Role of tumor necrosis factor α in endothelial-mesenchymal transition in vitro / 中华烧伤杂志

<p><b>OBJECTIVE</b>To observe the role of tumor necrosis factor α (TNF-α) in endothelial-mesenchymal transition (EnMT), and to explore the mechanism of fibrosis disease.</p><p><b>METHODS</b>Human umbilical vein endothelial cells (HUVEC) from umbilical cord of healthy fetus were isolated by enzymatic digestion and identified by immunofluorescence assay. The third to fifth generations of cultured HUVEC in logarithmic phase were harvested and seeded in 12-well plates and 6-well plates, and they were divided into control group (ordinary culture without any stimulation), 5, 10, 25, 50, and 100 ng/mL TNF-α groups (5, 10, 25, 50, 100 ng/mL of TNF-α was respectively added into the nutrient solution) according to the random number table, with three samples in each group. After being cultured for 72 hours, the cell morphology was observed under inverted phase-contrast microscope; the expression levels of coagulation factor VIII and α smooth muscle actin (α-SMA) were detected by immunofluorescence assay, and the ratios of numbers (absorbance values) of cells with expression of both factors were calculated. The mRNA expression levels of cadherin, α-SMA, and type I collagen were detected by RT-PCR (denoted as gray value ratio). Data were processed with one-way analysis of variance and LSD test.</p><p><b>RESULTS</b>(1) The shape of primary HUVEC was round, short-spindle, or flat, and cells grew vigorously in cobblestone appearance after passages. After being subcultured for 1, 2, 3, 4, 5 passage (s), the positive rate of coagulation factor VIII of HUVEC was respectively (85.5 ± 1.8)%, (88.1 ± 5.0)%, (93.6 ± 3.7)%, (92.9 ± 4.8)%, (89.5 ± 1.1)%, and they were significantly higher than that of primary HUVEC [(81.4 ± 3.8)%, with F values all equal to 7.481, P values all below 0.05]. (2) As compared with that in control group, the appearance of cells in 5, 10, 25, 50, and 100 ng/mL TNF-α groups was gradually transformed from round, short-spindle, or flat shape to long-spindle shape with reduced intercellular junction and larger intercellular gap along with the increase in the concentration of TNF-α. (3) The ratios of numbers and the absorbance values of coagulation factor VIII and α-SMA double positive cells in control group (0.055 ± 0.015, 0.078 ± 0.017) were significantly lower than those in 5, 10, 25, 50, and 100 ng/mL TNF-α groups (0.257 ± 0.106, 0.280 ± 0.129, 0.505 ± 0.059, 0.817 ± 0.035, 0.929 ± 0.101 and 0.437 ± 0.040, 0.456 ± 0.097, 0.496 ± 0.082, 0.787 ± 0.131, 0.885 ± 0.087, with F value respectively 45.009, 50.099, P values all below 0.01). (4) The expression levels of cadherin mRNA in 5, 10, 25, 50, and 100 ng/mL TNF-α groups were 0.70 ± 0.05, 0.63 ± 0.06, 0.60 ± 0.10, 0.45 ± 0.16, and 0.26 ± 0.14, and it was significantly lower in the latter four groups than in control group (0.83 ± 0.03, with F values all equal to 11.593, P < 0.05 or P < 0.01). The mRNA expression levels of α-SMA and collagen I in 5, 10, 25, 50, and 100 ng/mL TNF-α groups were 0.45 ± 0.10, 0.51 ± 0.16, 0.49 ± 0.12, 0.60 ± 0.09, 0.76 ± 0.03 and 0.38 ± 0.18, 0.45 ± 0.15, 0.52 ± 0.12, 0.66 ± 0.17, 0.76 ± 0.20, and they were significantly higher in the latter three groups than in control group (0.37 ± 0.14, 0.31 ± 0.12, with F value respectively 7.839, 2.898, P < 0.05 or P < 0.01).</p><p><b>CONCLUSIONS</b>TNF-α can obviously promote EnMT in a dose-dependent manner. EnMT may be another significant source of myofibroblasts that contributes to fibrotic tissue in scar formation.</p>

