mRNA expression: Con, 0.435±0.151%; TGF-β1, 1.271±0.195%; TGF-β2, 1.244±0.261%; and DEX, 1.217±0.233%; p<0.01). β-actin or GAPDH was included as a loading control. E, The relative expression of CXCR4 in PHTM cells was quantified by densitometry, and the data are presented as histograms. All the results were confirmed in three independent experiments. The error bars represent the standard deviation of the mean (n = 3). The asterisks indicate statistically significant differences between the control and experimental cells (*p<0.01).

Article Snippet: Primary human trabecular meshwork (PHTM) cells (Cat. No. 6590, ScienCell, San Diego, CA, USA; see http://www.sciencellonline.com for details) were cultured in TM cell growth medium (TMCM, Cat. No. 6591, ScienCell), which contains basic medium (BM, ScienCell), 2% fetal bovine serum (FBS, Cat. No. 0010, ScienCell), 1% TM cell growth supplement (Cat. No. 6592, ScienCell) and 1% penicillin/streptomycin solution (P/S, Cat. No. 0503, ScienCell).

Techniques: Immunohistochemical staining, Staining, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Expressing, Incubation, Cell Cycle Assay, Western Blot, Control, Standard Deviation

Journal: PLoS ONE

Article Title: Tetramethylpyrazine (TMP), an Active Ingredient of Chinese Herb Medicine Chuanxiong, Attenuates the Degeneration of Trabecular Meshwork through SDF-1/CXCR4 Axis

doi: 10.1371/journal.pone.0133055

Figure Lengend Snippet: A, Cells were treated with TGF-β1 (5 ng/mL) for 48 h in the presence of TMP (100 μM), AMD3100 (10 μg/mL) or PBS. RT-PCR and western blot analyses indicated that CXCR4 expression was up-regulated in PHTM cells by TGF-β1. However, this up-regulation was counteracted by TMP or AMD3100 (CXCR4 protein expression: Con, 0.24±0.031%; TGF-β1, 0.918±0.009%; TGF-β1+AMD3100, 0.681±0.135%; TGF-β1+TMP, 0.422±0.045%; p<0.01. CXCR4 mRNA expression: Con, 0.663±0.282%; TGF-β1, 1.192±0.195%; TGF-β1+AMD3100, 0.496±0.166%; TGF-β1+TMP, 0.434±0.037%; p<0.01). B, The relative expression of CXCR4 in PHTM cells was quantified by densitometry, and the data are presented graphically. C, Immunofluorescence staining revealed that CXCR4 expression in GTM cells was down-regulated by TMP. D, The western blot analysis indicated that CXCR4 protein expression in GTM cells was markedly down-regulated by TMP. All the results were confirmed in three independent experiments. The error bars represent the standard deviation of the mean (n = 3). #Statistically significant differences between the control and TGF-β1; *statistically significant differences between TGF-β1 and TGF-β1+TMP, TGF-β1+AMD3100 (#p<0.05; *p<0.05).

Article Snippet: Primary human trabecular meshwork (PHTM) cells (Cat. No. 6590, ScienCell, San Diego, CA, USA; see http://www.sciencellonline.com for details) were cultured in TM cell growth medium (TMCM, Cat. No. 6591, ScienCell), which contains basic medium (BM, ScienCell), 2% fetal bovine serum (FBS, Cat. No. 0010, ScienCell), 1% TM cell growth supplement (Cat. No. 6592, ScienCell) and 1% penicillin/streptomycin solution (P/S, Cat. No. 0503, ScienCell).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Immunofluorescence, Staining, Standard Deviation, Control

Journal: PLoS ONE

Article Title: Tetramethylpyrazine (TMP), an Active Ingredient of Chinese Herb Medicine Chuanxiong, Attenuates the Degeneration of Trabecular Meshwork through SDF-1/CXCR4 Axis

doi: 10.1371/journal.pone.0133055

Figure Lengend Snippet: A, TMP inhibits the TGF-β1-stimulated cytoskeleton arrangements in PHTM cells compared with AMD3100 treatment. The photomicrographs captured using a confocal microscope show that actin stress fibers and lamellipodial protrusions formed in PHTM cells upon TGF-β1 treatment, and this was inhibited by TMP and AMD3100. B, Images were captured by a confocal laser-scanning microscope (Zeiss LSM 510), and F-actin content was quantified using Zeiss LSM 510 examiner software to evaluate stress-fiber formation. F-actin content: Con: 40.50±3.45; TGF-β1: 78.17±6.08; TGF-β1+AMD3100: 65.17±9.06; TGF-β1+TMP, 57.00±8.05; #p<0.05, *p<0.05). C, Immunofluorescence for TSP-1 in cultured PHTM cells after treatment with TGF-β2 (5 ng/mL) for 48 h in the presence of TMP (100 μM) or AMD 3100 (10 μg/mL) or PBS. After treatment with TGF-β2, the intensity of staining for TSP-1 was considerably, an effect that was markedly reduced after treatment with TMP or AMD3100. D, Real-time PCR analyses indicated that the expression levels of CXCR4, FN and Col 1α were up-regulated in PHTM cells upon TGF-β2 treatment. However, this phenomenon was counteracted by TMP or AMD 3100 treatment (Relative fold change of CXCR4: TGF-β2, 7.16±1.16-fold; TGF-β2+AMD3100, 3.76±0.17-fold; TGF-β2+TMP, 3.14±0.17-fold; p<0.05. Relative fold change of Col 1α: TGF-β2, 3.63±0.67-fold; TGF-β2+AMD3100, 1.80±0.16-fold; TGF-β2+TMP, 1.68±0.16-fold; p<0.05. Relative fold change of FN: TGF-β2, 5.37±0.42-fold; TGF-β2+AMD3100, 3.25±0.15-fold; TGF-β2+TMP, 2.71±0.22-fold; p<0.05. All data are presented compared with the control). All results were confirmed in three independent experiments. The error bars represent standard deviation of the mean (n = 3). # Statistically significant differences between the control and TGF-β; *statistically significant differences between TGF-β and TGF-β+TMP, TGF-β+AMD3100 (#p<0.05; *p<0.05).