Effect of granulocyte-macrophage colony-stimulating factor on the mammalian target of sirolimus signaling pathway in the wound of rat / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) on wound healing and mammalian target of sirolimus (rapamycin) signaling pathway in rats.</p><p><b>METHODS</b>Fifty SD rats were divided into control group (n = 25) and treatment group (n = 25) according to the random number table. All rats were inflicted with 2 cm × 2 cm full-thickness skin wound on the back. Recombinant human GM-CSF gel (10 µg/cm(2)) was applied onto the wounds in treatment group, and the actual quantity was 1 × 10(-4) µg/cm(2). Gel vehicle (10 µg/cm(2)) without any medicine was applied onto the wounds in control group. The treatment was conducted once a day up to the day of wound healing. Five rats from two groups were sacrificed on post injury day (PID) 1, 3, 5, 7, 14 respectively to observe and determine the wound healing rate. Wound tissue samples were collected at the former 4 time points to observe the histopathological changes with HE staining, and to detect the content of GM-CSF with enzyme-linked immunosorbent assay, and the expression levels of GM-CSF, CD31, and the mTOR signal pathway associated molecules P70S6K, phosphorylated (p-) P70S6K, 4E-BP1, p-4E-BP1, mTOR, p-mTOR with Western blotting. Data were processed with t test.</p><p><b>RESULTS</b>(1) Wound healing rates in control group and treatment group were close on PID 1 (t = 0.307, P > 0.05). Wound healing rate in treatment group was obviously higher than that in control group on PID 3, 5, 7, and 14 (with t values from 2.704 to 4.030, P < 0.05 or P < 0.01). (2) Compared with those in control group, more abundant granulation tissue was observed in treatment group, in which an increase in the number of microvessels and obvious proliferation of keratinized epithelial cells in wound margin were observed at each time point. (3) The content and the protein expression level of GM-CSF peaked on PID 3 in two groups, and they were (720.9 ± 0.9) pg/mL, 2.45 ± 0.10 in control group and (910.5 ± 1.3) pg/mL, 2.80 ± 0.48 in treatment group. The content of GM-CSF in treatment group was significantly higher than that in control group at each time point (with t values from 105.743 to 298.971, P values all equal to 0.000). The protein expression level of GM-CSF in treatment group was significantly higher than that in control group on PID 1, 5, and 7 (with t values from 4.070 to 5.275, P values all below 0.01). (4) The expression level of CD31 in treatment group was obviously higher than that in control group on PID 1, 3, and 7 (with t values from 7.237 to 26.401, P values all below 0.01). (5) The expression levels of mTOR and p-mTOR in treatment group were significantly higher than those in control group at each time point (with t values from 2.921 to 23.143, P < 0.05 or P < 0.01). In treatment group, the expression level of P70S6K was obviously higher than that in control group on PID 3, 5, and 7 (with t values from 2.950 to 5.275, P < 0.05 or P < 0.01), and the expression level of p-P70S6K was significantly higher than that in control group on PID 1, 3, and 7 (with t values from 3.307 to 22.793, P < 0.05 or P < 0.01). In treatment group, the expression level of 4E-BP1 was significantly lower than that in control group on PID 1, 3, and 5 (with t values from 2.449 to 6.431, P < 0.05 or P < 0.01), but the expression level of p-4E-BP1 was significantly higher than that in control group on PID 1, 3, and 7 (with t values from 5.522 to 11.613, P values all below 0.01).</p><p><b>CONCLUSIONS</b>GM-CSF can promote wound healing in rats by activating mTOR signaling pathway through phosphorylating mTOR proteins and its downstream signal molecules P70S6K and 4E-BP1.</p>