Article Snippet: Primary human trabecular meshwork (PHTM) cells (Cat. No. 6590, ScienCell, San Diego, CA, USA; see http://www.sciencellonline.com for details) were cultured in TM cell growth medium (TMCM, Cat. No. 6591, ScienCell), which contains basic medium (BM, ScienCell), 2% fetal bovine serum (FBS, Cat. No. 0010, ScienCell), 1% TM cell growth supplement (Cat. No. 6592, ScienCell) and 1% penicillin/streptomycin solution (P/S, Cat. No. 0503, ScienCell).

Techniques: Microscopy, Laser-Scanning Microscopy, Software, Immunofluorescence, Cell Culture, Staining, Real-time Polymerase Chain Reaction, Expressing, Control, Standard Deviation

Journal: PLoS ONE

Article Title: Tetramethylpyrazine (TMP), an Active Ingredient of Chinese Herb Medicine Chuanxiong, Attenuates the Degeneration of Trabecular Meshwork through SDF-1/CXCR4 Axis

doi: 10.1371/journal.pone.0133055

Figure Lengend Snippet: A, The number of migrative cells was determined using transwell assay. The data showed that TGF-β1 significantly increased the cell migration speed and this effect was markedly reduced by TMP or AMD3100 treatment. B, Histogram representing the number of migrative cells per 40x field. Migration cell numbers per field: Con, 30.33±3.06; TGF-β1, 50.17±6.71; TGF-β1+AMD3100, 36.00±2.50; TGF-β1+TMP, 29.17±0.76; p<0.05. C, Representative phase contrast images demonstrating wound-induced PHTM cell migration at 12 h and 24 h. The scratch wound-healing assay indicated that TMP significantly decreased the migration of PHTM cells accelerated by TGF-β1, compared to the control. Further, the inhibition of cell migration by TMP was more effective than AMD3100. D, Histogram representing the relative wound area per 20x field. All results were confirmed in three independent experiments.

Article Snippet: Primary human trabecular meshwork (PHTM) cells (Cat. No. 6590, ScienCell, San Diego, CA, USA; see http://www.sciencellonline.com for details) were cultured in TM cell growth medium (TMCM, Cat. No. 6591, ScienCell), which contains basic medium (BM, ScienCell), 2% fetal bovine serum (FBS, Cat. No. 0010, ScienCell), 1% TM cell growth supplement (Cat. No. 6592, ScienCell) and 1% penicillin/streptomycin solution (P/S, Cat. No. 0503, ScienCell).

Techniques: Transwell Assay, Migration, Wound Healing Assay, Control, Inhibition

Journal: PLoS ONE

Article Title: Tetramethylpyrazine (TMP), an Active Ingredient of Chinese Herb Medicine Chuanxiong, Attenuates the Degeneration of Trabecular Meshwork through SDF-1/CXCR4 Axis

doi: 10.1371/journal.pone.0133055

Figure Lengend Snippet: A, The early apoptosis of PHTM cells was quantified by flow cytometry after Annexin V and propidium iodide staining. The data showed that TMP had no effect on the incidence of early apoptosis in PHTM cells (Apoptosis: Control, 2.06±0.26%; TMP200, 2.11±0.172%; TMP400, 2.04±0.092%. Death: Control, 2.27±0.25%; TMP200, 2.31±0.171%; TMP400, 2.34±0.17%). B, The percentages of apoptotic and dead PHTM cells are presented in histograms. C, The viability of PHTM cells after treatment with TMP at different concentrations for 48 h was determined using MTT assays, and the data are presented as the percent survival compared with negative controls. TMP treatment did not affect the viability of PHTM cells. All the results were confirmed in three independent experiments. The error bars represent the standard deviation of the mean (n = 3). The asterisks indicate statistically significant differences between the control and experimental cells (*p<0.05).

Article Snippet: Primary human trabecular meshwork (PHTM) cells (Cat. No. 6590, ScienCell, San Diego, CA, USA; see http://www.sciencellonline.com for details) were cultured in TM cell growth medium (TMCM, Cat. No. 6591, ScienCell), which contains basic medium (BM, ScienCell), 2% fetal bovine serum (FBS, Cat. No. 0010, ScienCell), 1% TM cell growth supplement (Cat. No. 6592, ScienCell) and 1% penicillin/streptomycin solution (P/S, Cat. No. 0503, ScienCell).

Techniques: Flow Cytometry, Staining, Control, Standard Deviation